Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
Louisiana State University Louisiana State University
LSU Digital Commons LSU Digital Commons
LSU Historical Dissertations and Theses Graduate School
1991
In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells. In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells.
John Kevin Thibodeaux Louisiana State University and Agricultural & Mechanical College
Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses
Recommended Citation Recommended Citation Thibodeaux, John Kevin, "In Vitro Culture of Bovine Uterine and Oviduct Epithelial Cells." (1991). LSU Historical Dissertations and Theses. 5279. https://digitalcommons.lsu.edu/gradschool_disstheses/5279
This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected].
IN FO R M A T IO N TO U SE R S
This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.
The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely afreet reproduction.
In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.
Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.
University Microfilms International A Bell & Howell Information C om pany
300 North Z eeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600
Order N um ber 9219579
In vitro cu ltu re of bovine u terine and oviduct epithelial cells
Thibodeaux, John Kevin, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1991
U M I300 N. Zeeb Rd.Ann Aibor, MI 48106
IN VITRO CULTURE OF BOVINE UTERINE AND OVIDUCT EPITHELIAL CELLS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in the
Department of Dairy Science
by
John Kevin Thibodeaux
B.S., University of Southwestern Louisiana, 1986 M.S., Louisiana State University, 1989
December, 1991
ACKNOWLEDGEMENTS
The author wishes to thank his major professor, Dr. J.D. Roussel for his
guidance, understanding and support during his graduate study. Thanks are also
extended to Dr. R.A. Godke, minor professor, for his helpful discussions in preparing
this dissertation. Both have provided the confidence and training necessary for me to
develop as a scientist.
Appreciation is also extended to members of the graduate committee, Dr. R.W.
Adkinson, Dr. G.F. Amborski, Dr. L.L. Goodeaux and Dr. R.H. Gough for their
assistance and critical review of this manuscript. Appreciation is also extended to Dr.
Y. Menezo for demonstrating various cell culture techniques used in these experiments
and for the excellent discussions over the past few years.
A very special thanks are extended to fellow graduate students, James R.
Broussard and Mike W. Myers for their assistance with animals and conducting
experiments. The author is also grateful to the following Research Associates, John
Moreau, Department of Dairy Science and Richard Denniston, Department of Animal
Science, for laboratory assistance, Marylin Dietrich, Department of Veterinary
Microbiology and Parasitology, for flow cytometry analysis and Lora Younger,
Department of Veterinary Anatomy, for help with electron microscopy studies.
The author is deeply grateful to his wife, Holly Thibodeaux, for being my
biggest supporter and demonstrating unlimited patience during the long hours of
conducting experiments during the past few years.
Finally, this dissertation is dedicated to my recently bom daughter, Sydney
Paige Thibodeaux, who has provided an enormous joy in my life and may one day
achieve an accomplishment such as this.
TABLE OF CONTENTSPage
ACKNOWLEDGEMENTS ................................................................... ii
LIST OF TABLES ............................................................................... via
LIST OF FIGURES ............................................................................ ix
ABSTRACT .......................................................................................... xi
INTRODUCTION ....................... 1
LITERATURE REVIEW ....................................................................... 3
In Vitro Co-Culture of Mammalian Embryos ................................. 3
Trophoblastic Vesicle Co-Culture ........................................... 3
Uterine Cell Co-Culture ......................................................... 5
Oviductal Cell Co-Culture ..................................................... 8
Granulosa Cell Co-Culture ..................................................... 13
Chick Embryo Co-Culture ..................................................... 16
Other Co-Culture Systems ..................................................... 17
In Vitro Culture of Epithelial Cells ................................................. 18
Uterine Epithelial Cells ......................................................... 18
Oviduct Epithelial Cells ........................................................ 23
Summary............................................................................................. 27
EXPERIMENT I : A METHOD FOR IN VITRO CELLCULTURE OF SUPERFICIAL BOVINE UTERINEENDOMETRIAL EPITHELIUM ........................................................ 29
Introduction ...................................................................................... 29
Materials and Methods ..................................................................... 30
Materials ................................................................................. 30
Procedure ................................................................................ 31
iii
TABLE OF CONTENTS (cont’d)Page
Results ............................................................................................. 35
Discussion ....................................................................... 38
Suppliers .......................................................................................... 39
EXPERIMENT I I : EFFECTS OF STAGE OF THE BOVINE ESTROUS CYCLE ON IN VITRO CHARACTERISTICS OF UTERINE AND OVIDUCT EPITHELIAL CELLS ......................... 40
Introduction ..................................................................................... 40
Materials and Methods .................................................................... 41
Experimental Animals and Treatment Groups........................ 41
Isolation of Epithelial Cells ................................................... 42
In Vitro Characteristics........................................................... 43
Freezing Epithelial Cells ....................................................... 44
Electron Microscopy............................................................... 44
Immunocytochemistry............................................................ 44
Cell Cycle Analysis ............................................................... 45
Progesterone Levels ............................................................... 45
Statistical Analysis ................................................................ 45
Results .............................................................................................. 46
In Vitro Characteristics .......................................................... 46
Cell Freezing ......................................................................... 47
Electron Microscopy............................................................... 47
Immunocytochemistry............................................................ 48
Cell Cycle Analysis ............................................................... 48
Progesterone Levels ............................................................... 48
iv
TABLE OF CONTENTS (cont’d)Page
Discussion . . .......................................................................................... S3
EXPERIMENT m : THE EFFECT OF CULTURE MEDIUM AND TEMPERATURE ON IN VITRO GROWTH AND PROLIFERATION OF UTERINE AND OVIDUCT EPITHELIAL CELLS ............................................................................. 57
Introduction ......................................................................................... 57
Materials and M ethods......................................................................... 58
Cell Source................................................................................. 58
Isolation of Epithelial Cells ...................................................... 58
Experiment 1 ............................................................................. 59
Growth Curve and Cell Cycle Analysis ............... 60
Experiment 2 ............................................................................. 61
Statistical A nalysis..................................................................... 61
Results .................................................................................................. 62
Experiment 1 ............................................................................. 62
Experiment 2 ............................................................................. 69
Discussion............................................................................................. 73
EXPERIMENT IV : MORPHOLOGICAL EVALUATION OFBOVINE UTERINE AND OVIDUCT EPITHELIAL CELLSUSING IMAGE ANALYSIS .................................................................. 76
Introduction .......................................................................................... 76
Materials and M ethods......................................................................... 77
Experimental Anim als............................................................... 77
Isolation of Epithelial Cells ...................................................... 77
Morphological Assessments and Optical Density ................... 79
Electron Microscopy.................................................................. 79
v
TABLE OF CONTENTS (cont’d)Page
Statistical Analysis................................................................... 80
R esults................................................................................................. 80
Optical D ensity......................................................................... 80
Electron Microscopy................................................................. 81
Discussion............................................................................................ 87
EXPERIMENT V : IN W7R0-MATURATTON, IN VITRO- FERTILIZATION AND CLEAVAGE OF BOVINE OOCYTES INCUBATED IN DIFFERENT CULTURE M EDIA........................... 89
Introduction ......................................................................................... 89
Materials and M ethods........................................................................ 90
Recovery of Oocytes ................................................................ 90
Oocyte Maturation and Fertilization......................................... 90
Fixation of Oocytes.................................................................. 91
Oviductal Cell Culture.............................................................. 91
Embryo Co-culture................................................................... 92
Statistical Analysis................................................................... 92
R esults................................................................................................. 92
Discussion............................................................................................ 98
EXPERIMENT VI : CO-CULTURING IVF-DERTVED BOVINE EMBRYOS WITH OVIDUCT EPITHELIAL CELLS ORIGINATING FROM DIFFERENT DAYS OF THE ESTROUS CYCLE.................. 101
Introduction......................................................................................... 101
Materials and M ethods........................................................................ 103
Experimental Embryos.............................................................. 103
Isolation and Culture of Oviductal Cells ............................... 103
vi
TABLE OF CONTENTS (cont’d)Page
Experiment 1 ............................................................................. 104
Experiment 2 ............................................................................. 105
Statistical A nalysis..................................................................... 106
Results ................................................................................................... 106
Experiment 1 ............................................................................. 106
Experiment 2 ............................................................................. 107
Discussion............................................................................................. 113
CONCLUSIONS ...................................................................................... 116
REFERENCES.......................................................................................... 119
VITA ..................................................................................................... 133
LIST OF TABLES
Table Page
1. Effects of the estrous cycle on in vitro evaluations of uterine and oviduct epithelial cells(least squares means±SE) ........................................................ 49
2. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curvein different m edia...................................................................... 68
3. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve atdifferent temperatures .............................................................. 72
4. Fertilization and cleavage rates of bovine oocytescultured in different media ...................................................... 94
5. Development rates of cleaved oocytes 48 h afterfertilization ............................................................................... 95
6. Developmental rates and viability of cleavedembryos following in vitro culture for 6 days ........................ 96
7. Development of 2- to 4-cell bovine embryos with oviduct epithelial cells isolated between d 4 to 6or d 14 to 16 of the bovine estrous cycle................................. 110
LIST OF FIGURES
Figure Page
1. A growth curve of uterine epithelial cells during thethird subculture conducted for an 8-day interval ................... 37
2. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture isolated from the same animal on d 4 to 6 of the estrous cycle. Notethe differences in cilia across the cell surface (2,700 x ) ...... SO
3. Scanning electron micrograph of oviductal (a) and uterinep>) epithelial cells at the third subpassage. These are cells isolated from the same animal as Figure 2 (20,000 x) ........ 51
4. Mean percent of cells proliferating isolated from different stages of the estrous cycle. Values representuterine and oviductal cells combined ..................................... 52
5. A growth curve on uterine epithelial cells conductedfor 8 days in different culture media ..................................... 64
6. A growth curve on oviduct epithelial cells conductedfor 8 days in different culture media ..................................... 65
7. An example of flow cytometry analysis of the cell cycle.Total events are the number of cells analyzed with individual quadrants gated based on distinct cell populations. Quadrants III indicates the percent ofthe cell population in a resting state whereas Quadrant IV indicates a proliferative state of the cell cycle ....................... 66
8. Growth rates of uterine and oviduct epithelial cells for 8 days of incubation. All culture media treatment groups were combined and presented as mean values foruterine and oviductal ce lls...................................................... 67
9. Growth rates for uterine epithelial cells culturedfor 8 days at 37°C or 39°C ................................................... 70
10. Growth rates for oviduct epithelial cells culturedfor 8 days at 37°C or 39°C ................................................... 71
11. An example of procedures used for harvesting oviduct epithelial cells. The same procedurewas used to isolate uterine epithelial cells ............................. 78
ix
LIST OF FIGURES (coal’d)
Figure Page
12. Mean optical density readings for uterine and oviductal cells combined for the five samplereplicates .............................................................. :............... 82
13. Mean optical density readings for uterine and oviductal cells combined during primary cultureand following the first and third subpassages.......................... 83
14. Mean optical density readings of good, flair and poor quality ceils for uterine andoviductal cells combined........................................................ 84
15. Examples of good (a) and poor (b) quality bovine oviductal cells stained with Giemsa stain followingin vitro culture (20 x ) ............................................................ 85
16. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture(oviductal cells=20,000 x and uterine cells=20,000 X ) 86
17. Percent embryo viability following in vitro culture in medium alone or co-culture withoviduct epithelial cells............................................................ 97
18. An example of free-floating clusters of oviductepithelial cells used during embryo co-culture ...................... 109
19. Proteins secreted by oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle.Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean countsfrom three replicates............................................................... I l l
20. Cellular proteins in oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle.Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean countsfrom three replicates............................................................... 112
x
ABSTRACT
A reliable procedure was developed to harvest purified populations of bovine
uterine epithelial cells from the luminal endometrium that allowed cells to be
maintained in vitro for multiple subpassages. This method allowed adequate numbers
of cells for in vitro culture studies to evaluate uterine secretion rates, uterine-embryo
interactions and a new embryo co-culture system.
In a second study, cells were isolated from the uterus and oviducts collected
from cattle on day of estrus, on d 4 to 6, d 8 to 10 and d 14 to 16 of the cycle to
monitor growth and development following incubation in vitro. The highest percent
cell viability and attachment during primary culture was noted for cells isolated on the
day of estrus or between d 4 to 6 for both uterine and oviductal cell populations.
The effects of different culture media and incubation temperatures on growth
and proliferation of uterine and oviduct epithelial cells in vitro were evaluated. Cells
incubated in CMRL and MB2 had the highest growth rates, however, there were no
differences in proliferation rates among media evaluated. There were no differences
in growth and proliferation rates between different incubation temperatures (37°C vs
39°C).
Morphological observations of cell integrity and quality were made using image
analysis during primary culture and following the first and third subpassages. Optical
density readings were influenced by cell subpassage and quality. Cell quality
following in vitro culture could be effectively monitored using image analysis.
Bovine oocytes were matured and fertilized in vitro in different culture media.
Culture medium did not influence fertilization or cleavage rates. Early embryo
development was enhanced following incubation in CMRL and TCM media. Embryo
viability percent improved when morulae were maintained on oviductal cells compared
with those cultured in medium alone.
Bovine embryos derived from in W/ro-fertilization procedures were co-cultured
with oviductal cells isolated between cycle d 4 to 6 or 14 to 16 and proteins secreted
by oviductal cells monitored. Cycle stage oviductal cells were isolated did not
influence developmental rates or proteins secreted. However, co-culture with oviduct
epithelial cells were superior to culture in medium alone.
IN T R O D U C T IO N
The use of embryo transfer methodologies in research has evolved from simple
embryo collection and transfer to recipient females in early years to the more technical
and complex embryo biotechnologies. These new embryo biotechnology procedures
encompass a wide range of laboratory techniques such as cryopreservation, micro
manipulation, in vitro fertilization (TVF), gene insertion and embryonic cloning. The
development of IVF procedures for hum animals in the late 1980’s (48,94) has
provided increased numbers of laboratory-derived embryos available for research
purposes. The access to large numbers of early-stage embryos has increased research
efforts in the areas of embryo bisection methodology (63), DNA insertion (69) and
nuclear transfer techniques (120,130,163) for farm animals.
For many of these new embryo biotechnologies to become successful, embryos
will have to be maintained and develop in vitro prior to recipient transfer. In addition,
the chances of successful pregnancies increase in farm animals if embryos develop to
the morulae or blastocyst-stages prior to their transfer. In most instances this requires
maintenance of early-stage embryos in vitro for up to 7 days. However, the specific
requirements for in vitro development of early-stage embryos of domestic animals are
still not fully understood (see review by 167). In 1965, Cole and Paul (32) first
reported that a high percentage of mouse embryos developed and hatched from their
zona pellucida when cultured in vitro with a feeder layer of irradiated HeLa cells.
This initial study provided a basis for embryo co-culture methodology currently in use
today. The development of embryo co-culture systems using various types of "helper
cells" has provided more efficient means of maintaining embryos in vitro for extended
periods with minimal reductions in viability (see review by 124). In addition, embryo
2
co-culture systems may offer the unique advantage of improving pregnancy rates of
marginal-quality embryos prior to recipient transfer.
The development of embryo co-culture systems has resulted in the emergence
of three different types of co-culture systems (trophoblastic vesicles, cellular
monolayers and chick embryo co-culture). In virtually all instances, co-culture
systems have improved embryo development over that of incubating similar embryos
in culture medium alone. The present hypotheses explaining the beneficial effects of
embryos co-culture systems are that monolayers remove embryotoxic substances from
the medium surrounding the developing embryo, secrete embryotropic factor(s) to
enhance embryo development and/or a combination of both (see review by 8,124).
Although the beneficial effects of co-culture systems have been demonstrated
by many different laboratories in recent years, there are still many biochemical
components and interactions of cell monolayers that are not fully understood.
Developmental capacity of individual embryos in vitro can be enhanced with co
culture, however, most scientists agree that optimum success rates have likely not been
achieved. The variability of success rates using similar co-culture systems across
studies and among different laboratories can be attributed to the cells of the monolayer
or the environmental conditions that the cells are maintained during incubation. It
would seem logical that conditions altering monolayer cell growth or characteristics
while maintained in vitro may ultimately affect embryotropic factor(s) produced during
co-culture. This review will discuss current in vitro co-culture systems used for
mammalian embryos and in vitro culture studies conducted on bovine epithelial
monolayers of uterine and oviductal origin.
L IT E R A T U R E R E V IE W
In Vitro Co-Culture of Mammalian Embryos
Trophoblastic Vesicle Co-Culture. In the mid-1980’s French scientists (27,72)
developed a unique embryo co-culture system using trophoblastic tissue segments of
d 12 to d 14 elongating bovine concentuses. The trophectoderm of elongating cattle
and sheep blastocyst were sectioned and incubated in vitro. These trophoblastic
segments subsequently formed spherical vesicles (trophoblastic vesicles), and were
suggested to secrete luteotropic and embryo development promoting factors (72).
Camous et al. (27) investigated the ability of one- to eight-cell bovine embryos
to overcome the eight- to 16-cell block stage in vitro by co-culturing with d 13 or d
14 bovine trophoblastic vesicles. In this study, 46% of the one- to eight-cell embryos
developed to the morula stage when cultured with trophoblastic vesicles for 3 to 4 days
in Menezo’s B2 medium (MB2). In contrast, <20% of similar stage embryos reached
the morula-stage when embryos were cultured in the control MB2 medium alone. In
a later study, Heyman et al. (73) co-cultured one- to eight-cell bovine embryos with
d 14 bovine trophoblastic vesicles and 46% reached the morula stage compared with
only 18% reaching the morula stage in control medium. In a second experiment, 55
one-cell embryos were co-cultured with trophoblastic vesicles and 44 % cleaved beyond
the eight-cell stage compared with only 13% of the one-cell embryos cultured in
medium alone. In addition, when one-cell ovine embryos were co-cultured with d 12
sheep trophoblastic vesicles 75% of the embryos reached the morula stage compared
with 35% of the embryos cultured in control medium alone.
Camous et al. (27) and Heyman and Menezo (71) have suggested that tropho
blastic vesicles may provide important metabolic component(s) (e.g. lipids), that are
3
required for normal embryo cleavage in utero. In addition, it was proposed that these
embryotropic factors are secreted into the culture medium, and that direct contact
between the developing embryo and trophoblastic cells during the co-culture was not
necessary to elicit this response. Heyman et al. (73) further verified that direct
embryo-trophoblastic vesicle contact was not necessary by showing that one- to two
cell bovine embryos could develop to the 16-cell stage by culturing in conditioned
medium harvested from bovine trophoblastic vesicles.
In a more recent study, Pool et al. (119) attempted to improve the efficiency
of trophoblastic vesicle co-culture for farm animals by, individually placing morula-
stage bovine embryos into the lumen cavity of bovine trophoblastic vesicles during
incubation. It was noted that embryos placed inside trophoblastic vesicles during
culture resulted in fewer grade 1 and 2 embryos (36%) following 60 h of culture
compared with that of similar embryos individually co-cultured outside of the
trophoblastic vesicles (69%). The authors suggested that the decreased viability of
embryos placed inside trophoblastic vesicles was possibly due to the level of
embryotropic factors being too concentrated inside the vesicle during incubation and/or
the metabolic by-products of the confined embryo accumulated to a toxic level in the
lumen of the vesicle during co-culture. It was concluded that placement of embryos
inside trophoblastic vesicles was less effective when compared with the conventional
co-culture method using trophoblastic vesicles (27,72).
Although initial reports gave positive results with trophoblastic vesicle co
culture in cattle, Rexroad and Powell (127) reported no beneficial effects with
trophoblastic vesicle co-culture when evaluated with ovine embryos. These conflicting
results may be due to the day the conceptus was harvested for trophoblastic vesicle
production. In the latter study, d 14 elongated ovine embryos for producing
5
trophoblastic vesicles resulted in lower cleavage rates for co-culture, similar to the
one-cell embryos cultured in medium alone. This study differed from the reports by
French scientists (72) when d 12 ovine blastocyst were used for trophoblastic vesicle
production for the in vitro co-culture of ovine embryos. Correspondingly, no embryo
tropic effect from trophoblastic vesicle co-culture was noted when d IS caprine
blastocyst were used for trophoblastic vesicle production for co-culture with early-stage
caprine embryos (16). The latter authors suggested that trophoblastic vesicles from
d 15 caprine blastocyst were too moiphologically advanced during incubation to
enhance development of blastocyst from two- to eight-cell goat embryos.
Based on the results reported with trophoblastic vesicle co-culture, it appears
that trophoblastic vesicles should be prepared following d 10 and prior to d 15 of
development in the cow, sheep and goat for maximum embryotropic activity during
in vitro embryo co-culture (16,27,72,73,119,127). The potential for biochemical
involvement of trophoblast cells and the developing embryo has recently been
reviewed (71).
Uterine Cell Co-Culture. Many different co-culture systems are now being
developed to enhance embryonic growth and development of mammalian embryos in
vitro. Initial sources of cells for co-culture were of reproductive origin, used in hopes
of mimicking the in vivo uterine environment. Early co-culture systems incorporated
monolayers of fibroblasts derived from reproductive tissues of different animal
sources. Fibroblast monolayers for embryo co-culture were prepared following three
to five subpassages of cells after their initial outgrowth from endometrial explants
(86,155). In one of the earliest reports co-culturing bovine embryos, Kuzan and
Wright (86) incubated morula-stage embryos in vitro and reported a higher percentage
of embryos developed to hatched blastocyst on either uterine or testicular fibroblast
monolayers compared with similar stage embryos cultured in medium alone.
In a later study, Allen and Wright (3) reported that early-stage porcine
embryos (four-cell to morula) had greater developmental rates following co-culture on
porcine endometrial monolayers compared with embryos cultured in medium alone.
Bovine uterine fibroblast co-culture monolayers have also been shown to be capable
of enhancing in vitro development of porcine embryos (85,87).
Voelkel et al. (155) further reported beneficial effects of uterine fibroblast co
culture with bovine demi-embryos. Improved viability of demi-embryos was evident
on uterine fibroblast monolayers after 72 h of co-culture compared with corresponding
demi-embryos cultured in medium alone. Also, beneficial effects of the fibroblast
monolayer were thought to be necessary during the in vitro repair process of the
micromanipulated demi-embryos. It was suggested by Kuzan and Wright (86) that
fibroblast monolayers could be releasing embryo growth factor(s) into the culture
medium or possibly removing toxic substances from the medium, thus resulting in
enhanced in vitro development.
With renewed interest in fibroblast co-cultures, Wiemer and co-workers
(158,159,163) developed a fetal uterine cell monolayer culture system that incorpo
rated uterine fibroblasts derived from near-term («270 days) bovine fetuses. This fetal
bovine uterine fibroblast co-culture system has been shown to enhance in vitro devel
opment of bovine (160), equine (163) and human (161,162) embryos over that of
culturing similar embryos in medium alone.
A majority of the studies reported on co-culturing embryos in uterine cell
monolayer systems have used primarily fibroblast cells originating from reproductive
tissue. In more recent studies, epithelial or epithelial-like cells for embryo co-culture
have been isolated from the uterine endometrial lining of the cow (148), goat (121)
and rhesus monkey (65). Prichard et al. (121) first used uterine and oviductal cells
to co-culture two- to four-cell goat embryos through the in vitro developmental block.
Although adequate hatching rates were noted on uterine cell monolayers (63%),
significantly greater hatching rates were noted with oviductal cell co-culture (87%).
Also, sequentially transferring another group of embryos from oviductal cell mono
layers to uterine cell monolayers (to mimic the in vivo environment) during the
incubation interval was not found to be beneficial over that of oviductal cell co-culture
(73 %). Blakewood et al. (14) have also reported caprine uterine epithelial monolayers
enhanced post-thaw development of bovine embryos when co-culturing prior to
freezing over that of medium alone.
Recently, a larger scale study was conducted evaluating uterine, oviductal and
granulosa cell co-culture systems on IVF-derived bovine embryos (77). These three
different cell co-culture systems were also evaluated in different combinations to
evaluate enhancing effects between cell types during embryo co-culture. The
percentage of hatched blastocyst obtained when cultured, with granulosa cell, oviductal
cell, uterine cells, granulosa and oviductal cells, granulosa and uterine cells, oviductal
and uterine cells and a combination of granulosa, oviductal and uterine cells ranged
from 58 to 73%. The addition of granulosa cells to uterine or oviductal cell co-culture
systems provided the highest hatching rates for IVF-derived bovine embryos. The
bovine uterine cell co-culture system resulted in the lowest percentage of hatched
blastocyst (58%) following incubation. Unexpectedly, these findings suggested other
cell types were better for culturing bovine embryos in vitro than cells derived from
uterine tissues.
The recent development of a nonsurgical embryo recovery technique in rhesus
monkeys has allowed access to early-stage embryos for subsequent culture research
(64). Goodeaux et al. (65) reported increased development and hatching rates of
rhesus monkey embryos co-cultured on rhesus uterine epithelial cells compared with
culture in medium alone (63% vs 25%, respectively). Furthermore, co-culture of
rhesus embryos on rhesus uterine epithelial cell monolayers followed by nonsurgical
transfer has resulted in the birth of a live transplant offspring. It was also shown in
this study that rhesus uterine epithelial monolayers were also capable of supporting
development of morula-stage cattle embryos.
Oviductal Cell Co-Culture. Although positive results have been reported with
the use of uterine cells as a co-culture system for later-stage embryos, oviductal cell
monolayers have been shown to be more effective in enhancing the development of
early-stage mammalian embryos in vitro. Over two decades ago, Biggers et al. (10)
noted that two-cell mouse embryos would develop through the in vitro developmental
block stage when embryos were cultured in explanted mouse oviducts maintained in
culture medium. Whittingham (157) later suggested that only the ampullar region of
the murine oviduct was capable of maintaining embryo development in vitro.
It has also been reported that oviductal cells of various mammalian species
were capable of stimulating embryonic development in different animal species.
Earlier studies have demonstrated the capabilities of the rabbit oviduct to maintain or
enhance in vivo embryonic development in mouse, sheep, cow, pig, goat and horse
embryos (see reviews by 8,18).
Rexroad and Powell (125) were among the first to report the use of oviduct
cell monolayers for short-term culture of early-stage ovine embryos. In addition,
Gandolfi and Moor (57) evaluated the developmental potential of pronuclear-stage
9
ovine embryos co-cultured on both oviductal epithelial and uterine fibroblast
monolayers. Both of these monolayer systems were capable of supporting embryo
development to the early blastocyst stage. After 6 days of incubation, 42% of the
ovine embryos developed into expanded blastocyst using oviductal cell co-culture
compared with only 5% co-cultured on uterine fibroblast monolayers. The transfer
of embryos co-cultured on oviductal cell monolayers resulted in higher pregnancy rates
in recipient sheep compared with those co-cultured on fibroblast monolayers.
Oviductal cell monolayers have also been used successfully for in vitro co
culture of early-stage bovine embryos (37,39,41). In an initial study, Eyestone et al.
(43) reported higher developmental rates of five- to eight-cell bovine embryos cultured
for 4 to 5 days in an oviductal cell co-culture system compared with culture medium
alone. In a subsequent study, Eyestone and First (41) evaluated the effects of ovi
ductal cell co-culture on embryonic growth by comparing this culture system with that
of conditioned medium from oviductal cells. A higher percentage of morula- and
blastocyst-stage embryos resulted when IVF-derived one-cell bovine embryos were co-
cultured on oviductal cell monolayers compared with that of culture in medium alone
(22% and 3%, respectively). In contrast, when embryo development on oviduct cell
co-culture was compared with that of oviductal-cell conditioned medium, in vitro
developmental rates for bovine embryos were similar (22% and 22%, respectively).
Furthermore, embryos transferred to recipient animals following co-culture in
conditioned medium resulted in a 67% pregnancy rate.
McCaffrey et al. (97) evaluated the requirements of oviductal monolayer-
embryo contact and age of monolayer during co-culture using one- to four-cell bovine
embryos. Oviductal monolayers were prepared for co-culture 3 days prior to embryo
recovery. These monolayer systems were used with or without a microporous (.4 /im)
10
membrane inserted between the monolayer cells and embryo during culture. Morulae
or blastocyst resulting were similar following co-culture on monolayers prepared either
on the day of embryo recovery (69%) or 3 days earlier (62%). As was indicated in
a previous report (41), direct contact between the embryo and monolayer co-culture
system did not improve developmental rates in this study.
Recently, Ellington et al. (39) compared in vitro embryo development rates of
early-stage bovine embryos co-cultured on oviductal monolayers in a simple medium
(CZB) with that of in vivo development within the reproductive tract of the cow. In
this study, embryos obtained 40 to 48 h post-estrus were co-cultured in vitro for S
days, however, the developmental rate and number of nuclei per embryo did not differ
from embryos developed in vivo. This finding suggests that the oviductal cell co
culture system with a simple serum-free medium can function to enhance development
of bovine embryos. In addition, Ellington et al. (38) compared in vitro development
of one- to two-cell bovine embryos cultured in either a simple (CZB) or complex
(CMRL-1066 and Ham’s F-10) medium with bovine oviductal cells. Higher develop
ment of early-stage embryos in CZB with oviductal cells occurred compared with
similar embryos co-cultured in either of the complex media. Frozen-thawed oviductal
cells used for co-culture also produced similar rates of blastocyst development resulted
compared with fresh monolayers cultured in CZB medium (32 and 39%, respectively).
Significantly fewer blastocyst were obtained when the CZB oviductal-cell conditioned
medium (24%) was used in place of oviductal cells. It was concluded that a simple
serum-free medium would adequately support in vitro development of one-cell bovine
embryos to the blastocyst stage in the presence of oviductal cells.
In a recent study, Ellington et al. (37) co-cultured one- to two-cell bovine
embryos on oviductal qrithelial cells in a simple medium (CZB) or in the oviducts of
an intermediate host rabbit prior to transferring the cultured morulae and blastocyst to
recipient animals. The pregnancy rate for embryos co-cultured on oviductal cells was
57% compared with 58% for similar embryos cultured in rabbit oviducts. It was
concluded that oviductal epithelial cell co-culture systems supported embryo
development in vitro to obtain an adequate pregnancy rate, even when a simple culture
medium (without protein supplementation) was used for in vitro culture of bovine
embryos.
Recently, extensive studies have been conducted with oviductal cell monolayers
to promote in vitro development of IVF-derived bovine embryos (17,40,52,84,133)
and IVF-derived chimeric embryos (82). In each case, oviductal cell monolayers were
more effective than culturing embryos in medium alone. In addition, Aoyagi et al.
(4) evaluated the ability of six different culture systems to promote blastocyst develop
ment for IVF-derived bovine embryos. The co-culture systems included cumulus
cells, oviductal epithelial cells, trophoblastic vesicles, amniodc sac cells and in vivo
culture in rabbit oviducts. No difference was reported in blastocyst development
among oviductal cells, trophoblastic vesicles and amniotic sac cells, with develop
mental rates ranging between 39 and 51%. These culture systems were more effective
for stimulating blastocyst development in vitro than when bovine cumulus cell co
culture, culture in rabbit oviducts and culture medium alone were used to evaluate
embryo development in vitro.
Oviductal cell monolayers are also capable of stimulating in vitro development
of early-stage pig embryos. White et al. (156) reported enhanced development of two-
to 16-cell pig embryos co-cultured on porcine oviductal cell monolayers. In this
study, in vitro development was evaluated during culture with either medium alone,
porcine oviductal cells, porcine endometrial fibroblasts or a bilayer of porcine
12
endometrial fibroblast and oviductal cells. The highest percentage of hatched
blastocyst were noted with co-culture of porcine oviductal cells and a bilayer of
oviductal and fibroblasts cells (54% and 61 %, respectively). It was concluded that
oviductal cell co-culture provided necessary factors to overcome the four-cell block in
the pig.
Oviductal cell monolayers capable of supporting development of sheep
embryos to the blastocyst stage in vitro have been found to secrete proteins similar to
those found in ovine oviductal fluid (58). Two main classes of proteins were found
to be secreted by sheep oviductal cells; one secreted throughout the cycle (MW range
of 10,000 to 100,000) and those exhibiting cyclic variations in secretion. The latter
class of proteins is further divided into a 92,000 MW protein and a 46,000 MW
protein. Both proteins were secreted during the first 4 days of the cycle but the
96,000 MW protein then declined sharply, thereafter. In addition, the proteins
produced during the first 4 days of the ovine estrous cycle have the ability to bind to
the zona pellucida (59).
A present hypothesis suggests that oviduct epithelial cell feeder-layers enhance
the development of preimplantation embryos by one or more mechanisms. First,
oviductal monolayers may secrete specific growth factors (e.g. protein, peptides) that
are required by early-stage embryos to maintain normal rate of development (58,59).
Another possibility is that monolayers have the ability to remove toxic components that
are detrimental to the embryo from the surrounding medium during co-culture (for
review see 8). In addition to secreting embryotropic components and/or removing
embryotoxic substances from the medium, Bavister (8) has proposed that both cell
types may lower oxygen tension in the immediate vicinity of the embryo to enhance
development. It was suggested that the major difference between uterine fibroblast
13
and oviduct epithelial cell co-culture systems is that fibroblast feeder-layers are capable
of removing embryotoxic components, reducing oxygen tension, thereby, improving
embryo development over that of culture medium alone but are unable to secrete
embiyotropic factors. In contrast, it was concluded that oviductal cell feeder layers
apparently have the ability to remove embryotoxic components, lower oxygen tension
in addition to having embryotropic capabilities.
Results thus far suggest that oviductal cell monolayers are the most effective
co-culture system for early-stage farm animal embryos while fibroblast monolayers are
adequate for development of later-stage embryos (morulae to blastocyst) but less
effective for developing earlier stage embryos through the hatched blastocyst stage
(124). Due to the limited number of studies available evaluating embryo culture on
uterine epithelial cells, it is unclear whether development of early-stage embryos is
dependent upon the source of cells (uterus vs oviduct) for co-culture.
Granulosa Cell Co-Culture. Research efforts have now demonstrated the
importance of bovine granulosa/cumulus cells for the in vitro maturation of bovine
oocytes obtained from abattoir ovaries. Critser and First (34) suggested that
granulosa/cumulus cells are not only beneficial for in vitro maturation of oocytes but
maybe important for the development of IVF-derived embryos to the morula and
blastocyst stages. Faundez et al. (46) have reported that in vitro fertilization rates of
bovine oocytes were higher with granulosa cells than when similar oocytes were
exposed to IVF procedures without the aid of granulosa cells. In addition, the highest
fertilization rates resulted when cumulus-intact bovine oocytes were incubated on
granulosa cell monolayers prior to and during the IVF procedure.
In an initial study, Goto et al. (66) reported successful bovine transplant preg
nancies were obtained following in vitro co-culture of IVF-derived embryos with
14
bovine cumulus cells for 6 or 7 days. In a similar study using granulosa cell co
culture, Goto et al. (67) reported that 25% of in vz'rro-matured and fertilized bovine
oocytes reaching the eight-cell stage following 3 to 4 days of incubation, with 21%
reaching the morula and blastocyst stages following IVF procedures.
Fukuda et al. (55) have also used cumulus cell monolayers to maintain IVF-
derived cattle embryos in vitro. Following co-culture on cumulus monolayers, six
frozen-thawed blastocyst were transferred into three recipients, with one recipient
producing twin offsprings. In addition, seven fresh IVF-derived blastocyst were
transferred to six recipients and two recipients delivered live calves. This study
demonstrated the ability of cumulus cell monolayers to produce live offspring from
both fresh and frozen-thawed IVF-derived embryos.
More recently, Zhang et al. (172) used a different procedure for in vitro
fertilization of bovine oocytes and reported 54% cleavage rates and 41% of the total
oocytes placed into culture reaching the morula stage of development using a cumulus
cell co-culture system. In addition, a bovine cumulus cell co-culture system was
reported to be effective for in vitro development of porcine IVF-derived embryos
(171). Younis and Brackett (170) reported similar results using a bovine cumulus cell
co-culture system on IVF-derived bovine embryos. In addition, Berg and Brem (9)
noted significantly higher rates of development of IVF-derived bovine embryos to
morulae and blastocyst (32%) using granulosa cell co-culture compared with oviductal
epithelial cell co-culture (17%).
In 1990, Japanese researchers evaluated the proportion of inner cell mass
(ICM) cells of bovine blastocyst fertilized in vitro and in vivo (76). In this study,
IVF-derived bovine embryos were co-cultured on cumulus cell monolayers or placed
in rabbit oviducts for 8 to 12 days following fertilization. The proportion of ICM and
15
trophectoderm cells of IVF-derived embryos was compared between an in vitro and
two in vivo culture systems by a differential fluorochrome staining technique after 7
days. In this study, the highest proportion of ICM was noted with in vivo culture for
early (25%), expanded (21%) and hatched (27%) blastocyst in the rabbit oviducts.
There was a significantly lower proportion of ICM noted with IVF-derived in vitro-
cultured (cumulus cells) embryos for early (16%), expanded (15%) and hatched (13%)
blastocyst when compared with those cultured in an in vivo system. In addition, there
was a significantly lower proportion of ICM noted with IVF-derived in vivo cultured
(rabbit oviducts) embryos for early (23 %) and expanded (21 %) blastocyst but not with
hatched blastocyst (17%) compared with the in vitro system. It was concluded that
the differential staining of ICM and trophectoderm nuclei provided an effective method
for evaluating embryo development in an in vitro co-culture system.
Nakao and Nakatsuki (109) recently compared bovine trophoblastic vesicle and
cumulus cell co-culture systems for the in vitro culture of IVF-derived bovine
embryos. Embryos developing to the morula stage were similar when co-cultured on
either trophoblastic vesicles (17%), cumulus cells (19%) or co-cultured with both
vesicles and cumulus cells (16%). However, in this study there was no enhanced
embryotropic effect evident when both cell types were combined in a single culture
unit.
Although most results indicate that granulosa cell co-culture systems are
similar to other co-culture systems, the major advantage of the granulosa cell culture
system is that the cells are available for harvesting from the follicle at the time of
oocyte collection. This provides a simple and cost efficient co-culture system without
lowering success rates of in vitro culture systems.
16
Chick Embryo Co-Culture. The first report of using the amniotic cavity of
developing chick embryos to culture mammalian embryos was reported by Blakewood
et al. (12). Blakewood and Godke (11) embedded pronuclear-stage mouse embryos
in agarose and injected embryos into the amniotic cavity of a 96 h-old chick embryo
incubated at 37eC. In the initial experiment (13), pronuclear mouse embryos from
two different lines were placed in the amniotic cavity of chick embryos for 72 to 96
h of incubation. Following incubation, significantly more embryos developed into
hatching blastocyst within both strains of mice compared with those in culture medium
alone. Blakewood et al. (14) additionally reported success using the chick embryo co-
culture system to enhance post-thaw development of precompaction-stage bovine
morulae. Further studies have demonstrated that the chick embryo amnion was
capable of supporting development of bovine morulae (IS) and two- to eight-cell goat
embryos (16).
Blakewood et al. (16) demonstrated the ability of the chick embryo co-culture
system to promote development of two- to eight-cell goat embryos through the in vitro
block stage. Both the fetal bovine uterine fibroblast monolayer and chick embryo co
culture systems resulted in significantly more expanded and hatched blastocyst
compared with embryos incubated in medium alone following 72 h of incubation. In
a second experiment, two- to eight-cell caprine embryos were maintained on uterine
fibroblast monolayers or in the amniotic cavity of the chick embryo for 96 h. It was
reported that no early-stage goat embryos developed to the expanded or the hatched
blastocyst stages when co-cultured on the fetal uterine monolayer or with the control
medium. However, 86% of the embryos co-cultured in the chick embryo amnion
reached the expanded blastocyst stage and 82% of the embryos developed to the
hatched blastocyst stage in vitro. In addition, four recipient goat females received
17
surgically transplanted chick-embryo co-culture morulae, two maintained pregnancies
to term and a total of six live-transplant offspring (50% of all embryos transferred)
were bom. These findings further indicate that the developing chick embryo has
potent embryotropic properties that are evident with mammalian embryos across
species.
Other Co-Culture Systems. Glass et al. (61) evaluated different types of
helper cells (L-cells, liver, JLS-V11 and teratocarcinoma cells) for culturing mouse
embryos to the hatching stage and reported no differences among cell types. More
recently, Overskei and Cincotta (86) noted adequate success rates (83%) when two-cell
mouse embryos were co-cultured in vitro on hamster hepatocyte monolayers. In
addition, Hu et al. (75) reported excellent development of two-cell mouse embryos co-
cultured on a established cell line of Buffalo Rat liver cells. It was noted that Buffalo
Rat liver cells produced better results during in vitro culture than did similar embryos
incubated on mouse oviductal cells.
Recently, bovine fetal spleen (BFS) cell and chicken embryo fibroblast (CEF)
monolayers were used for the in vitro co-culture of morula-stage mouse (79) and
bovine (80) embryos. The BFS and CEF monolayers produced more hatched bovine
embryos (75 and 83%, respectively) than did culture in medium alone (45%). In
addition, Kim et al. (81) compared these two monolayer co-culture systems with a
bovine cumulus cell co-culture system using IVF-derived bovine embryos. The best
success with development and hatching occurred when using bovine cumulus cells
compared with BFS and CEF monolayers.
Ouhibi et al. (113) evaluated development of one-cell mouse embryos co-
cultured on reproductive tract cells (oviduct epithelium) or established cell lines
derived from cells other than of reproductive origin (kidney cells). The oviductal cell
18
co-culture treatments consisted of mouse oviductal organ cultures, mouse oviductal
cells and bovine oviductal monolayers consisting of both polarized and unpolarized
cells. The highest percentage of morulae and blastocyst were obtained from embryos
placed in mouse oviductal organ cultures (77%). The percentage of morula- and
blastocyst-stage embryos resulting from mouse oviductal, bovine unpolarized and
polarized monolayers were 67%, 48% and 14%, respectively. Also, Vero unpolarized
and polarized monolayers had lower development rates (8% and 4%, respectively)
compared with MDBK unpolarized and polarized cells (74% and 21 %, respectively).
Overall, maintenance of cell polarization resulted in lower development during mono
layer co-culture. From this study, it appears that the established Vero cell line was
not suitable for development of mouse embryos.
These studies indicate that various cell lines and cell monolayers developed
from cells other than from adult and fetal reproductive tissue are capable of supporting
development of early-stage embryos for up to 72 h during in vitro culture. The major
advantage of established cell line co-culture systems is a pathogen-free cell line with
embryotropic capabilities and high viability and growth of cells following repeated
subpassages.
Id Vitro Culture of Epithelial Cells
Uterine Epithelial Cells. The development of an effective uterine epithelial
cell culture system may improve embryo co-culture systems for IVF-derived embryos.
In addition, this in vitro culture system would allow researchers to investigate
prostaglandin release, protein synthesis, early embryo development, embryo/uterine
interactions and implantation using uterine cell monolayers or explanted tissue. The
majority of research efforts have concentrated on isolation and culture of epithelial or
19
stromal (fibroblast) cells from laboratory and farm animal species. It has been
suggested that vital communications occur in vivo between stromal and epithelial cells
to construct the complexity of the uterus (62). These communications support the
integrity or continued secretion of embryotropic factor(s) by cells. Secretion of
embryotropic factor(s) by monolayers is continued even though an in vitro culture
system results in some degree of transformation or removal of the in vivo system.
Research efforts have utilized membrane inserts for in vitro culture of epithelial cells
to retain polarity of the cells and create a more in vivo type co-culture system
(113,131,133,156). However, these systems have not proven beneficial over standard
culture conditions for enhancing embryonic development.
Several studies have been conducted to evaluate the action of progesterone and
estrogen on protein synthesis of uterine epithelial cells in vitro. In one study (30),
endometrial sections obtained from guinea-pigs were digested and isolated cells
incubated in CMRL-1066 medium in vitro. Following 3 days of incubation, cells were
incubated with estradiol- 17B and progesterone for 48 h and the confluent monolayers
radiolabelled with 3SS-methionine. It was reported that progesterone supplemented in
the culture medium modified the patterns of proteins compared with those proteins
from monolayers incubated with estradiol-17B. It was further suggested that a single
protein (MW 19,000) was the major contributor to the antagonist effects of prog
esterone on estradiol-17B action, but the protein disappeared when both progesterone
and estradiol-17B was supplemented in the incubation medium. From this study, it
was concluded that the action of steroid hormones on uterine epithelial cells does not
require the presence of stromal cells. In addition, progesterone altered the synthesis
of proteins when combined with estradiol-17B and affected both cellular and secreted
proteins.
20
In the guinea-pig, progesterone induced secretory morphological changes in
epithelial cells maintained in vitro (1,2). In addition, the effects of estradiol-17B and
progesterone on progesterone receptors have been previously reported for guinea-pigs
(100,101,102,142,143). In ovariectomized guinea-pigs, estrone sulphate induced an
increase in progesterone receptors (104). In a later study, estrone sulphate was
reported to induce modifications in the shape and formation of microvilli (1). Further
investigations revealed that estrone sulphate induces the sulphation of proteins and
more specifically increased the sulphate incorporation into the cellular proteins (143).
The authors concluded that estrone sulphate acts with progesterone to preferentially
enhance the secretion of sulphated proteins.
Rickets et al. (129) digested portions of rabbit uterine mucosa in attempts to
characterize cell types used during in vitro culture systems. Both epithelial and
stromal cells were isolated and provided evidence that the source of uteroglobulin
proteins are derived from epithelial cells in rabbits. The roles of estrogens in
endometrial cell proliferation have also been previously investigated in the rabbit (33).
A possible role of estrogen in regulation of uterine cell function using in vitro culture
systems was suggested. Estrogen administered to ovariectomized rabbits resulted in
increased proliferation rates of quiescent epithelial cells. In addition, estrogens
increased cell migration towards the uterine lumen whereas progesterone was suggested
to promote development of new uterine glands.
As in rabbits, estrogen stimulates uterine epithelial cell proliferation in adult
ovariectomized mice (123,141). Estrogen also regulates the secretion of uterine
proteins along with morphological and functional differentiation of the uterine
epithelium (96). The roles of estrogen in regulating mouse uterine cell activity
appears to be mediated via epidermal growth factor (EGF) receptors and increased
21
levels of EGF peptides (106). Tomooka et al. (151) also reported stimulatory effects
of EGF on mouse uterine epithelial cells maintained in vitro. An increase in cell
number was associated with supplementation of EGF to culture medium. However,
this increase did not occur when nerve growth factor (NGF), multiplication-stimulating
activity (MSA), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)
or somatomedian-C was added to incubation medium. Tomooka et al. (151) also
demonstrated the ability of mouse uterine cells to maintain EGF receptors in vitro and
EGF had stimulatory roles in cell proliferation. The presence of EGF receptors were
also detected in rat uterine cells (105) and EGF levels increased under the influence
of estrogens (106).
As in laboratory animals, protein synthesis by the endometrium is influenced
by ovarian steroids in the ewe. Estrogen treatment of ovariectomized ewes resulted
in increased uptake of 35S-methionine into both cellular and secreted proteins and
progesterone increased the proportion of newly synthesized proteins in both luminal
and glandular epithelial cells (136).
Using an in vitro culture system, Salamonsen et al. (136) suggested possible
roles of ovarian steroids on uterine protein secretion in the ewe. Both estrogen and
progesterone act synergisdcally, with estrogen stimulating overall synthesis of proteins
and progesterone promoting the increased synthesis of secretory proteins. In a
subsequent study, Salamonsen et al. (137) provided evidence that ovine blastocyst
influence uterine epithelial cells on d 13 of pregnancy by increasing the overall protein
synthesis and secretion of specific proteins by uterine cells.
lim ited studies have been reported on the in vitro culture of uterine epithelial
cells in the cattle. Recently, Munson et al. (108) reported methods for establishing
endometrial epithelial cell lines derived from both adult and fetal bovine uteri. For
adult uteri, layers of intercarunclar endometrium were removed and minced into small
fragments. Tissue fragments were separated from small clusters of cells by filtration
and centrifugation. In contrast, the fetal uteri were filled with collagenase and
incubated for 1 h at room temperature. Attempts to produce cell lines by digestion
with trypsin were unsuccessful. However, three adult and one fetal cell line were
established using collagenase digestion techniques. It was reported that fetal cell lines
were more viable than adult lines following subpassages and cry opreservation. In
addition, fetal lines survived longer in culture than adult cell lines (10 and four
subpassages, respectively). These initial experiments provided the basis for the in
vitro culture of bovine endometrial epithelial cells derived from adult and fetal uteri.
The in vitro culture of human endometrial cells has recently received attention
due to the increased efforts in human IVF procedures. The human endometrium
undergoes morphological and biochemical changes during the menstrual cycle and
pregnancy (35,168). These changes have prompted many researchers to develop in
vitro culture systems for endometrial tissue to study interactions between epithelial and
stromal cells.
Several studies have provided culture techniques for maintaining endometrial
tissue in vitro with varied success (47,83,152). Varma et al. (154) cultured human
endometrial tissue in vitro in an attempt to characterize and study growth properties
of various cell types. Different cell types were obtained following digestion of
endometrial tissue with glandular tissue and individual cells separated and placed into
culture. In addition, explants of endometrial tissue were placed in culture dishes to
promote fibroblast outgrowth. Extensive electron microscopy studies revealed three
cell types proliferating during primary culture: epithelial cells, stromal cells (epithelial-
like) and fibroblasts. Both stromal and fibroblast cells could be subcultured up to 20
23
passages in vitro, however, epithelial cells only proliferated during primary culture and
could not be subpassaged.
In a subsequent study by the same laboratory, Dorman et al. (36) indicated that
in vitro cultures of endometrial stromal cells exhibited aggregation following estrogen
and progesterone treatment. In addition, the plating efficiency of subcultured cells was
highly variable (32% to 75%). The addition of estrogen and progesterone to culture
medium did not enhance plating efficiency. Varma et al. (154) and Dorma et al. (36)
concluded that extensive characterization of endometrial cell lines used in in vitro
culture systems is required to understand basic morphological changes the uterus
exhibits throughout the menstrual cycle and pregnancy.
In an earlier study, Liszczak et al. (93) obtained epithelial monolayers from
human endometrial samples by separation of stromal cells by cloning cylinders.
Cultures of epithelial cells were incubated in steroid-free medium in the presence of
10"6 M estradiol-17B, 10"6 M progesterone or a combination of both for 7 days. No
alterations in cell morphology were noted from any steroid treatments. Furthermore,
criteria for classification of epithelial cells were conducted using electron microscopy.
These criteria were the presence of structures such as junctional complexes,
perinuclear microfilaments and microvilli surrounded by glycocalyx. In addition,
structures of secretory cells were abundant rough endoplasmic reticulum, golgi
apparatus and the presence of membrane-bound electron-dense bodies in the cytoplasm.
Oviduct Epithelial Cells. Recently, Ouhibi et al. (112) reported procedures
for isolation and in vitro culture of oviductal epithelial cells derived from the mouse,
rabbit, cow and humans. Epithelial cells were isolated by filling oviducts with .25%
trypsin solution and incubating at 37°C for 45 min. Following the incubation period,
cell clusters were disrupted by repeated pipetting and layered on Percoll gradients.
24
Harvested cells were seeded in culture dishes and incubated in MB2 medium with the
addition of 10 ng/ml serotonin. Serotonin added to the culture medium was suggested
to provide necessary components for cell attachment and differentiation and has
selective properties against fibroblast proliferation.
Quhibi et al. (112) reported that bovine oviductal monolayers were easily
obtained and could be subcultured for 14 to 18 passages before the appearance of an
in vitro crisis phase. Similarly, rabbit oviductal monolayers could be maintained for
10 passages before the appearance of altered growth and morphology. However,
limited numbers and slower growth rates of human oviductal cells were noted
compared with other species. In addition, monolayers could be maintained up to the
sixth passage. It was reported that establishment of mouse monolayers were pH
dependent and growth was only initiated in MEM in place of MB2 medium. In
addition, all attempts to obtain subpassaged monolayers were unsuccessful in this
study.
French scientists have also used radiolabelled methionine to evaluate protein
secretions by monolayers during in vitro culture as an indication of cell quality.
Ouhibi et al. (113) evaluated protein secretions by subpassaged oviductal monolayers
of different species using radiolabelled methionine incorporation and noted that
monolayers maintained high protein secretion rates following 96 h in culture.
Evaluation of radiolabelled protein secretion by monolayers during in vitro culture will
likely provide vital information on embryotropic factors produced by monolayers
during embryo co-culture. Identifying the factors that alter or directly influence cell
monolayers of embryo co-culture systems would provide the key to establishing the
most efficient co-culture systems for mammalian embryos.
Joshi (78) conducted extensive transmission electron microscopy and immuno-
cytochemical studies on oviduct epithelial cells isolated from the cow. Epithelial cells
were isolated by inflating oviducts with .1% collagenase and incubating at 37°C for
90 min. Harvested cells were incubated in DMEM and Ham’s F-10 medium and they
reported isolating both ciliated and nonciliated secretory cells. Using electron
microscopy, ciliated cells were characterized by presence of fewer endoplasmic
reticulum that secretory cells, many mitochondria, free ribosomes, polyribosomes and
microtubules. Attached ciliated cells lost their cilia following 4 to 5 days during in
vitro culture. However, free floating ciliated cells maintained cilia for 10 to 12 days.
These results provided specific criteria for evaluation of two specific cell types
obtained from the bovine oviduct and further verified epithelial origin of cells by
immunocytochemical studies.
Using similar culture techniques as previously reported (78), Hishinuma et al.
(74) evaluated growth and monolayer culture of bovine oviduct epithelial cells and
similar characteristics were noted. In this study, the addition of lO*5 and 10*9 M
estradiol-17B did not affect ciliary activity or growth of oviductal cells. Furthermore,
the authors noted different types of secretory cells maintained in vitro, those with
homogenous matrix and those possessing lamellar structures within the matrix.
Limited studies have been reported in the scientific literature on the in vitro
culture of human epithelial cells. In a recent study, Henriksen et al. (70) described
methods for the in vitro culture of human oviduct epithelial cells. Epithelial cells were
harvested by dissection of mucosa pieces obtained from the ampullary region of
oviducts. Tissue pieces were minced thoroughly with scissors the resuspended in
RPMI 1640 medium supplemented with 20% bovine calf serum. Following a 48 h
incubation period, the levels of calf serum in medium were lowered to 10%.
26
In this study, extensive election microscopy and immunocytochemical studies
were conducted using monoclonal antibodies to cytoskeleton proteins for character
ization of cell types. The plating efficiency of cultures ranged between 10 to 20%
within 48 h after initial seeding. Growth curves of cells during primary culture
revealed increases in the number of cells for 7 days followed by stationary periods of
cell growth. However, cultures could be maintained for 6 to 8 wks before
degeneration of cells. It was noted that cells isolated from human oviducts were of
epithelial origin as judged by presence of microvilli, typical desmosomes, cilia and
positive staining for cytokeratin antigens. It was further suggested that the low plating
efficiency noted may be attributed to the ciliary activity of cells. Furthermore,
pretreatment of culture vessels with fibronectin did not improve plating efficiency. It
was concluded that the specific origin of cells during in vitro culture must be based
on electron microscopy and presence of cytokeratin antigens to fully distinguish
between epithelial and fibroblast cell types.
Bongso et al. (20) successfully established epithelial monolayers isolated from
ampullary regions of human oviducts. Monolayers could be established regardless of
donor age or phase of the menstrual cycle, and it was reported that epithelial
monolayers were maintained for four to six subpassages. However, monolayers were
transformed to fibroblast-like cells following repeated subpassages. Although scanning
microscopy was used, extensive characterization of cell types using immunocyto-
chemistry was not performed.
Oki et al. ( I ll) evaluated the effects of age and stage of menstrual cycle of
donor subjects on performance of human oviduct epithelial cells in vitro. Oviductal
cells were obtained by treatment with collagenase then incubated in TCM-199 with
10% fetal calf serum. Survival of oviductal monolayers were determined following
27
7 days of incubation. Monolayer survival rates of 78%, 50% and 100% was reported
for subject age groups between 20-39 yr, 40-59 yr and over 59 yr, respectively. In
addition, cell survival rates for menstruating and post-menopausal women were 63%
and 83%, respectively. Although there were no differences in survival rates of
monolayers, the highest percentage of confluent cultures were obtained in the younger
age groups (44%) and for menstruating women (25%). It was further stated that there
were no differences in monolayer survival for cells obtained from women in follicular
and luteal stages of the menstrual cycle. However, the highest percentage of confluent
monolayers were obtained in the follicular phase opposed to luteal phase of the
menstrual cycle (40% and 17%, respectively). In addition, the authors reported
growth of human oviductal cells in vitro was only successful up to the third
subpassage.
Summary
Repeatable techniques for producing in v?Y/t>-fertiIized farm animal embryos
have made the early-stage embryo more accessible for biochemical and morphological
study (67,95,172). This accessibility to laboratory-derived embryos has illustrated the
need for culture systems that have the ability to promote normal in vitro development
of early-stage farm animal embryos. For farm animals, the ability to produce larger
numbers of early-stage embryos in the laboratory would offer little benefit without
developing an in vitro culture system that would allow the embryos to progress
through the in vitro block stage. Since acceptable pregnancy rates in cattle are not
obtained unless embryos of the morulae and blastocyst stages of development are
transferred, the need for a simple, efficient culture systems becomes increasingly more
important.
28
With recent advances made in embryo culture systems for farm animals, it
appears that in vitro incubation problems may have been partially alleviated with the
helper cell co-culture systems, that permit embryonic growth and development through
the in vitro block stage to morulae and blastocyst while in culture.
The use of oviductal cell co-culture systems have improved in vitro develop
ment of embryos in nearly all studies conducted. However, there still remains
variability in results between studies. Based on in vitro studies of uterine and oviduct
epithelial cells, culture conditions and cell source needs to be investigated in an effort
to explain differences in co-culture systems. Furthermore, specific characterization
of cell types used in co-culture systems are needed to evaluate results based on
different cell types.
The evaluation of factors that directly affect epithelial monolayers themselves
may provide the ultimate embryo co-culture system. The best possible monolayer
becomes increasingly more important due to the fact that the epithelial cell co-culture
system appears to be the source of embryotropic secretions during co-culture.
E X P E R IM E N T I
A METHOD FOR M VITRO CELL CULTURE O F SUPERFICIAL BOVINE UTERINE ENDOMETRIAL EPITHELIUM
Introduction
Efforts to culture early stage bovine embryos in vitro have met with limited
success (167). However, the use of various co-culture systems, including fetal uterine
(161,163) and adult uterine (86,118,155) fibroblasts, oviduct epithelial cells
(41,57,122), trophoblastic vesicles (27,73,119,127), and shell-less chick embryos (13)
have been shown to enhance early development of mammalian embryos in vitro.
Although embryo development benefits from co-culture, the establishment of an
adequate uterine epithelial cell in vitro culture system provides an attractive method
to evaluate uterine epithelial cell secretory capacity during the estrous cycle. In
addition, this model could be used to study early embryonic signals produced by the
uterus and the conceptus during early embryonic development in farm animals.
Isolation and culture of uterine endometrial epithelial cells has been previously
reported in mice (89,151), rats (62), guinea-pigs (1,28,29,30), rabbits (33,107), pigs
(3), sheep (136,137), rhesus monkeys (65), humans (31) and the cow (108). Although
epithelial cells have been isolated from the uterine endometrium of the cow and other
species, these procedures often rely on dissecting endometrial tissue samples for
placement directly in culture or digesting sections of endometrial tissue to obtain cells
for culture. A major drawback of utilizing these techniques is the possible contamin
ation of the culture with uterine stromal cells.
Uterine stromal cells may, themselves, have a secretory roll in the functional
uterus (62). However, to effectively conduct studies on epithelial cell secretory
capacity and/or interactions with embryos, a pure cell population is needed. The
29
30
objective of this study was to develop a rapid and efficient procedure for recovering
and maintaining a monolayer of uterine lumen epithelial cells to study uterine secretory
activity and uterine cell to embryo interactions. This procedure was developed based
on methods previously reported for recovery of oviductal cells from various species
(112). Using the procedure reported herein, only the superficial cell layers of the
uterine endometrium are removed without requiring digestion of excised endometrial
tissue samples.
Materials and Methods
I. MATERIALS
A. Equipment
1. C02 incubator, model 3158, Forma Scientific1
2. Laminar-flow hood, Nuaire2
3. Inverted microscope, phase contrast, Diaphot, Nikon3
4. Centrifuge, table top, model TJ-6, Beckman4
5. Programmable cell freezer, Planer Biomed Kryo 10 Series5
6. Peristaltic pump, no. XX80 000 00, Millipore6
7. Temperature-controlled water bath, 50°C
B. Culture media and chemicals
1. Tissue culture medium-199 (TCM-199), no. M0393, Sigma7
2. Dulbecco’s phosphate-buffered saline (PBS), no. D5773, Sigma7
3. Dulbecco’s phosphate-buffered saline (PBS), without Ca2+ and Mg2+, no.
D5773, Sigma7
4. Heat-treated fetal bovine serum (FBS), Gibco8
5. Penicillin-streptomycin (100 X ), Gibco®
6. Fungizone (100 X ), E.R. Squibb9
7. Trypsin (10 X ), .25%, no. T5650, Sigma7
8. Sodium bicarbonate, no. S5761, Sigma7
9. Serotonin, no. H4511, Sigma7
10. Dimethyl sulfoxide (DMSO), no. D2650, Sigma7
11. Ultrapure water, MilliQ system6
12. Red blood cell lysing agent, no. RT7757, Sigma7
13. EDTA, no. E8008, Sigma7
C. Supplies
1. Tissue culture flasks, 25 cm2 vented, Costar 305610
2. Tissue culture flasks, 75 cm2 vented, Costar 337610
3. Centrifuge tubes, 15 ml, Costar 321510
4. Pasteur pipettes, borosilicate glass, no. 033-76111
5. Cryovials, no. 368632, Nunc12
6. Sterile plastic petri dish, 100 x 15 mm, no. 1012, Falcon13
7. Hemacytometer, Brightline, Reichert14
8. Syringes plastic, 10 ml, no. 88291, Air-Tite15
9. Nalgene sterilization filter units, no. 125-002016
10. Glass slides, 7.5 X 2.5 cm, no. 166-645, Coming11
11. Hypodermic needles, 18 gauge x 3.75 cm length
12. Stainless steel forceps, small
13. Stainless steel scissors, medium
14. Stainless steel hemostats, medium
H. PROCEDURE
A. Preparation of media and solutions
32
1. Prepare TCM-199 and PBS according to the directions of the manu
facturer using ultrapure water.
2. Adjust the pH to 7.3 and filter medium (.2 jxm filter).
3. Store medium at 4°C until use.
4. Prepare serotonin by diluting in 10 ml of sterile PBS and freezing in
1 ml aliquots at -20°C.
5. Combine TCM-199 (450 ml), FBS (50 ml), serotonin (.5 ml) and
penicillin-streptomycin (.5 ml).
6. This results in a final concentration of 10 ng serotonin, 100 units
penicillin and 100 jig streptomycin/ml of culture medium (TCM-199).
7. Combine 500 ml PBS (without Ca2+ and Mg2+) with 1.0 ml penicillin,
1.0 ml streptomycin and .5 ml fungizone.
8. This results in a final concentration of 200 units penicillin, 200 jig
streptomycin and .25 jig fungizone/ml of PBS which is used to transport
uterine cornua to the laboratory.
9. Add .2 mg/ml EDTA to .25% trypsin solution.
10. Dilute .25% trypsin solution with sterile PBS (without Ca2+ and Mg2+)
to achieve .025% trypsin solution.
B. Isolation of epithelial cells
1. Uterine cornua are aseptically collected from the abattoir and placed on
ice in sterile PBS (without Ca2+ and Mg2+) containing 200 units
penicillin, 200 ng streptomycin and .25 pg fungizone/ml PBS.
2. Intact uterus and cornua are placed under a laminar flow hood and the
connective tissue removed.
3. The trypsin solution (.25%) is placed in a 37°C water bath prior to use.
33
4. Separate uterine homs from uterus and flush each horn with 5 ml of
warm trypsin solution (.25%) to remove debris with 10 ml syringe and
18 gauge needle.
5. Clamp one end of the uterine horn with a pair of hemostats and fill with
warm trypsin (.25%) solution until inflated and then clamp the proximal
end.
6. Repeat procedure for second uterine horn.
7. Place the fluid-filled uterine horn in sterile petri dish with a small volume
of PBS (without Ca2+ and Mg2+) and incubate for 45 min at 37°C.
8. Remove hemostats and recover the trypsin solution from each uterine
horn into a second sterile petri dish.
9. Flush both uterine horn with fresh trypsin solution (.25%).
10. Open uterine homs longitudinally with scissors and gently scrape the
lumen surface (once) with a sterile glass slide.
11. Separate epithelial clusters in the petri dish by repeated pipetting with a
pasteur pipette.
12. Place trypsin solution containing the harvested cells into 15 ml centrifuge
tubes and add equal volumes of culture medium (TCM-199) to inactivate
trypsin.
13. Centrifuge harvested uterine epithelial cells at 200 X g for 10 min.
14. Remove supernatant and resuspended pellet in 1 ml of red blood cell
lysing agent for 1 min.
15. Add 10 ml of fresh culture medium (TCM-199) to red blood cell lysing
agent and centrifuge at 200 x g for 10 min.
16. Wash the epithelial cells three times with fresh TCM-199.
34
17. Assess cell viability by trypan blue exclusion as reported by Freshney
(51).
18. Resuspend epithelial cells in 5 ml of TCM-199 and place in 25 cm2
flasks.
19. Culture the cells at 37°C in a atmosphere of 95% air and 5% C02.
20. Replace TCM-199 at 48-h intervals during the incubation period.
C. Subculture and cryqpreservation of uterine epithelial cells
1. After cells reach 90% confluency remove culture medium, add 3 ml of
.025% trypsin solution.
2. After 2 min remove trypsin solution, add fresh trypsin solution (.025%)
and incubate at 37°C and 5% C02.
3. Observe for cell rounding and for cells loosening from the bottom of the
culture flask 5 min).
4. Shake the flask gently to remove loosely adhered cells and then pipette
solution into 15 ml centrifuge tubes with pasteur pipette.
5. An equal volume of TCM-199 is added to the centrifuge tube to
inactivate trypsin.
6. Pellet cells by centrifugation at 200 X g for 10 min, and seed new 25
cm2 flasks at a density of 1 x 106 viable cells/flask with 5 ml of TCM-
199.
7. At the third subculture, prepare the cell suspension as described for
subculturing above.
8. Prepare the freezing solution consisting of TCM-199 supplemented with
30% FBS, 10% DMSO, 100 units penicillin and 100 Mg streptomycin/ml.
9. Dilute cells to 1 X 106 viable cells/ml in the freezing solution.
35
10. Place 1 ml aliquots of cells into cryovials and place in a programmable
freezer.
11. Cool cells in programmable freezer with a curve of 5°C/min to
-20°C, hold for 5 min, then decrease l°C/min to -30°C, then decrease
2°C/min to -60°C.
12. Transfer subpassaged epithelial cells to liquid nitrogen for storage.
D. Developing a growth curve
1. At the third subculture, trypsinize the epithelial cells as previously
outlined.
2. Place 2.5 x 10s cells/flask in 15 sterile 25 cm2 flasks.
3. On d 1, 2, 4, 6 and 8 of culture, triplicate 25 cm2 flasks are trypsinized
using the same procedures, except use .25% trypsin solution to ensure
removal of all cells.
4. Under 10 x power determine number of cells with a hemacytometer.
Evaluate cell viability (51).
Results
Uterine cornua were recovered from 10 adult, mixed-breed cows in various
stages of the estrous cycle. Using this cell harvesting method, the mean number of
cells isolated per uterine horn was 3.3 x 106 cells. When placed in culture flasks,
uterine epithelial cells adhered to the plastic surface and began to form monolayers
within 48 h. Ciliated and nonciliated epithelial cells were noted in the culture flasks
during primary culture. Confluent monolayers were obtained within 5 to 7 days after
initial seeding. Cilia were not evident in cultures after the first subculture. Viability
of uterine cells at different subcultures ranged from 87 to 92%. Uterine epithelial
36
cells were subcultured for seven to eight subcultures before exhibiting a decrease in
cell viability, attachment efficiency and vital cell characteristics. Decreased growth
rate, enlarged cells with a flattened appearance and vacuolization of cytoplasm were
associated with epithelial cell death.
Cells thawed rapidly at 37°C (after 60 days of storage) were placed in fresh
TCM-199. Good post-thaw cell viability and cell attachment were noted during in
vitro culture. The mean (±SE) post-thaw viability and attachment efficiency were
88.8±.8% and 88.5±2.2%, respectively. Cells could be cultured for four
subpassages following thawing before becoming flattened in appearance and losing
viable cell characteristics.
A growth curve for these uterine epithelial cells is presented in Figure 1. The
mean values presented include the number of cells for five replicates. Uterine
epithelial cells exhibited a slight decrease in cell number during the first day in culture
but exhibited a log increase in cell growth for 7 days. A stationary phase did not
appear during the culture period examined. On d 8 of culture there was still an
increase in the number of cells. The mean (±SE) number of cells were: 2 .7 ± .l,
3.7±.2, 8.3±.6, 11.7±1.7 and 13.6±.5 x 10s for d 1, 2, 4, 6 and 8 of culture,
respectively.
37
Num ber o f Days in Culture
Figure 1. A growth curve of uterine epithelial cells during the third subculture conducted for an 8-day interval.
38
Discussion
Munson et al. (108) previously established endometrial epithelial cells derived
from adult pregnant cows (d SO to 120 of gestation). In this study, epithelial cells
were successfully established by both explants and collagenase digestion techniques.
Establishment of epithelial monolayers during primary culture was not possible, in this
case, most likely due to stromal cell contamination. Efforts were made to remove
stromal cells contaminants after the first subpassage with difficulty as noted by these
researchers (108).
The major advantage of the procedure reported herein is the ability to obtain
epithelial monolayers during the primary culture free from stromal cell contamination.
This is possible by harvesting and trypsinizing superficial lumen of the uterus, instead
of obtaining cells from explants or collagenase digestion.
Munson et al. (108) reported procedures for removal of fibroblast cells from
epithelial monolayers at the first subpassage. It was noted that trypsinized fibroblasts
detached from the culture surface more readily than epithelial cells. We also found
that warm (37°C) trypsin solution (.025%) placed on cultured cells for 1 min
effectively removes any fibroblasts without removing epithelial cells. Gently shaking
the flasks also helps to remove any loosely adhered fibroblast cells. This can be
incorporated into the epithelial cell procedures at each subpassage. The use of
serotonin in the culture medium also helps maintain monolayers of epithelial cells. It
has been previously reported that serotonin has selective properties against fibroblast
proliferation in vitro (112).
Uterine epithelial monolayers have been used to enhance embryo development
of mammalian embryos in vitro. However, the identification of embryotropic factor(s)
produced by endometrial cell monolayers during co-culture is still unknown (124). The
39
ability to establish and maintain uterine epithelial cells in vitro may be a potential
model to study their secretory capacity and biochemical interactions between the
embryo and uterine endometrium.
With the procedure reported herein, uterine epithelial cells isolated from the
adult bovine uterine endometrium developed and proliferated in culture. Uterine
epithelial cells harvested by this method provided excellent monolayers virtually free
of fibroblast cell contamination. The findings while developing this method for our
laboratory indicate that uterine epithelial cells may be isolated and in vitro cultured
with good success rates. Since this system is simple and efficient it may be used in
the future to provide a model to study uterine secretory capacity, embryo interactions
with the uterus and a new embryo co-culture method.
Suppliers
1 Forma Scientific, Inc., Marietta, OH2 Nuaire Inc., Plymouth, MN3 Advanced Scientific, Inc., Chalmette, LA4 Beckman Instruments, Inc., Palo Alto, CA5 T.S. Scientific, Peakasie, PA6 Millipore, Bedford, MA7 Sigma Chemical Co., St. Louis, MO8 GIBCO Laboratories, Grand Island, NY9 Pfizer, Inc., New York, NY
10 E.R. Squibb, Princton, NJ11 Costar, Cambridge, MA12 Curtin Matheson Scientific, Inc., Houston, TX13 Vangard International, Inc., Neptune, NJ14 Falcon, Oxnard, CA13 Reichert Scientific Instruments, Buffalo, NY16 Air-Tite of Virginia, Inc., Virginia Beach, VA17 VWR Scientific, Rochester, NY
E X PE R IM E N T H
EFFECTS OF STAGE OF THE BOVINE ESTROUS CYCLE ON IN VITRO CHARACTERISTICS OF UTERINE AND
OVIDUCT EPITHELIAL CELLS
Introduction
Oviductal epithelial cells of bovine and ovine origin have also been used for
in vitro co-culture of early-stage cow (41), goat (121) and sheep (126) embryos. In
each instance, the co-culture system used has proven to be effective in supporting
development of embryos through the characteristic in vitro block stages. These
findings suggest the existence of unidentified growth factor(s) produced by the cells
of monolayers that have embryotropic properties, perhaps via the secretion of specific
peptides and/or proteins (124). Studies have shown that ovarian hormones influenced
proliferation, ciliation and secretory processes of the oviduct epithelium in the rabbit
(50), monkey (25,110) and cow (98). Although various studies have used epithelial
monolayers for co-culture of early stage embryos (41,57,126), these studies have not
addressed possible differences in cells obtained for culture during different stages of
the estrous cycle and their effects on developing a successful embryo co-culture
system.
Salamonsen et al. (136) reported differences in appearance of endometrial
epithelial cells during primary culture when ovariectomized ewes were pre-treated with
different levels of progesterone and estrogen prior to cell harvest. Also, the hormonal
environment of the endometrium at the time of harvest resulted in a difference in
growth characteristics of human stromal endometrial cells during primary culture (49).
Changes in uterine epithelial glands were also reported following the administration
of progesterone to estradiol-primed ovariectomized animals (21,22). Progesterone has
40
41
also been reported to induce morphological changes in cultured uterine epithelial cells
obtained from the guinea pig (1,2).
Steroid treatment in sheep influenced in vivo incorporation of radiolabelled
methionine into cellular and secreted proteins of epithelial cells (136). In addition to
changes in cell morphology, cyclic changes in hormonal levels during the estrous cycle
may result in differences in cell characteristics. Differences in cell characteristics due
to the stage of the estrous cycle may ultimately have an effect on embryonic growth
in vitro when these cells are used in an in vitro co-culture system. The objective of
this study was to compare in vitro morphology, growth and monolayer foundation of
bovine uterine and oviduct epithelial cells when collected at different days of the
estrous cycle.
M aterials and Methods
Experimental Animals and Treatment Groups. A group of cyclic Holstein
heifers (range in estrous cycle length of 17 to 23 days), all in good body condition and
ranging in body weight from 350 to 400 kg, were used in this study. Heifers were
maintained on native grass pasture and provided daily with hay and com silage.
During the spring months heifers were synchronized with prostaglandin
(Lutalyse®; Upjohn Co., Kalamazoo, MI) then equally and randomly assigned within
day of the estrous cycle to one of four treatment (Trt) groups (n = 5/treatment). Only
heifers exhibiting standing estrus were used in the experiment. Treatment groups were
assigned by days of the estrous cycle (estrus — d 0): d 0 (Trt A), d 4 to 6 (Trt B), d
8 to 10 (Trt C) and d 14 to 16 (Trt D). Heifers in Trt A had one or more medium-
size follicles present on ovaries, plasma progesterone levels below 1 ng/ml and often
had visible mucus on the vulva. Females in Trt B, C and D had either a developing
42
corpus luteum or palpable corpus luteum and £ 2.0 ng/ml plasma progesterone at the
time the reproductive tracts were harvested.
Reproductive tracts were collected from animals at slaughter on respective
estrous cycle treatment days. Tracts were placed on ice immediately after collection
in PBS (without Ca2+ and Mg2+) containing 200 units penicillin, 200 Mg streptomycin
and .25 Mg Amphotericin-B/ml and transported to the tissue culture laboratory. The
time from the slaughter of animals until reaching the laboratory was «1 h.
Isolation of Epithelial Cells. Tissue Culture Medium-199 (Sigma) with 10%
heat-treated fetal bovine serum (FBS), 100 units penicillin, 100 fig streptomycin
(Gibco), 10 Mg serotonin (Sigma), .1 ml L-glutamine and .1 ml Na pyruvate/ml
(Gibco) of culture medium (TCM-199) was used as the base medium throughout the
study.
Uterine and oviductal cells were isolated and cultured with modifications of the
procedure previously reported for uterine (148) and oviduct (112) epithelial cell
cultures in cattle. With this procedure, a 10 cm section was removed from each
uterine hom 3 cm posterior to the utero-tubal junction and 3 cm posterior of the
ampulla for oviducts, then trimmed free of mesentery. Uterine horn and oviduct
sections were flushed with 5 ml of warm .25% trypsin supplemented with .2 mg/ml
EDTA to remove debris then filled with fresh trypsin solution, clamped and incubated
at 37°C for 45 min. The trypsin solution was recovered and the tissue flushed a
second time with fresh .25% trypsin. Uterine horns and oviducts were dissected
longitudinally and the lumen surface gently scraped once with a sterile glass slide.
Cells isolated from each section were combined and epithelial clusters separated by
repeated pipetting. The trypsin solution was inactivated by the addition of the base
culture medium (1:2 v/v). The entire procedure was carried out under a laminar flow
43
hood at room temperature.
Harvested cells were centrifuged at 200 x g for 10 min, the supernatant
removed and the cellular sediment resuspended in 1 ml of red blood cell lysing agent
(Sigma) for 1 min. Fresh TCM-199 was added (10 ml) and the suspension centrifuged
at 200 x g for 10 min. The supernatant fluid was removed and the cellular sediment
resuspended in TCM-199 (1 ml). A small portion of cells were assigned to electron
microscopy and the remaining cells placed in 25 cm2 vented tissue culture flasks
(Costar) with 5 ml of TCM-199. All cultures were maintained at 37°C in an
atmosphere of 95% air and 5% C02 and culture medium changed at 48-h intervals.
Subculturing confluent monolayers were conducted by trypsinization with .025%
trypsin supplemented with .2 mg/ml EDTA. Flasks were incubated at 37°C for 5 to
10 min until cells became rounded in shape and detached from the bottom of the flask.
The trypsin was inactivated by the addition of TCM-199 (1:2 v/v). Cells were
centrifuged, the supernatant removed then resuspended in fresh TCM-199 and seeded
at 1:2 ratios for each treatment group.
In Vitro Characteristics. Prior to initial seeding, the number of cells recovered
was determined on a hemacytometer and viability assessed by the trypan blue dye
exclusion method (51). Percent cell attachment was determined by removing the
culture medium and counting the number of cells that did not attach during the initial
48 h (51). The number of cells, percent viability and attachment were also determined
at the first and third subpassages. Histological evaluations of cells stained with
Giemsa stain (51) were made during primary culture and first and third subpassages.
During primary culture and after the first and third subpassage, samples of cells were
fixed for scanning and transmission electron microscopy. After the third subpassage,
cells were frozen in liquid nitrogen.
44
Freezing Epithelial Cells. Cells from the third subpassage were placed in 2 ml
cryovials (Nunc, Naperville, EL) at a concentration of 1 x 106 viable cells/vial and
frozen in TCM-199 adjusted for a final concentration of 30% FBS and 10% DMSO.
Cells were frozen in a Planer Biomed Kryo 10 Series programmable freezer using a
curve of 5°C/min to -20°C (held for 5 min), l°C/min to -30°C then 2°C/min to
-60°C and then transferred and stored in liquid nitrogen.
Electron Microscopy. For scanning electron microscopy, uterine and oviductal
cells were seeded in one well of a 24-well Transwell9 tissue culture plate (Costar).
At confluency, monolayer cultures were rinsed with PBS, fixed in 3% glutaraldehyde
in sodium-cacodylate buffer (. 1 M, pH 7.4) for 1 h, washed twice in buffer, post
fixed in 1% osmium tetroxide in sodium-cacodylate buffer (.1 M, pH 7.4) and
dehydrated in a dilution gradient of ethanol:water (30, SO, 70, 80, 90 and 100%
ethanol). Cells were critical-point dried then coated with gold-palladium and evaluated
with a Cambridge Stereoscan 150 scanning electron microscope.
For transmission electron microscopy, uterine and oviductal cells were seeded
in two-well plastic chamber slides (Lab-Tek). At confluency, monolayer cultures were
rinsed with PBS, fixed in 3% glutaraldehyde in sodium-cacodylate buffer (.1 M, pH
7.4) for 1 h, washed twice in buffer, post-fixed in 1 % osmium tetroxide in sodium-
cacodylate buffer (.1 M, pH 7.4). Samples were then dehydrated in a dilution
gradient of ethanol:water (30, 50, 70, 80, 90 and 100% ethanol), infiltrated in an
alcohol-Epon mixture and embedded in Epon and evaluated with a Zeiss-10
transmission electron microscope.
Immunocvtochemistrv. Verification of uterine and oviductal cell origin using
immunofluorescence staining was performed as previously described (113). Cells were
fixed in paraformaldehyde, permeabilized with Triton X-100 then incubated with
45
bovine monoclonal anti-cytokeratin (clone K 8.13; Sigma) diluted 1:20 and finally
incubated with FITC-conjugated mouse and-IgG (1:50). Negative controls consisted
of a fibroblast cell line and positive controls were a MDBK cell line. Culture
preparations were examined using a Zeiss microscope equipped for FTTC excitation.
Cell Cycle Analysis. Immediately after cell isolation, 1 x 106 cells from the
remaining portion of the reproductive tract were obtained from animals in each
treatment. Cell cycle analysis was conducted using flow cytometry as described by
Lakhanpal et al. (88). Samples were fixed in 2% paraformaldehyde, then in 95%
ethanol, treated with 100 Mg RNase (Boehringer Mannheim Biochemicals, Indinapolis,
IN) and DNA stained with 5 ng/ml of propidium iodide (Sigma). Flow cytometry
was performed on a FASC 440 (Becton-Dickinson, San Jose, CA) fluorescence-
activated cell sorter that was configured for red fluorescence analysis that was linearly
amplified. Cells were exposed to 488 nm argon line and fluorescence emission was
detected using a 630/22 bandpass filter for propidium iodide detection. Data from
flow cytometry was analyzed using Consent-40 software (Becton-Dickinson) and the
percent of a cell population in a resting (Gap 1 or Gap 0) or proliferative (S, Gap 2
and mitosis phase) state of the cell cycle recorded (68).
Progesterone Levels. Bovine blood samples were collected via venipuncture
48 h prior to and immediately before transport to the abattoir. Blood samples were
collected in EDTA coated vacuum tubes, plasma harvested and stored at -20°C until
later assayed for progesterone according to procedures previously reported (150).
Statistical Analysis. The number of cells isolated, percent viability and
attachment were analyzed using a completely randomized split plot design with
repeated measures in time (60). Main effects within the model included treatment,
heifer within treatment, tissue type (uterine or oviduct), subpassage and important
46
interactions. Significant differences between least square means were determined by
preplanned comparisons of means.
Results
In Vitro Characteristics. The mean number (±SE) of cells isolated for uterine
and oviductal tissue were 3.3±.8 and 4 .4 ± .8 x 106, respectively. The mean number
of cells recovered from.uterine tract was not significantly different (P>.05) across
days of the estrous cycle or between uterine or oviductal cells. The percent viability
and attachment for each treatment group during primary culture are summarized in
Table 1. There was a reduction (P< .005) in percent viability of uterine cells in Trt
C and D compared with Trt A and B. For oviductal cells, Trt B had higher (P < .01)
percent viability than those of other treatments. There was a marked (PC .0001)
reduction in percent attachment in both uterine and oviductal cells noted for Trt C and
D during primary culture compared with that of Trt A and B.
Uterine and oviductal cells in Trt A and B adhered to the culture surface and
proliferated rapidly within 24 h after initial seeding. Confluent monolayers were most
often obtained within 5 to 6 days following the start of incubation. However, since
there were significantly fewer cells that attached in Trt C and D, confluent monolayers
were obtained in 10 to 14 days. The cells that attached to the culture surface began
to curl around each other and form colonies within a few h after seeding. Cells grew
in swirls composed of cohesive groups of polygonal cells. These characteristics were
observed for all treatment groups. Based on daily observations and Giemsa staining,
there were no noted differences in morphology between uterine and oviductal cells or
between treatment groups for day of the estrous cycle. Populations of ciliated and
nonciliated cells were isolated from both the uterus and oviducts. Ciliated cells could
47
be detected in cultures until 4 to 5 days after seeding but were not noted after the first
trypsinization (subpassage).
The cells that attached and proliferated in culture exhibited increases in percent
cell viability (PC.0001) and percent cell attachment (PC .0001) after the first and
third subpassages. There were ho significant differences in viability among treatment
groups for both uterine or oviductal cells following the first subpassage. Although
there were significant improvements in attachment efficiency following the first
subpassage, Trt C had a significantly lower percent attachment for oviductal cells than
did Trt B (PC .01) and Trt D (PC .05). However, after the third subpassage, there
were no significant differences in percent cell viability and cell attachment between
treatment groups for either uterine or oviductal cells.
Cell Freezing. After a minimum of 60 days, cells were thawed at 37°C and
washed three times in TCM-199. Percent post-thaw viability and attachment for
uterine and oviductal cell were not significantly different. Therefore, all data for
uterine and oviductal cells across all treatment groups were combined and expressed
a single percent. The mean (±SE) post-thaw percent viability and attachment were
88.8±.8 and 88.5±2.2, respectively.
Electron Microscopy. Scanning electron microscopy revealed numerous
bundles of cilia present on the cell surface of uterine and oviduct cells during primary
culture. Oviductal cells contained more dense patches of cilia present on cell surface
compared with uterine cells obtained from the same experimental animal (Figure 2).
Only microvilli were present on the culture surface at the first subpassage, indicating
a loss of cilia from the primary cultures (Figure 3). In many cells evaluated,
microvilli could be detected on the apical surface of the cell for all subpassages inves
tigated along with numerous mitochondria, desmosomes and microfilaments. These
48
characteristics were noted for both uterine and oviduct cells. Secretory granules could
be detected in specific cells at the first and third subpassages along with small clusters
of rough endoplasmic reticulum and some lipid inclusions.
Immunocvtochemistry. The presence of keratin filaments were detected on
both uterine and oviductal cultures during primary culture and the third subpassage.
The immunofluorescent detection of cytokeratin was easily observed in the MDBK
established cell line but absent for chick embryo fibroblast cell lines. These results
along with electron microscopy data confirm both uterine and oviductal cell cultures
to be of epithelial cell origin.
Cell Cvcle Analysis. The mean percent cell proliferation rates for treatments
A, B, C and D for both uterine and oviductal cells combined were 22.7, 7.6,22.1 and
17.7%, respectively (Figure 4). The coefficient of variation for each sample obtained
from cell cycle analysis ranged from 5.6 to 11.4 for all experimental animals
combined. The lowest percent cell proliferation observed in Trt B corresponds with
the highest percent cell attachment during primary cultures.
Progesterone Levels. The mean progesterone levels 48 h prior to and at
slaughter were .14 and .38 for heifers in (Trt A), .05 and 1.0 (Trt B), 9.91 and 10.34
(Trt C) and 7.59 and 5.26 (Trt D) ng/ml, respectively. Estrus detection records and
the progesterone values verified that the reproductive tract cells were sampled from
estrual, early, middle and late luteal phases of the estrous cycle.
49
Table 1. Effects of the estrous cycle on in vitro evaluations of uterine and oviduct epithelial cells (least squares means ±SE)
Treatment
ItemA
dOB
d 4-6C
d 8-10D
d 14-16
Primarv Culture Percent Cell Viability
Uterine Oviductal
Percent Attachment Uterine Oviductal
80.6±2.5*79.2±2.5*
84.6±3.9d79.7±3.9d
87.7±2.5b88.4±2.5b
87.5±3.9d87.2±3.9d
76.5 ±2.5*77.5 ±2.5*
10.8±3.9*27.3±3.9*
68.8±2.5* 74.1 ±2.5*
10.5±3.9C18.4±3.9®
Third Subpassaee Percent Cell Viability
Uterine Oviductal
Percent Attachment Uterine Oviductal
89.0±4.087.8±4.0
88.1 ±6.2 81.9±6.2
91.3±4.092.8±4.0
91.5±6.2 90.1 ±6.2
95.3±4.093.2±3.2
82.7±6.285.8±6.2
89.3±4.083.9±4.0
91.7±6.282.4±6.2
■*b,eSuperscripts with different letters within rows and tissue type differ (P< .05). ^'Superscripts with different letters within rows and tissue type differ (P< .0001).
Figure 2. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture isolated from the same animal on d 4 to 6 of the estrous cycle. Note the differences in cilia across the cell surface (2,700 x).
Figure 3. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells at the third subpassage. These are cells isolated from the same animal as Figure 2 (20,000 x).
52
26 r2 4 -
T rea tm en t Groups
Figure 4. Mean percent of cells proliferating isolated from different stages of the estrous cycle. Values represent uterine and oviductal cells combined.
53
Discussion
There were no noted differences in morphology of uterine and oviduct epithelial
cells isolated from different stages of the estrous cycle. Stage of the estrous cycle
dramatically affected epithelial cell monolayer foundation in this study. Other
researchers (30,136) have suggested that progesterone may also act by interfering with
patterns of proteins synthesized and/or released by the reproductive tract epithelial
cells in vivo or in vitro. The marked reductions in percent attachment of uterine and
oviductal cells from animals in the mid to late luteal phases of the estrous cycle, may
be attributed to several factors such as physiological state of the cell (68), cell mor
phology and/or unknown factors.
Differences noted in cells harvested during mid to late luteal stages of the
estrous cycle may be attributed to fewer attachment factors present on the cell surface
during the late luteal phase of the estrous cycle. Another possible explanation for
reduced attachment may be cells during the late luteal phase have attachment factors
on the cell surface that are more sensitive to trypsinization. Removal of attachment
factors such as E-cadherin (S), present on the cell surface, would account for reduced
attachment and this sensitivity to trypsin may only be detected after removal in vivo.
Also, the highest proportion of cells in a proliferative state of the cell cycle (Trt C and
D) corresponded with the lowest percent cell attachment. These suggestions are
strengthened by the fact that after the first subpassage the percent attachment
increased, and this was similar for all treatment groups. There appears to be a
selection process occurring during primary culture with only cells that have the
greatest capacity for in vitro growth being maintained following subpassages.
Previous reports (74,78) indicate that three different cell types are routinely
isolated from oviducts, consisting of ciliated, nonciliated and columnar cells. Only
54
the nonciliated cells are generally classified as secretory cells. All three cell types
could be detected in the present study with both ciliated and nonciliated cells attaching
to the culture surface and rapidly proliferating. Although experimental animals in the
mid to late luteal stages of the estrous cycle had low percent attachments both ciliated
and nonciliated cells were detected in the culture dish. This suggests that the low
percent attachment is not specific to ciliated or nonciliated cells. These findings
suggest that epithelial cells examined in this experiment had similar morphology and
growth with observations previously reported (74,78). The results of the present study
also showed that most of the cilia activity was lost for both uterine and oviductal cells
during the first 4 to 5 days of incubation.
Loss of ciliation in cultures may be attributed to one or more factors. First,
changes in pH of culture medium may result in loss of cilia during primary cultures
or ciliated cells are not capable of dividing and there is a selection against these cells
during subpassages. Possibly, the ciliation process is the end point of differentiation
and cannot be easily induced in an in vitro system. Therefore, only secretory cells and
uncompleted ciliated cells existed during subpassages.
In this study it was noted that cells isolated from «10% of the animals became
vacuolated during incubation and appeared to enter a "crisis” phase. This was not
associated with any particular subpassage, hormonal status of animals at collection or
for cell type (uterine cells or oviductal cells). It was noted that if oviductal cells
began to enter a "crisis" phase then uterine cells from that same animal exhibited
similar patterns in vitro. This occurrence suggests individual animal variation that may
be due to the previous endocrinological history and/or present status of the animal.
There may be a limited number of cell cycles that are highly dependent upon each
animals response to circulating hormones. This model of programmed cell death may
55
be directly modulated by the previous endocrinological pattern of the animal. This
suggestion is further strengthened with electron microscopy revealing normal cell
morphology during subpassages and little damage or deteoriation of cells due to
trypsinization. Also, viable cell morphology and function were evident in the high
viability and percent attachment following the third subpassage.
Several preliminary trials conducted at this laboratory have suggested that once
cells begin vacuolation, this process associated with degeneration could be reversed by
increasing the serum content of the culture medium from 10 to 20% and replacing half
of the culture medium at 48-h intervals. Perhaps increasing the serum content
provides more growth factors to help reduce the cell degeneration process. The
vacuoles observed with degeneration in this study appeared to differ from lipid
containing vacuoles observed in healthy cells by Witkowska (166) and healthy cells in
this study (TEM evaluations).
Epithelial cell monolayers are now being used to enhance development of early
stage embryos in vitro. However, identification of embryotropic factors produced by
helper cell monolayers during co-culture are still unknown (124). When using primary
cultures of uterine or oviduct epithelial cells in an embryo co-culture system the source
of cells needs consideration. It has been reported, however, that subpassaged cells are
equally as effective or even more effective in some cases than primary cultures for co
culture (113,121). In this study, subpassaged cells exhibited the same characteristics
regardless of stage of estrous cycle uterine and oviductal cells were harvested. There
appears to be a selection process occurring for subpassaged cells harvested from the
mid to late luteal stage of the estrous cycle. This selection process may ultimately
affect embryonic development in an embryo co-culture system. Finally, it appears that
individual animal to animal variation may be reduced by the use of subpassed
monolayers to provide more uniform co-culture systems producing embryotrqpic
factors. Further experiments are needed to identify specific factors produced by cell
monolayers during embryo co-culture that result in enhanced in vitro development of
embryos.
E X PE R IM E N T m
THE EFFECT OF CULTURE MEDIUM AND TEMPERATURE ON IN VITRO GROWTH AND PROLIFERATION OF UTERINE
AND OVIDUCT EPITHELIAL CELLS
Introduction
Many studies have been conducted on uterine and oviduct epithelial cells using
different culture media to maintain in vitro growth and development. Epithelial cells
from uterine or oviductal origin have been isolated from the sheep (136), mouse (151),
rabbit (129) and guinea pig (28) and maintained in vitro in a variety of media such as
Dulbecco’s Modified Eagles medium (DMEM), a mixture of DMEM and Ham’s F-12
(HF12), DMEM and CMRL-1066 (CMRL), respectively. Also, a variety of media
has been used to study epithelial cell-embryo interactions with bovine uterine and
oviduct epithelial cells in in vitro culture systems. Excellent growth of bovine
epithelial cells has been obtained with Menezo’s B2 medium (MB2) (112), TCM-199
(148), DMEM (74) and a mixture of DMEM and HF12 (78).
In addition to establishing monolayer characteristics, oviductal cells are also
used for co-culturing embryos using various media. Oviduct epithelial cells have been
used for in vitro co-culture of early-stage cattle (40) and sheep (126) embryos. In
addition, uterine epithelial cells have been used for in vitro co-culture of
preimplantation embryos of rhesus monkeys (65). In each instance, the co-culture
system has proven effective in supporting some degree of development through the
characteristic in vitro block stages (for review see 124). A variety of culture media
have been used to establish oviductal cells prior to embryo co-culture experiments.
In the cow, culture medium used to maintain monolayers prior to and during embryo
co-culture consisted of TCM-199 (41,57,117), Ham’s F-10 (37), HF12 (14), CMRL
57
58
(37) and MB2 (116,138).
Recently, in vitro fertilization studies have used oviductal cell monolayers to
enhance embryonic growth following fertilization (40,82,84,133). In vitro fertilization
procedures are routinely conducted at 39 °C to more closely resemble body temperature
of the cow (38.9°C). The possibility exists that altering standard incubation
temperature of cell culture from 37°C to 39 °C may affect growth and embryotropic
factor production by epithelial cells used in embryo co-culture experiments. Although
various experiments have adjusted culture conditions in maintaining epithelial
monolayers for embryo co-culture, the authors are aware of no studies that have been
conducted to address if culture media and incubation temperature affect growth and
longevity of epithelial cells maintained in vitro. The optimum culture conditions for
growth of epithelial cells used in embryo co-culture systems could ultimately enhance
the embryotropic factor(s) secreted by monolayers during co-culture.
The objective of this study was to evaluate cell growth patterns and percent of
cells proliferating for uterine and oviduct epithelial cells incubated in different media
and temperatures.
Materials and Methods
Cell Source. Reproductive tracts from 10 mature cyclic cattle were obtained
from a local abattoir. Reproductive tracts were placed on ice immediately after
collection in phosphate-buffered saline without Ca2+ and Mg2+ (PBS) containing 200
units penicillin, 200 Mg streptomycin and .25 fig/ml Amphotericin B. The time from
slaughter until reaching the laboratory was »1 h.
Isolation of Epithelial Cells. The culture medium used to initiate cell growth
was Tissue Culture Medium-199 (Gibco) supplemented with 10% heat-treated fetal
59
bovine serum (FBS), 100 units penicillin, 100 /Jg streptomycin, 10 Mg serotonin
(Sigma) and . 1 ml L-glutamine/ml of medium (TCM).
Uterine and oviductal cells were isolated and cultured with modifications to
procedures previously reported by this laboratory (146,148). In summary, uterine
horn and oviduct sections were flushed with 5 ml of warm .25% trypsin supplemented
with .2 mg/ml ETDA to remove debris then filled with the trypsin solution, clamped
and incubated at 37°C for 45 min. The trypsin solution was recovered and the tissue
flushed a second time with warm flesh trypsin solution. Uterine horns and oviducts
were dissected longitudinally and the lumen surface gently scraped once with a sterile
glass slide. Epithelial clusters were then separated by repeated pipetting and the
trypsin solution inactivated by the addition of culture medium (1:2 v/v). Harvested
cells were then washed three times in TCM.
Cells were seeded in 25 cm2 tissue culture flasks with 5 ml of TCM. All
cultures were maintained at 37°C in an atmosphere of 95% air and 5% C02 and
culture medium replaced at 48-h intervals. Subculturing confluent monolayers were
conducted by trypsinization with .05 % trypsin supplemented with .2 mg/ml EDTA.
Flasks were incubated at 37°C for 5 to 10 min until cells became rounded in shape
and detached from the bottom of the flask. The trypsin was inactivated by the
addition of TCM (1:2 v/v). Cells were resuspended in flesh TCM and seeded at ratios
of 1:2.
Experiment 1.
The objective of this experiment was to evaluate the effects of five different
culture media on growth and percent of proliferating cells for uterine and oviduct
epithelial cells maintained in vitro. Reproductive tracts from five cyclic cows were
60
used in this experiment. At the second or third subpassage, uterine and oviductal cells
were trypsinized as previously described. Cells originating from each animal were
allotted within animals to five treatment groups. Treatment groups consisted of cells
incubated in TCM (Trt A), CMRL (Trt B), MEM (Trt C), MB2 medium (Trt D) or
HF12 (Trt E). All culture medium except MB2 (Biomerieux, France) was purchased
from Gibco Laboratories and supplemented with 10% FBS, 100 units penicillin and
100 Mg streptomycin/ml of medium. All cultures were maintained at 37°C in an
atmosphere of 95% air and 5% C02 and culture medium replaced at 48-h intervals.
Growth Curve and Cell Cvcle Analysis. Growth characteristics for uterine and
oviductal cells were determined by initially plating cells at a density of 2.5 X 10s
cells/flasks in ten 25 cm2 flasks/treatment. Cells were allowed to attach and initiate
growth for 24 h before the first cell count. At designated intervals duplicate flasks
from each treatment group were trypsinized with .25% trypsin solution and the
number of cells/flask counted on a hemacytometer (51). The number of cells from
each treatment and examinations of the cell cycle were determined on d 1, 2, 4, 6 and
8 of incubation.
Immediately following cell counts, the percent of the cell population in a
proliferative state of the cell cycle was determined using flow cytometry (88). Cells
from each treatment group were washed twice in PBS then fixed in 2% para
formaldehyde (Fisher Scientific Co., Fair Lawn, NJ) for 3 min at room temperature.
Cells were washed with PBS, centrifuged, the supernatant fluid removed, and the
pellet resuspended in PBS then incubated in 95% ethanol for 3 min. Samples were
washed twice in PBS then incubated for 30 min in 100 fil of PBS-EDTA and 100 Mg
of RNase (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a 37°C water
bath. Finally, samples were resuspended in 200 m! PBS-EDTA with 5 Mg/ml of
61
propidium iodide for DNA staining. Flow cytometry was performed on a FASC 440
(Becton-Dickinson, San Jose, CA) fluorescence-activated cell sorter. Cells were
excited with the 488 nm argon line and fluorescence emission detected using a 630/22
bandpass filter for propidium iodide detection.
The percent of the cell population in a resting state or proliferating state
recorded and data analyzed using Consent 40 software (Becton-Dickinson). Cells in
a resting state were composed of Gap 1 or Gap 0 while cells in a proliferating state
of the cell cycle were composed of S, Gap 2 and mitosis phases (68).
Experiment 2.
The objective of this experiment was to evaluate the effects of two incubation
temperatures on growth and percent of cells proliferating for uterine and oviduct
epithelial cells maintained in vitro. Based on the success of the culture medium in the
first experiment, CMRL supplemented with 10% FBS, 100 units penicillin and 100
tig streptomycin/ml of medium was used throughout the second experiment. Data for
fitting growth curves and cell cycle analysis were collected on the same experimental
days as in Experiment 1. However, treatment groups consisted of cells incubated at
37®C (Trt A) or 39°C (Trt B).
Statistical Analysis. A total of three replicates of duplicate flasks from each
animal were used to construct the growth curve for each experiment. The number of
cells and percent of cells proliferating were analyzed using a RBD split plot design
with repeated measures in time (60). Statistical comparisons of least squares means
were conducted using preplanned comparisons. For the analysis, cells originating
from each animal served as blocks to account for variation between animals.
62
Results
Experiment 1.
Uterine and oviductal epithelial cells attached and initiated growth in all culture
media evaluated. A growth curve of uterine epithelial cells is shown in Figure 5.
There was a slight decrease in cell numbers on the first day of incubation followed by
a log increase in growth for 5 days. On d 6 of incubation, Trt B had significantly
(P< .01) more cells than all other treatment groups. However, on d 8 of incubation,
Trt B and D had significantly (P < .0001) more cells than Trt A, C and E. There were
no significant differences between Trt A, C or E on d 6 or 8 of incubation. Cells
incubated in Trt D exhibited slow growth rates during the first 6 days of culture but
had a high growth rate at the end of the culture period. Cells incubated in Trt A, C
and E reached a stationary phase of growth by d 8 of incubation. However, cells
incubated in Trt B and did had not reach a stationary phase at the end of the culture
period. All treatment groups reached confluency by the end of the culture period.
There were no significant differences in cell growth between treatment groups
during the first 2 days of incubation for oviductal cells (Figure 6). However, Trt B
and C had significantly (PC .0001) more cells than Trt A, D and E on d 4 of
incubation. Cells incubated in Trt B had significantly (PC .0001) more cells than
other treatment groups on d 6 and 8 of incubation. Oviductal cells incubated in Trt
A, C and E exhibited stationary phases on d 6 of culture. However, cells in Trt B and
D were still exhibiting increases in cell growth at the end of the incubation period.
Oviductal cells cultured in Trt B reached confluency by d 6 of culture however, there
were still increases in cell numbers. All other treatments reached confluency by d 8
of incubation.
The percent of proliferating cells for each treatment were determined using
63
flow cytometry. All samples were collected, analyzed and results stored in the
computer prior to data processing. This allowed samples to be analyzed under similar
conditions and parameters. Gating of cell populations for analysis was conducted
based on histograms illustrating two distinct populations of cells (Figure 7). The
coefficient of variation for each peak observed during analysis was between 5 and 10%
for all samples. The mean percent of cells proliferating for uterine and oviduct
epithelial cells prior to the initiation of culture (d 0) were 50.2 and 49.6%,
respectively.
There was a decrease in percent of cells proliferating for uterine and oviduct
epithelial cells as the number of cells increased (Table 2). There was also an inverse
relationship between the number of cells and percent of cells proliferating. There were
no significant differences in the percent of cells proliferating between treatment groups
for any of the days in culture examined for both uterine and oviduct epithelial cells.
Those treatments with fewer cells at the end of the incubation period had slightly
higher percent of cells proliferating (Trt A, C and E). Epithelial cells incubated in Trt
B continued to increase in cell numbers even though percent of cells proliferating
indicated a resting state of development.
Oviduct epithelial cells gave evidence of growing more rapidly than uterine
epithelial cells. Therefore, all treatment groups were combined and growth of uterine
cells were compared to oviduct epithelial cells (Figure 8). There were no significant
differences between uterine and oviductal cells during the first 2 days of incubation.
However, there was a significant increase (PC .005) in the number of oviductal cells
compared with uterine cells for d 4, 6 and 8 of incubation.
64
inOx
jn*q3O
cuXIE3
2 5 r
20
15
10
CMRLMB2
V
TCM
MEM8 HF12
J. X X X81 2 3 4 5 6 7
Num ber of Days in Culture
Figure 5. A growth curve of uterine epithelial cells conductedfor 8 days in different culture media.
65
inO
x*w'm"55
a>x>E=3
5 0
4 5
4 0
3 5
3 0
2 5
2015
10
5
lT I | I I I I
0 1 2 3 4 5 6 7 80
V CMRL
MB2
MEM
HF12TCM
Number o f Days in Culture
Figure 6. A growth curve of oviduct epithelial cells conductedfor 8 days in different culture media.
66
64
48
32
Side SC (4)
16
Total E vents = 9835
Q uad ran ts a t [19,50]
1) 0 = 0.0%II) 0 = 0.0%
III) 5876 = 59.7%IV) 3959 = 40.3%
16 32Red Flu (3)
48 64
Figure 7. An example of flow cytometiy analysis of the cell cycle. Total events are the number of cells analyzed with individual quadrants gated based on distinct cell populations. Quadrant m indicates the percent of the cell population in a resting state whereas Quadrant IV indicates a proliferative state of the cell cycle.
67
Oviduct2 5
20o
U terusx
tn"a>O«*-o1_a)s iE3
2 3 4 - 5 6 7 810Number o f Days in Culture
Figure 8. Growth rates of uterine and oviduct epithelial cells for 8 days of incubation. All culture media treatment groups were combined and presented as mean values for uterine and oviductal cells.
68
Table 2. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve in different media
Davs in CultureItem d 1 d 2 d 4 d 6 d 8
Trt A (TCM) Uterine Cells Oviductal Cells
37±344±6
36±4 31 ±4
48±4 25 ±4
23±3 23 ±4
24±3*22±4
Trt B (CMRL) Uterine Cells Oviductal Cells
37±340±4
42 ±4 37±3
37±324±4
21 ±3 14±3
15±3,,b15±3
Trt C (MEM) Uterine Cells Oviductal Cells
36±4 34 ±4
33±4 41 ±4
33 ±4 30±4
17±417±2
16±4'*b17±2
Trt D (MB2) Uterine Cells Oviductal Cells
37±336±4
32±530±4
36±531±4
22 ±3 20±4
19±3,,b17±3
Trt E (HF12) Uterine Cells Oviductal Cells
33±5 35 ±6
33 ±5 35 ±6
22±5 28 ±5
16±3 22 ±4
15±3b21±4
*,b Mean number of cells within columns with different superscripts differ (PC .05).
69
Experiment 2.
In this experiment, cells were cultured in CMRL medium to determine
maximum effects of incubation temperature on growth and percent of cells
proliferating for uterine and oviduct epithelial cells. There were no significant
differences between treatment groups for growth of uterine (Figure 9) or oviduct
(Figure 10) epithelial cells. Similar growth patterns were noted for uterine and
oviductal cells in this experiment as obtained for Trt B in Experiment 1. Also, there
were no significant differences in the percent of cells proliferating among temperature
treatment groups (Table 3). Oviductal cells incubated under these conditions exhibited
rapid cell growth and could be maintained for several subpassages without
degeneration of cell quality associated with cell death.
70
■nOx
m"oO
0)XIE3
2 5
20
15
10
39 C
37 C
O
O
1 2 3 4 5 6 7Number o f Days in Culture
8
Figure 9. Growth rates for uterine epithelial cells incubatedfor 8 days at 37°C or 39°C.
71
6 039 C
5 5
5 0 37 C4-5
4 0
3 5
3 0
2 5
20
B_l_
0 1 2 3 4 5 6 7 8Number o f Days in Culture
Figure 10. Growth curves for oviduct epithelial cells incubatedfor 8 days at 37°C or 39°C.
72
Table 3. Mean percent of cells proliferating for uterine and oviduct epithelial cells during a 8-day growth curve at different temperatures
Davs in CultureItem d 1 d 2 d 4 d 6 d 8
Trt A (37 °C) Uterine Cells Oviductal Cells
39±338±6
33 ±4 36±3
19±4 15±3 36±3 21±3
15±315±3
Trt B (39°C) Uterine Cells Oviductal Cells
40±338±3
29±337±3
18±3 16±3 32±3 22±3
16±317±3
73
Discussion
All culture media evaluated in this study adequately supported growth of
uterine and oviduct epithelial cells. Several cell populations were maintained for three
or four subpassages on each medium tested. Also, uterine and oviductal cells were
maintained at 39 °C for three or four subpassages. There were no adverse effects
detected due to culture medium or temperature.
Uterine and oviduct epithelial cells exhibited characteristic growth patterns.
Freshney (51) described cell growth as having a short lag period after seeding,
followed by a log increase in cell numbers for several days followed by a stationary
phase and subsequently a decline phase. Epithelial cells cultured in CMRL (Trt B)
and MB2 (Trt D) medium did not reach a stationary or decline phase in cell numbers
during the culture period examined. Other culture media had reached a stationary
phase of growth following 8 days in incubation. These results indicate that CMRL
and MB2 medium were still maintaining growth after other media were staring to enter
a decline phase. But for uterine cells, MB2 medium seems to combine a selection of
subpopulations as observed by a two step growth. However, the initial seeding density
may have affected subsequent cell growth during the culture period. Those cells
incubated in MB2 medium exhibited low growth rates during the initial days of
incubation. Ouhibi et al. (112) suggested that optimal seeding densities for bovine
oviduct epithelial cells to be 1 x 106 cells/25 cm2 flask when maintained in MB2
medium.
All cells in culture maintained the same general appearance and characteristics
regardless of medium used for culture. However, as oviductal cells incubated in
CMRL (Trt B) reached confluency, growth of fibroblast-like cells were noted in
culture flasks. After 8 days of incubation, the swirl-like morphology of these cells did
not overgrow epithelial cells. Cells originated from the same source cultured in other
media for extended periods of time did not have the same characteristics. Ouhibi et
al. (112) have reported that serotonin added to the culture medium was selective
against fibroblast proliferation. However, in this experiment oviductal cells cultured
in CMRL supplemented with serotonin still resulted in these growth patterns.
Extensive immunofluorescence studies were conducted using cytokeratin antibodies to
determine the true origin of fibroblast-like cells. Immunofluorescence staining
revealed these fibroblast-like cells to be true epithelial cells. Merchant (99) reported
similar characteristics for prostate epithelial cells maintained in vitro. The specific
alterations in cell morphology was termed dysplastic and Merchant (99) further stated
that the morphological alterations in cell characteristics was not a transformation form
epithelial to fibroblastic cell types. Perhaps the rapid cell growth noted with CMRL
medium may induce a more accelerated dysplastic cell state.
Under normal conditions cells enter a proliferative state of the cell cycle and
continue proliferating until a signal is received that growth conditions are sub-optimal.
These sub-optimal conditions result in cells entering an arrested state of growth and
may re-enter the cycle (proliferative) if conditions become favorable (68). In the
present study, the proliferative state consisted of those cells in a mitosis phase, gap 2
phase and a period in which DNA synthesis occurs (S phase). Our original hypothesis
for examining the cell cycle was that different culture conditions may provide a
optimum rate of DNA synthesis (and subsequently protein synthesis) after transcription
and translation at selected times during the culture period that may be beneficial during
embryo co-culture. However, there were no major differences in proliferation rates
during maximal increases in growth of uterine and oviductal epithelial cells.
In summary, all culture conditions examined adequately supported growth of
bovine uterine and oviduct epithelial cells. The maximum growth rates were obtained
when cells were cultured in CMRL and MB2 medium. Higher incubation temperature
routinely used for culture of IVF-derived embryos had no adverse affects on uterine
and oviduct epithelial cell growth and proliferation. Finally, flow cytometry provided
an efficient method to monitor the cell cycle of uterine and oviduct epithelial cells
during in vitro culture. The results provided here show the versatility of these cells
used for co-culture indicating that election of the best medium for embryo co-culture
should be based on the embryos metabolism.
E X PE R IM E N T IV
MORPHOLOGICAL EVALUATION OF BOVINE UTERINE AND OVIDUCT EPITHELIAL CELLS USING
IMAGE ANALYSIS
Introduction
The in vitro culture of early cleavage-stage mammalian embryos has met with
limited success and inconsistent results. Various embryo co-culture systems have been
used to enhance embryo development during in vitro culture. Cell types used to
increase embryonic development during co-culture include uterine fibroblasts
(86,87,163), trophoblastic vesicles (71,73,119) and uterine epithelial cells (65). In
addition, oviduct epithelial cells have been used for the in vitro co-culture of early-
stage cattle (37,38,41), goat (121), pig (156) and sheep embryos (126,127,128). In
each instance, the co-culture system used has proven to be effective in supporting
development of embryos through the characteristic in vitro developmental block stages
(124). These findings suggest the existence of unidentified factor(s) produced by these
cells that have embryotropic properties, perhaps via the secretion of specific peptides
and/or proteins (124). Furthermore, researchers now suggest that metabolism of
transport epithelium (113) and maintenance of cell polarity (116) are important during
embryo development in vitro.
Although various experiments have used epithelial monolayers for co-culture
of early stage embryos, these studies have not addressed possible differences in
embryotropic factor(s) produced by cells of different morphological qualities. The
objective of this study was to establish selection criteria for morphological assessment
of cell quality using a computer image analysis system. The development of a reliable
system to monitor cell quality in vitro may allow selection of the most competent
76
monolayers for embryo co-culture experiments.
77
Materials and Methods
Experimental Animals. Reproductive tracts were collected from five cyclic
cows immediately following slaughter. Tracts were placed in phosphate-buffered
saline without Ca2+ and Mg2+ (PBS) containing 200 units penicillin, 200 ng
streptomycin and .25 ng Amphotericin-B/ml on ice and transported to the laboratory.
Isolation of Epithelial Cells. Tissue Culture Medium-199 supplemented with
10% fetal bovine serum, 100 units penicillin, 100 ng streptomycin and 10 /zg
serotonin/ml of culture medium (TCM-199) was used throughout the study.
Uterine and oviductal cells were isolated and cultured with modifications of the
procedures previously reported by this laboratory (146,148). Briefly, uterine horns
and oviducts were removed, then trimmed free of mesentery tissue. Uterine horn and
oviduct sections were flushed with 5 ml of warm .25% trypsin then filled with fresh
trypsin solution, clamped and incubated at 37°C for 45 min (Figure 4.1). The trypsin
solution was recovered and tissues flushed a second time with fresh .25 % trypsin. The
luminal surface of uterine horns and oviducts were gently scraped once with a sterile
glass slide. Epithelial clusters were separated by repeated pipetting and the trypsin
solution inactivated by the addition of TCM-199 (1:2 v/v).
Harvested cells were then layered on Percoll gradients as previously described
(112). Following centrifugation, a small portion of harvested cells was assigned to
electron microscopy and remaining cells seeded in 25 cm2 tissue culture flasks with
5 ml of TCM-199. All cultures were maintained at 37°C in an atmosphere of 95%
air and 5% C02 and culture medium replaced at 48-h intervals. Subculture of
confluent monolayers were conducted by trypsinization with .05% trypsin. Culture
Epithelial Cell Preparation
Oviduct Inflated with .25% trypsin and Incubated 45 mln.
Cellscollected
Cells allowed to monolayerOviduct flushed with .25% trypsin C>
6
25 cm 1 flask
m onolayer trypslnlzed
W ashed 3 x resuspended in TCM-199
Figure 11. An example of procedures used for harvesting oviduct epithelial cells. The same procedure was used to isolate uterine epithelial cells.
79
flasks were incubated at 37°C for 5 to 10 min until cells became rounded in shape and
detached from the bottom of the culture vessel. Cells were then centrifuged, the
supernatant removed, and the cellular pellet resuspended in fresh TCM-199 and seeded
in new culture flasks at a 1:2 ratio.
Morphological Assessments and Optical Density. At designated subpassages,
confluent 25 cm2 flasks of uterine (n = 8) and oviduct (n = 13) epithelial cells were
fixed and stained with Giemsa stain to assess cell quality (51). Morphological
assessment of cell integrity and quality and optical density readings were made on cells
stained with Giemsa during primary culture (n = 6) and following the first (n — 6)
and third (n = 9) passages at confluency. For visual morphological assessment, cells
were assigned quality scores ranging from 1 to 3 (1 = good to 3 = poor cell quality).
Morphological scores were based on the presence of vacuoles, with a score of 1
having 10% or less of the culture surface containing visible vacuoles, 2 with 10% to
50% of the culture surface containing vacuoles and 3 with greater than 50% of the
culture surface containing vacuoles. All morphological evaluations were carried out
blind without regard to tissue type or passage. The formation of vacuoles within a
culture vessel has been associated with the onset of cell degeneration, which differed
from the presence of lipid inclusions (166).
Optical density readings were determined using a silicon-intensified target
camera and a computer based image analysis system (Micromeasure Version 3.0;
Analytical Imaging Concepts). All evaluations were conducted concurrently with light
source settings of 74 (lower threshold) and 255 (upper threshold).
Election Microscopy. For scanning electron microscopy, uterine and oviductal
cells were seeded on microporous membrane inserts in a 24-well Transwell9 tissue
culture plate (Costar). At confluency, monolayers were rinsed with PBS, fixed in 3%
80
glutaraldehyde in sodium-cacodylate buffer (.1 M, pH 7.4) for 1 h, washed twice in
buffer, post-fixed in 1 % osmium tetroxide in sodium-cacodylate buffer (. 1M, pH 7.4)
and dehydrated in a dilution gradient of ethanol: water (30, 50, 70, 80, 90 and 100%
ethanol). Cells were critical-point dried, then coated with gold-palladium and
evaluated with a scanning electron microscope (Cambridge Steieoscan 150).
Statistical Analysis. A total of five optical density readings for 100 by 100 pm
regions within each flask of cells w oe determined and analyzed using analysis of
variance. Main effects in the model included cell type (uterine or oviductal), passage
and cell quality score. Significant differences between least squares means were tested
using predicted differences.
Results
Optical Density. Cell type (uterine or oviductal) did not significantly (P > .05)
influence optical density readings. Therefore, for further comparisons, mean values
represent combined uterine and oviductal cell populations. In addition, there were no
significant (P> .05) differences between sample replicates, indicating that repeatable
measurements were obtained (Figure 12).
Comparisons were made between cells during primary culture and following
the first and third subpassages to determine optical density readings following repeated
subpassage. There was a significant increase (PC .05) in optical density following
each passage (Figure 13). Mean (±SE) optical density readings for combined uterine
and oviductal cell populations during primary culture and following the first and third
passage were 139.7±4.1, 154.5±3.6and 173.3±3.2, respectively.
Optical density readings were then compared with morphological evaluations
of cell quality. In this case, optical density was significantly (PC .01) influenced by
81
cell quality. There was an increase in optical density as cell quality scores increased.
The mean (±SE) optical density for good (1), fair (2) and poor (3) quality cells were
137.0±3.6, 159.7±3.4 and 170.8±3.9, respectively (Figure 14). An example of
good, fair and poor quality cells are shown in Figure IS.
Electron Microscopy. Jn this study, there were no noted differences in
morphology of uterine and oviductal epithelial cells when micrographs were evaluated.
Scanning electron microscopy revealed numerous bundles of cilia present on the cell
surface of uterine and oviduct cells during primary culture. Oviductal cells also
contained more dense patches of cilia present on the cell surface when compared with
uterine cells (Figure 16). However, only microvilli were observed on the uterine and
oviductal cell surface following the first passage. Furthermore, results from electron
microscopy in this study did verify the visual classification of cell quality.
Opt
ical
D
ensi
ty
82
170 r
160
150
140
130
120
1 1 0
1002 3 4
S a m p le R ep l ica tes
Figure 12. Mean optical density readings for uterine and oviductal cells combined for the five sample replicates.
83
180 r
■S''tocoo"ooQ .O
_ a ,b ,c (P < .0 1 )
100 LPrim ary First
Cell P a s s a g e
c
Third
Figure 13. Mean optical density readings for uterine and oviductal cells combined during primary culture and following the first and third subpassages.
84
2*coca>ooa
CLo
180 i-
-| 7 Q _ a ,b ,c (P < .0 5 )
160 -
150
140 -
130 -
120 -
110
100 LGood Fair Poor
M orphologica l Cell Quality
Figure 14. Mean optical density readings of good, fair and poor quality cells for uterine and oviductal cells combined.
85
Figure 15. Examples of good (a) and poor (b) quality bovine oviductal cells stained with Giemsa stain following in vitro culture (20 X ).
Figure 16. Scanning electron micrograph of oviductal (a) and uterine (b) epithelial cells during primary culture (oviductal cells=20,000 X and uterine cells =20,000 X ).
87
Discussion
The morphological characteristics of uterine and oviduct epithelial cells noted
visually and with electron microscopy in this study were similar to those described in
the reports of others (74,78,112). Furthermore, basic uterine and oviductal epithelial
cell characteristics were maintained throughout the culture period.
A increase in optical density readings became evident following repeated
subpassages and was noted in both uterine and oviductal cells. Changes in epithelial
cells from a tight polygonal monolayer to a more flattened appearance may account
for the increasing density readings following subsequent subpassages. Although
bovine uterine and oviduct cells can be maintained in vitro for nine to 13 subpassages,
there are changes in cell shape noted following three subpassages (112,146). The
increasing optical density readings following subpassage may only be a reflection of
cell shape. This suggestion is further supported by reports of subpassaged oviductal
monolayers effectively supporting embryonic development compared with monolayers
of primary cultures (121). In addition, oviduct epithelial monolayers maintained high
protein secretion rates during extended in vitro culture following repeated passages
(113).
The increased optical density readings noted for poor quality cells were
associated with the high degree of vacuolation that resulted in a lower incorporation
of Giemsa stain. In contrast, good quality cells had increased quantities of stain
incorporated into the cellular material resulting in lower optical density readings. The
formation of vacuoles that coincides with cell degeneration may not be a result of
trypsinization (112) but rather a reflection of individual animal variation (146).
The results reported in this study provide a basis for continued investigations
on cell quality using image analysis. The use of a computer-based method of cell
evaluation may allow researchers to fully investigate changes in cellular structure
during in vitro culture. Although oviduct epithelial cell cultures are proven to
stimulate early embryonic development of farm animal embryos in vitro, there are
limited studies investigating sources of variation in cells used for embryo co-culture.
The investigation of factors that influence monolayers during co-culture may reveal an
optimal epithelial cell embryo co-culture system.
E X PER IM EN T V
IN VTTRO-MATURATION. IN VITRO-FERTILIZATION AND CLEAVAGE OF BOVINE OOCYTES INCUBATED
IN DIFFERENT CULTURE MEDIA
Introduction
During the last decade, there has been increased research interest in new
embryo biotechnology procedures (e.g. in vitro fertilization, nuclear transfer, gene
insertion). In recent years, research has focused on in vitro fertilization of oocytes to
supply supplemental embryos for research at low cost. This increased interest in in
vitro fertilization of bovine oocytes has prompted investigations aimed at enhancing
in vitro maturation, fertilization and subsequent development (6,7,19,24,54,90,92).
Aspects of in vitro fertilization for mammalian oocytes can be evaluated in several
excellent review articles (7,91,167).
Various in vitro fertilization techniques for bovine oocytes have been previously
reported by different laboratories for in vivo matured (24,139) and in vitro matured
oocytes (94,95,115,140,169). Although in vitro fertilization methodologies have been
improved in recent years, development beyond the bovine eight- to 16-cell in vitro
developmental block stage (41) has met with limited success. The use of oviductal
cells (41,53,95) and cumulus cell (67,172) co-culture systems have provided adequate
development of in vfrro-fertilized bovine oocytes to morulae and blastocyst stages.
Efforts are now being made to evaluate various culture media on the in vitro
fertilization efficiency of bovine oocytes. Most reports to date have focused on the
addition of hormones to the medium or using granulosa/cumulus cells for maturation,
fertilization and early cleavage of cattle oocytes. In a recent report, van de Sandt et
al. (153) evaluated eight different culture media for maturation, fertilization and
89
90
subsequent development of mouse oocytes. In this study, differences were reported
among treatment media for pre- and post-implantation development. Furthermore, it
was proposed that improved embryonic development following oocyte maturation
could be influenced by oocyte maturation medium.
Recently, researchers in this laboratory have reported that in vitro incubation
of bovine uterine and oviduct epithelial cells in different culture media and
temperatures resulted in varied responses in cell growth rates (147,149). In addition,
cells incubated in CMRL medium resulted in cell morphological alterations following
several subpassages. Monolayers of epithelial cells would begin to exhibit fibroblastic-
like characteristics. However, cytokeratin staining revealed the origin to be true
epithelial cells.
The objective of this study was to evaluate culture media previously used in
bovine uterine and oviduct epithelial cell culture experiments and to compare these
media for in vitro fertilization and development of cattle follicular oocytes.
Materials and Methods
Recovery of Oocytes. Ovaries were collected from heifers at a local abattoir
and transported to the laboratory in Dulbecco’s phosphate-buffered saline (DPBS)
(Gibco) at £25 °C. Oocytes were aspirated from 3 to 8 mm diameter follicles using
a 20 gauge needle and a 5 ml syringe. Recovered cumulus-oocyte complexes were
transferred into 15 ml conical centrifuge tubes and allowed to settle («5 min).
Supernatant fluid was then aspirated from tubes and remaining oocytes were washed
three times with DPBS with 10% fetal bovine serum (FBS).
Oocyte Maturation and Fertilization. Standard procedures for in vitro
maturation and fertilization of bovine oocytes are those developed by this laboratory
91
(172). Oocytes with intact, unexpanded cumulus oophorous (90) were randomly
distributed into four-well plates (Nunc) containing culture treatment medium. Oocytes
were incubated for 24 to 26 h at 39°C and 5% C02 in Tissue Culture Medium-199
(TCM) (Gibco), CMRL-1066 (CMRL) (Gibco) or Menezo’s B2 medium (MB2; API
Biomerioux) all supplemented with 10% FBS. Following the maturation period,
oocytes were washed with B-O medium (23) supplemented with 20 mg/ml bovine
serum albumin (BSA; Sigma) and transferred to 30 /d drops. Two units of frozen-
thawed semen was washed twice in B-O medium with 10 mM caffeine sodium
benzoate. Sperm cells were exposed to .1 /iM Ca2+ ionophore A23187 (Sigma) for
1 min to aid in capacitation then placed in microdrops of B-O medium containing
oocytes. Oocytes (20 to 25) and capacitated sperm cells were incubated for 5 to 6 h
in B-O medium with BSA containing l.S X 106 motile sperm/ml. Following the
fertilization interval, oocytes were washed in treatment medium and transferred into
four-well plates then incubated for an additional 48 h. Following the 48-h post
insemination interval, cumulus cells attached to oocytes were removed by gently
teasing with a 27 gauge needle. Oocytes were evaluated with a Nikon inverted
microscope (40-100 X ) to assess cleavage rates. Cleavage was defined as those
oocytes completing one or more cell divisions.
Fixation of Oocvtes. Oocytes were fixed and stained to determine fertilization
rates 18 h following insemination. Oocytes were mounted on glass slides and fixed
in acetic acid:ethanol (1:3) for 48 h, stained with 1% orcein in acetic acid and
examined for fertilization using a phase-contrast and Nomarski interface-contrast
microscope.
Oviductal Cell Culture. Bovine oviduct epithelial cells were isolated from
oviducts of heifers obtained at slaughter and maintained in vitro as previously reported
92
(146,148). Isolated epithelial cells were incubated in treatment media (TCM, CMRL
and MB2) and were used for co-culture following the first subpassage. Oviductal cells
were isolated, pooled together then cultured in respective treatment medium until
initiation of co-culture experiments. All media were supplemented with 10% FBS,
100 units penicillin and 100 /ig streptomycin/ml of treatment medium. Cells were
seeded in 25 cm2 tissue culture flasks (Costar) with 5 ml of TCM. All cultures were
maintained at 37°C in an atmosphere of 95% air and 5% C02 and culture medium
changed at 48-h intervals. Cells were seeded in 24-well tissue culture plates (Costar)
at a concentration of 1.0 x 10s viable cells/ml 48 h prior to co-culture. All wells had
reached ®90% confluency prior to initiation of co-culture experiments.
Embryo Co-culture. Two- to six-cell embryos (n = 180) obtained 48 h
following insemination from each treatment media (n = 60/medium) were randomly
allotted to respective treatment medium alone (n = 30/treatment) or co-cultured with
oviductal epithelial cells (n = 30/treatment). All cultures were maintained at 39°C
and 5% C02 with medium replaced at 48-h intervals. Embryos were co-cultured for
6 days (morulae stage of development) following in vitro fertilization.
Statistical Analysis. The proportion of oocytes that were fertilized and
completed at least one cell division 48 h following insemination were statistical
evaluated by the Chi-square test. In addition, treatment comparisons among groups
of embryos incubated in medium alone or with oviduct epithelial cells were compared
by the Chi-square test.
Results
The total number of fertilized oocytes consisted of those with two or more
pronuclei and/or with penetrated sperm. Those oocytes with only two pronuclei
93
present were further characterized (Table 4). There were no significant differences
(P> .05) between culture medium treatment groups for the total number of fertilized
oocytes. In addition, oocytes incubated in MB2 had significantly (P < .05) more
oocytes with only two pronuclei present compared with TCM medium. However,
there were no significant (P>.05) differences between treatment groups for the
number of oocytes completing at least one cell division 48 h following fertilization.
When oocytes were evaluated to determine cleavage rates, development stages*
of cleaved embryos were noted to evaluate if there were medium treatment differences
during early cell divisions (Table 5). There were significantly (P < .05) more two-cell
embryos in the MB2 treatment compared with TCM and CMKL treatments. Also,
there were more (P < .05) embryos £8 cells in the CMRL treatment compared with
MB2 medium.
The number of two- to six-cell embryos available for culture experiments were
75, 66 and 81 for TCM, CMRL and MB2 media, respectively. A total of 60
embryos/medium were randomly selected and further divided into culture in respective
treatment media alone (n = 30) or with oviduct epithelial cells (n = 30). Early-stage
embryos (n = 180) were cultured in vitro in respective treatment media or with
oviductal epithelial cells for 6 days following fertilization in hopes of enhancing
medium treatment effects (Table 6). However, no significant differences (P>.05)
were noted between treatment groups for embryos developing to the morula stage in
vitro. When culture medium treatment groups were combined, embryos co-cultured
on oviduct epithelial cells had more viable appearing embryos compared with similar
stage embryos maintained in media alone (Figure 17). There were 32% of morula-
stage embryos judged viable following in vitro culture in medium alone. In contrast,
70% of morula-stage embryos co-cultured on oviductal cells were judged viable.
94
Table 4. Fertilization and cleavage rates of bovine oocytes incubated in different media
TreatmentN o./group
Fertilization CleavageNo.
oocytesexamined
No. (%) oocytes_______ fertilized________
Total 2 Pronuclei
No.oocytescultured
No. (56)oocytescleaved
TCM 248 48 37(77%) 22(45 %)b 2 0 0 102(51%)CMRL 248 48 31(65%) 28(58%)b,c 2 0 0 104(52%)MB2 248 44* 33(75%) 30(68%)c 2 0 0 92(46%)
* Four oocytes were lost during fixation procedures.b,c Means within columns with different superscripts differ (P< .05).
95
Table 5. Developmental rates of cleaved oocytes 48 h after fertilization
Cleaved 2 cell 3 cell 4 cell 6 cell £8 cellTrt n n(% ) n (%) n (%) n(% ) n (%)
TCM 102 19(19%)* 8(8% ) 33(32%) 15(15%) 27(27%)*,bCMRL 104 18(17%)* 12(12%) 20(19%) 16(15%) 38(37%)bMB2 92 33(36%)b 15(16%) 24(26%) 9(10%) 11(12%)*
*,b Means within columns with different superscripts differ (PC .05).
96
Table 6. Developmental rates of cleaved embryos following in vitro culture for 6 days
Maturationmedium
No.cleaved Treatment
No.cultured
Morula n (%)
Viable embryos n (%)
TCM 102 TCM 30 12(40%) 3(10%)TCM+BOC* 30 13(43%) 10(33%)
CMRL 104 CMRL 30 14(47%) 5(17%)CMRL+BOC 30 17(57%) 11(37%)
MB2 92 MB2 30 12(40%) 4(13%)MB2+BOC 30 13(43%) 9(30%)
•BOC = bovine oviduct cells
Perc
ent
Viab
le
Em
bryo
s
97
100
90
SO
70
60
50
40
30
20
10
0
a ,b (P < .0 1 )
ControlMedium
b
OviductalCells
Figure 17. Percent embryo viability following in vitro culture in medium alone or co-culture with oviduct epithelial cells.
98
Discussion
Oocytes incubated in MB2 medium had more with two pronuclei present on
evaluation compared with TCM medium, however, this did hot affect the number of
oocytes completing one or more cell divisions. Oocytes maintained in CMRL medium
had greater level of morphological development at 48 h following fertilization
compared with IVF-derived embryos in MB2 medium. However, when embryos were
maintained in vitro for 6 days, development rates to the morula stage for embryos
incubated in CMRL or MB2 medium were similar. Similar in vitro fertilization and
cleavage rates of bovine oocytes have been recently reported (56,134).
Several studies have used various culture media to maintain oocytes or early-
stage bovine embryos in vitro for extended periods of time with adequate success rates.
Adequate in vitro development of early-stage bovine embryos have been reported
following incubation with MB2 (117) or CMRL (38) medium. In a early study, Fukui
et al. (54) evaluated different combinations of culture media, gonadotropins and
steroids supplemented in medium in an attempt to improve success rates for in vitro
maturation of bovine oocytes. The authors noted that the addition of steroids
(estradiol) was more critical for oocyte maturation than culture medium or
gonadotropins. However, the effects of steroids on oocyte maturation differed
depending on the culture medium selected.
More recently, Ellington et al. (38) compared in vitro development of one- to
two-cell bovine embryos incubated in either a simple serum-free (CZB) or complex
(CMRL or Ham’s F-10) medium with bovine oviduct epithelial cells. Similar
development rates (morula or blastocyst stages) were noted between three different
media following co-culture for 5 days. However, embryos incubated in Ham’s F-10
medium had lower embryo viability scores compared with those of CMRL and CZB
99
medium. In addition, CZB supplemented with insulin, transferrin and selenium (ITS)
was compared with CMRL containing serum and ITS. A higher percentage of morula
or blastocyst stage embryos were noted following incubation in CZB for S days in
vitro. Ellington et al. (38) concluded that a simple serum-free medium would support
in vitro development of early-stage bovine embryos following co-culture with oviduct
epithelial cells.
Differences in blastocyst development in mouse (153) and cow (56) embryos
incubated in vitro under different conditions suggest there are still mechanisms in early
embryonic development not fully understood. Although oviduct epithelial cell co
culture improved viability of morula-stage embryos in this study, the mode of action
of co-culture is still debatable. Researchers suggest somatic cell/embryo contact
necessitates beneficial effects (3) while others noted adequate success when using
conditioned medium (41,71). Although some researchers suggest somatic cells secrete
embryotropic factors that stimulate embryo development in vitro (57) others indicate
that a combination of embryotropic factor production and/or removal of toxic
substances occur (see review by 8).
The failure to detect differences between medium treatment groups may be
related to the length of in vitro incubation period. There were no developmental
differences noted between treatment groups even when embryos were co-cultured on
oviduct epithelial cells. However, this may be directly related to the reduction in
number of embryos per group by expanding treatment groups. Although there were
no differences in the number of embryos reaching the morula stage of development,
co-culture with oviductal cells improved the percent viability of embryos.
The present study demonstrated the ability of cell culture media evaluated to
stimulate development of in vzrro-matured, in vzVro-fertilized bovine oocytes to the
morula stage of development. In addition, co-culture with oviduct epithelial cells was
superior to culture in medium alone. Further studies are needed to substantiate
interactions between early embryonic development and in vitro culture conditions.
E X PER IM EN T V I
CO-CULTURING IVF-DERIVED BOVINE EMBRYOS WITH OVIDUCT EPITHELIAL CELLS ORIGINATING FROM DIFFERENT
STAGES OF THE ESTROUS CYCLE
Introduction
Bovine embryos of early cleavage stages do not readily develop beyond the
eight- to 16-cell developmental block under in vitro conditions (167). However,
researchers have recently been able to overcome the in vitro developmental block (42)
with the use of somatic cell co-culture with trophoblastic vesicles (27,71,73,119) and
oviductal tissue (38,41,57,128). Oviductal cells have been reported to enhance
embryo development in sheq> (57,125,126,127,128), goat (121,122,135), pig (156)
and cattle (38,39,97) embryos. In addition, medium harvested from oviductal cells
following 48-h of incubation will also support development of bovine embryos beyond
the eight- to 16-cell stage to the blastocyst and hatched blastocyst stages (41). The use
of oviductal tissue in embryo co-culture systems has received increased attention in an
effort to understand the mechanisms by which "helper" cells interact with embryos to
enhance in vitro development. Such studies have evaluated the use of fresh or
subpassaged monolayers (121,122). The use of subpassaged monolayers may provide
a more uniform and consistent co-culture system for mammalian embryos (113,121).
Other researchers have investigated using fresh and frozen-thawed monolayers for
embryo co-culture (38,127). In addition, researchers have demonstrated that oviductal
cell co-culture enhanced embryonic development when incubated in serum-free
medium. These studies (38,126,128) have provided the basis for noting that
embryotropic effects are directed by oviductal cells themselves and are not in part due
to serum effects.
101
102
Evaluation of specific factors that influence oviductal cells prior to and during
embryo co-culture may provide experimental evidence on the mechanism by which
embryotropic factors regulate embryonic development. These criteria become
increasingly important since the embryotropic properties of oviductal cells are
mediated perhaps via the secretion of specific proteins and/or peptides (see review by
124). The suggestion that the embryotropic effect of oviductal and uterine cells may
be hormonal dependent (128) has been further strengthened by a recent report from
this laboratory (146).
The latter report noted significant decreases in percent viability and attachment
of both uterine and oviduct epithelial cells in vitro following their daily exposure to
raised circulating progesterone in vivo. In this case, it suggested that the stage of
estrous cycle influenced the secretory capacity of both uterine and oviduct epithelial
cells while maintained in vitro. The possible secretion of specific proteins by
oviductal cells has been noted by reports of a estrus-specific protein present in the
ovine oviduct in vivo (144). In addition, Gandolfi et al. (58,59) detected a similar
molecular weight protein from in vitro cultured oviductal cells. The latter authors
further characterized in vitro secreted proteins into two groups: one secreted
throughout the estrous cycle and the second exhibiting cyclic patterns of secretion.
Based on the existing reports in the scientific literature, the objectives of the
present study was two fold. The first was to evaluate the ability of oviduct epithelial
cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle to support early in
vitro development of IVF-derived bovine embryos. The second objective was to
monitor proteins secreted by oviductal cells used for co-culture of embryos as an
indicator of cell secretory capacity. The evaluation of proteins secreted by oviductal
cell monolayers may provide an indication of the potential embryotropic effect during
103
embryo co-culture.
M aterials and Methods
Experimental Embrvos. Embryos were produced from in vzm?-matured and
in vz/ro-fertilized bovine oocytes with slight modifications of the procedure in use in
this laboratory (172). Briefly, cumulus intact oocytes were matured for 24 h in 500
Ml of Tissue Culture Medium-199 (TCM-199) supplemented with 10% fetal bovine
serum (FBS) (Hyclone Laboratories, Logan, UT) under paraffin oil at 39°C and 5%
C 02. Following maturation, oocytes were transferred to 50 /i 1 drops of B-O medium
(23) supplemented with 20 mg/ml BSA (Sigma). Two units of frozen-thawed semen
from a single dairy bull were washed twice in B-O medium with 10 mM caffeine
sodium benzoate (Sigma) and exposed to .1 pM Ca2+ ionophore A23187 (Sigma) for
1 min. A 50 jil portion of sperm cells containing 1.5 X 106 motile sperm cells/ml
were added to oocytes and incubated for an additional 5 h at 39°C and 5% C02.
Following the insemination interval, oocytes were washed twice and incubated in
TCM-199 with 10% FBS for an additional 42 h.
Isolation and Culture of Oviductal Cells. Bovine oviduct epithelial cells were
isolated with procedures similar to that previously reported at this laboratory (146).
Oviducts were obtained from animals immediately following slaughter and transported
to the laboratory on ice in Ca2+ and Mg2+ free phosphate-buffered saline (Gibco) with
200 units penicillin, 200 /Jg streptomycin and .25 fig Amphotericin-B/ml (PBS-AB).
Oviducts were trimmed free of mesentery tissue and washed three times with fresh
PBS-AB. Epithelial cells from both oviducts were then stripped with a pair of fine
forceps from the isthmic end distally to the infundibulum. Small clusters of ciliated
epithelial cells were recovered and resuspended in TCM-199 with 10% FBS, 100 units
104
penicillin and 100 Mg streptomycin/ml of medium (TCM). Oviductal cells were
washed three times with fresh medium by centrifugation (200 x g) and the cellular
pellet resuspended in 48 ml of TCM. The cell suspension was then incubated in 24-
well plates (1 ml suspension/well) at 39°C and 5% C02. Culture medium was
replaced at 48-h intervals and oviductal cells were used for co-culture experiments
within 48 h of initial seeding.
Experiment 1.
The objective of this experiment was to evaluate the efficiency of bovine
oviduct epithelial cells isolated from different stages of the estrous cycle to support in
vitro development of IVF-derived bovine embryos. Oviduct epithelial cells were
isolated and cultured from cyclic Holstein heifers between d 4 to 6 (n = 3) and d 14
to 16 (n = 3) of the estrous cycle (estrus = d 0). Embryos (two- to four-cell) were
randomly allotted («20 embryos/treatment/replicate) in suspension co-culture with cells
originating from either cycle d 4 to 6 (Trt A) or cycle d 14 to 16 (Trt B) 48 h
following cell isolation. Immediately prior to embryo co-culture, fresh medium
(TCM) was replaced on oviductal cell suspensions. The free-floating clusters of
epithelial cells, in this case, were allowed to remain in the culture wells (Figure 18).
A portion of oviductal cells often attached and would form monolayers («20%) during
the 48 h pre-incubation period. The control culture (Trt C) consisted of similar stage
embryos cultured in TCM. All cultures were maintained at 39°C and 5% C02 in 1
ml of TCM and a portion of culture medium (750 (A) replaced at 48-h intervals.
Embryos were evaluated for development to the morula, blastocyst and hatched
blastocyst stage on d 6, 8 and 10 following in vitro insemination, respectively.
105
Experiment 2.
The objective of this experiment was to monitor proteins secreted by oviduct
epithelial cells isolated from different stages of the bovine estrous cycle as an indicator
of cell secretory capacity. The oviduct epithelial cells were obtained from oviduct
tissue harvested between d 4 to 6 or d 14 to 16 used in Experiment 1. The same
culture conditions were used as in Experiment 1 with the following changes. Oviduct
epithelial cells were allowed to form confluent monolayers and all free-floating cells
and cell clusters were removed prior to the radiolabelling procedure. Oviductal cells
harvested from each female were seeded in eight wells of a 24-well tissue culture
plate.
Confluent monolayers were radiolabelled according to previously reported
procedures (113). Monolayers were washed twice and subsequently incubated for 2
h in methionine-free Dulbecco’s modified Eagle medium (Gibco) (DMEM-MET)
followed by a 90 min incubation period with DMEM-MET containing SO pCi of
[35S]methionine/ml of DMEM-MET. Radiolabelled [35S]methionine (Amersham Ltd)
had a specific activity of >1,000 Ci/mmol. Radiolabelling of monolayers were
conducted in 24-well plates (750 pi volume). Following a 90-min radiolabelling
period, monolayers were washed three times in DMEM and further incubated in TCM-
199 containing 10% FBS for an additional 48 h.
At 24- and 48-h intervals, aliquots of culture medium (500 pi) were harvested,
precipitated in .5 N perchloric acid. Free-labelled methionine was separated from the
bound methionine by centrifugation and the protein pellet counted by liquid
scintillation to determine 35S-methionine incorporation into secreted proteins. In
addition, cells were removed from the culture well, precipitated in .5 N perchloric
acid, centrifuged and the cellular pellet counted for 3SS-methionine incorporation into
106
the cellular proteins by liquid scintillation counting. Control samples consisted of
TCM-199 with 10% FBS alone and TCM-199 with 10% FBS and cells without
radiolabelling. In addition, control wells were treated with radiolabelled methionine
and washed to determine nonspecific activity. The amount of radiolabelled protein
secreted and the cellular protein content of oviductal cells were determined from each
of four wells at 24 and 48 h for each experimental animal across treatment days of the
estrous cycle. The number of samples for secreted proteins was 24 duplicates by cells
isolated from d 4 to 6 and d 14 to 16 of the estrous cycle.
Statistical Analysis. In Experiment 1, the proportion of embryos developing
to morula, blastocysts and hatched blastocyst were compared by the Chi-square
analysis. In Experiment 2, cpm of activity in secreted and cellular proteins were
compared across days of the estrous cycle using a split-plot design with repeated
measures in time (60) and a mixed model with animal as a random effect in the
model. Statistical comparisons of least square means were conducted with predicted
differences.
Results
Experiment 1.
There was a higher percentage (PC .05) of two- to four-cell embryos that
developed to morula stage when co-cultured on oviductal cells compared with that of
culture in medium alone (52% vs 32%, respectively). The percent of embryos
developing to the morula stage were similar for cells isolated between d 4 to 6 or
between d 14 to 16 (Table 7). Furthermore, developmental rates to the blastocyst
stage were higher with oviductal cell co-culture compared with culture in medium
alone (37% vs 9%, respectively) (PC .01). Similar results between d 4 to 6 and d 14
107
to 16 treatments were noted at the hatched blastocyst stage (14% and 15%,
respectively) and were higher than culture in medium (P < .01). In this experiment,
14 to 15% of the embryos hatched from their zona pellucida during co-culture while
no embryos hatched when incubated in medium alone.
Experiment 2.
All control samples exhibited low cpm indicating no nonspecific activity.
Aliquots of radiolabelled medium were harvested following the radiolabelling period
and counted to determine incorporation by monolayers. The percentage of
[35S]methionine incorporation based on total cpm averaged 16% and was similar for
both monolayer treatment groups. There was no differences noted among individual
wells within treatments, subsequently all data were combined for final analysis. The
stage of the estrous cycle from which cells were isolated did not influence the amount
of proteins secreted following 24 or 48 h of incubation in vitro (Figure 19). The mean
cpm (±SE) of secreted proteins following 24 h of incubation from cells isolated
between d 4 to 6 and d 14 to 16 were 128.9±10.3 and 111.2±10.3 x lOVlO6 cells,
respectively. In addition, similar secreted proteins were noted following 48 h of
incubation for d 4 to 6 and d 14 to 16 (116.6±10.3 and 130.8±10.3 x ltfVlO6 cells,
respectively). Epithelial cells isolated from both stages of the estrous cycle maintained
an extraded level of protein secretion following 48 h of in vitro culture.
For radiolabelled methionine incorporation into cellular proteins, stage of the
estrous cycle cells were harvested did not influence the amount of cellular protein
following 24 h of in vitro culture (P>.G5) (Figure 20). There was a significantly
lower (P<.05) amount of cell protein observed for cells isolated from d 14 to 16
compared with d 4 to 6 cells following 24 h of culture. Cells isolated between d 14
to 16 had significantly higher (P< .OS) amounts of cellular protein following 48 h of
culture compared with 24 h of incubation. The lower amount of cellular protein from
cells isolated between d 14 to 16 following 24 h of incubation may be attributed to a
higher turnover rate of protein synthesis and not necessarily a decrease in cell quality.
Figure 18. An example of free-floating clusters of oviduct epithelial cells used during embryo co-culture.
110
Table 7. Development of 2- to 4-cell bovine embryos on oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16
of the bovine estrous cycle (3 replicates) f
Treatment nMorula
n (%)BLST*n(% )
HBLST* n (%)
A (d 4-6) 65 35 (54%)* 25 (39%)c 9 (14%)cB (d 14-16) 65 32 (49%)' 23 (35%)c 10 (15%)cC (control) 65 21 (32%)b 6 (9% )d 0 ( 0%)d
-{-Evaluations on embryo development were conducted on d 6 (Morula), d 8 (BLST) and d 10 (HBLST) following insemination.*BLST= blastocyst; HBLST= hatched blastocysta,b Means within columns with different superscripts differ (Pc.05).c,d Means within columns with different superscripts differ (P< .01).
Ill
m"55o
ID
KJ
Eo.u
1 501 4 01 30120110100
9 08 07 0605 04 03 02010
0d 4 - 6 d 1 4 - 1 6
2 4 Hoursd 4 - 6 d 1 4 - 1 6
4 8 Hours
Figure 19. Proteins secreted by oviduct epithelial cells isolated between d 4 to 6 or d 14 to 16 of the estrous cycle. Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean counts from three replicates.
112
m"5o
co
O
X
EDlO
120
110100
9 0
8 0
7 0
6 0
5 0
4 0
3 0
20
10
0
_ a ,b (P < .0 5 )
d 4 - 6 d 1 4 - 1 6 2 4 Hours
d 4 - 6 d 1 4 - 1 64 8 Hours
Figure 20. Cellular proteins in oviduct epithelial cells isolated between d 4 to 6 and d 14 to 16 of the estrous cycle. Evaluations were conducted 24 and 48 h following in vitro culture. Data are mean counts from three replicates.
113
Discussion
In this study, protein secretions were evaluated to assess secretory patterns of
oviductal cells isolated between d 4 to 6 and d 14 to 16 of the estrous cycle. Proteins
secreted by oviductal cells in vitro in the present study were similar between both
stages of the estrous cycle. Gandolfi et al. (58,59) monitored in vitro protein
secretions by sheep oviductal cells and noted a small group of proteins secreted
throughout the estrous cycle and a larger group exhibiting cyclic variations. It was
further noted that in vitro secreted proteins, at a lower level, were similar to in vivo
secretion patterns.
The level of proteins secreted in the present study may be at a lower level,
which would have prevented detection of differences between stages of the estrous
cycle. However, the level of proteins secreted by oviductal cells in the present study
were similar to those reported for subpassaged oviductal monolayers (113). Oviductal
cells in the present study were maintained in vitro for approximately 7 days to reach
confluency prior to radiolabelling. Gandolfi et al. (58,59) also noted a reduction in
secretion of specific proteins following 3 days of in vitro incubation. In an earlier
study, Salamonsen et al. (136) noted a higher incorporation of radiolabelled
methionine into secreted and cellular proteins in ovariectomized ewes treated with
exogenous estrogen. The higher methionine incorporation into proteins secreted and
in cellular proteins were more pronounced than control ewes and those treated with
progesterone. It was suggested that exogenous treatment with estrogen influenced
incorporation of radiolabelled methionine into proteins and progesterone increased the
secretion of newly synthesized proteins.
The results reported in this study indicate that stage of the estrous cycle that the
oviductal cells are harvested had no overt effect on embryo development in vitro. In
114
addition, co-cultures with oviduct epithelial cells harvested from either early- and late-
luteal stages were superior for culture of bovine embryos over that for medium alone.
These results are in agreement with those recently reported by Eyestone et al. (44)
using oviductal-cell conditioned medium for culturing IVF-derived bovine embryos.
In the latter report, no difference in development of embryos was noted using medium
harvested from oviduct cells of cows either at estrus or in the luteal phase of the
estrous cycle. Furthermore, similar embryo development results have been reported
following in vivo culture within oviducts of sheep in estrus or during the luteal phase
or in an estrous and ovariectomized animals (45,103,164).
Previously, Thibodeaux et al. (146) reported significant differences in viability
and percent cell attachment of both uterine and oviduct epithelial cells harvested from
cattle on different days of the estrous cycle. It was noted that following the first cell
passage, percent cell attachment was similar for cells collected between different stages
of the cycle. In the present study, it appears that once cells begin to attach and grow
along with free-floating cells, both stages of the cycle investigated are capable of
enhancing embryo development in vitro. Based on a previous study (45) and the
results reported herein, indicate oviduct epithelial cells harvested from different stages
of the estrous cycle will enhance early-stage embryo development when incubated in
vitro. Secondly, clusters of free-floating unattached oviductal cells are capable of
stimulating embryo development.
Recently, researchers have demonstrated that oviductal cell co-culture combined
with a simple serum-free culture medium enhanced in vitro development of early-stage
sheep (128) and cattle (37,38) embryos. These studies noted that a serum-free
medium combined with oviductal cell co-culture would stimulate embryonic
development and resulted in transplant pregnancies. These reports further indicate that
115
oviduct cells are secreting embryotropic factors not dependant on the presence of
bovine serum. It has been suggested the embryotropic effects of oviductal cell co
culture may reflect their ability to maintain a required minimal amount of protein
secretion (58). Based on the findings thus far it appears that once cells are placed in
in vitro conditions a minimal amount of protein secretion is maintained resulting in
adequate embryonic development and co-culture of embryos with serum-free medium
does not alter the minimal amounts of proteins secreted.
Although there were no differences noted in developmental rates between
oviductal cells isolated from early- and late-luteal stages, several suggestions can be
made. Due to the efficiency of growth, increased ciliary activity, viability and
attachment, cells isolated from early luteal stages of the estrous cycle would be more
suitable candidates for embryo co-culture experiments. In this study, confluent
monolayers may not be necessary to obtain good embryo development in vitro. Free-
floating clusters of oviduct epithelial cells in primary culture may provide a more in
vivo type culture system for embryo development in vitro. This suggestion was
previously demonstrated with sheep embryos co-cultured with free-floating oviduct
cells (26). Finally, the simultaneous evaluation of embryo development and protein
secretions by monolayers provides an adequate evaluation of embryo co-culture
systems. These criteria may be used in future studies to explain possible variation in
monolayers used for embryo co-culture studies.
CO N CLU SIO N S
For almost a decade there has been a growing controversy on which was the
best type of helper cells to use for optimal embryo co-culture. Recently, positive
success has been reported using epithelial cells of sheep and cattle for embryo co-
culture over that of traditional uterine cell (e.g. fibroblast) co-culture systems.
Furthermore, the possibility exists that uterine and oviduct epithelial cells harvested
from different stages of the bovine estrous cycle may vary in developmental patterns,
cell attachment and secretory ability when used in an embryo co-culture system.
In order to maintain and evaluate epithelial cell activity in an embryo co
culture system, methodology was developed to maintain uterine and oviduct cells in
an epithelial cell state during in vitro incubation. In the first experiment, procedures
were established to isolate and maintain bovine uterine epithelial cells based on
modifications of methodology previously reported for bovine oviductal cells by French
scientists (112). A new method to maintain and culture bovine uterine epithelial cells
was then developed and evaluated in this laboratory. Immunofluorescence staining for
cytokeratin filaments verified that these procedures did maintain both uterine and
oviduct cells in an epithelial cell state during culture experiments.
When uterine and oviduct epithelial cells were harvested from heifers on day
of estrus, between d 4 to 6, d 8 to 10 or d 14 to 16 of the estrous cycle, there was a
difference in patterns of percent cell viability and cell attachment. The highest percent
cell viability and cell attachment resulted when uterine and oviductal cells were
harvested prior to d 6 of the cycle and the lowest percent viability and attachment
resulted when cells were isolated following d 8 of the estrous cycle. The possibility
existed that the secretory capacity of cells during in vitro culture may differ from cells
116
117
isolated on different stages of the estrous cycle. It was proposed that changes in
secretory capacity of these cells may lead to alterations in embryotropic capabilities.
Therefore, IVF-derived bovine co-cultured on uterine and/or oviductal cells harvested
from different stages of the estrous cycle may result in different developmental rates.
In vitro incubation conditions (culture medium) for maintaining uterine and
oviduct epithelial cells in the most favorable in vitro environment was evaluated. In
an experiment, four of the more established culture media were compared with a
standard tissue culture medium (TCM-199) for effects of in vitro cell growth and
proliferation. In addition, in vitro incubation temperatures routinely used for cell
culture experiments (37°C) was compared with incubation temperatures used in IVF
culture experiments (39°C). In summary, CMRL-1066 medium resulted in more
accelerated growth rates for uterine and oviduct epithelial cells but did not influence
the cell cycle when compared with the base culture medium. Also, incubation
temperatures evaluated influenced neither cell growth or the cell cycle.
A method of evaluating uterine and oviduct epithelial cell populations using
image analysis may provide a way to evaluate cell populations used for embryo co
culture. Using photometric measurements, changes in cell quality following repeated
subpassages were consistently identified. Thus, photometric image analysis may be
used in future studies to characterize changes in cell populations before cell monolayer
are used for embryo co-culture.
In uterine and oviduct epithelial cell culture experiments, CMRL-1066provided
a more suitable in vitro environment for uterine and oviductal cell growth. There
fore, CMRL-1066 was compared with Menezo’s B2 medium and the base culture
medium TCM-199 in a bovine IVF culture system. In this study there were no
differences in the media alone used to maintain bovine embryos to the morula stage
118
of development. However, using the three media with oviduct epithelial cells, a
marked improvement in embryo viability resulted following 6 days of in vitro culture,
verifying that epithelial cells are needed in addition to the medium for maximum
development of early-stage bovine embryos.
In an effort to establish if bovine epithelial cells would serve as an efficient
embryo co-culture system, IVF-derived bovine embryos were co-cultured with
oviductal cells harvested between d 4 to 6 or d 14 to 16 of the estrous cycle. In
addition to embryo co-culture, radiolabelled proteins secreted by oviductal cells were
monitored as an indicator of cell secretory capacity. Co-culturing IVF-derived bovine
embryos with oviductal cells isolated between d 4 to 6 or d 14 to 16 resulted in similar
developmental rates to the morula, blastocyst and hatched blastocyst stages.
Furthermore, proteins secreted by oviductal cells were not different between d 4 to 6
or d 14 to 16. Possibly, variability in cows within days of the estrous cycle may
account for there being no difference in embryo developmental rates among different
days of the bovine estrous cycle. The least amounts of animal variability, however,
was noted in cows with epithelial cells isolated between d 4 to 6 of the estrous cycle.
The experiments reported herein suggest that subpassaged monolayer cells
provided a more uniform population of cells than primary cultures for embryo co
culture studies. In addition, the use of subpassaged cells reduced the cell variation
observed during primary cultures as a result of host animal variation. However, co
culture with primary cultures of free-floating clusters of cells provided a more in vivo
type co-culture system for early-stage bovine embryos.
Further studies are needed to fully define the mechanisms by which uterine and
oviduct epithelial cells interact with early-stage embryos to allow development beyond
the eight- to 16-cell development block to the blastocyst stage.
R E FE R E N C E S
1. Alkhalaf, M., G. Chaminadas, A.Y. Propper, and G.L. Adessi. 1987. Compared effects of oestrogens and oestrone sulfate on the epithelial cell surface of guinea-pig endometrium (in vivo and in vitro studies). Exp. Clin. Endocrinol. 89:201-210.
2. Alkhalaf, M., G. Chaminadas, A.Y. Propper, and G.L. Adessi. 1987. Hormones and anti-hormones effects on cultured guinea-pig uterine epithelial surfaces. Pages 101-109 in The cell surface in signal transduction. E.Wagner,H. Greppin and B. Millet, ed. Nato Asi Series, Cell Biology, Springer, Berlin Heidelberg. New York, NY
3. Allen, R.L., and R.W. Wright. 1984. In vitro development of porcine embryos in coculture with endometrial cell monolayers or culture supernatants. J. Anim. Sci. 59:1657-1661.
4. Aoyagi, Y., Y. Fukui, Y. Iwazumi, M. Urakawa, and H. Ono. 1990. Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts. Theriogenology 34:749-759.
5. Babalola, G.O., C. Coutifaris, U. Chin, W.J. Nelson, C.A. Buck, and J.F. Strauss. 1990. Differential expression of cell adhesion proteins in human trophoblastic cells. Biol. Reprod. 42(Suppl. l):80.(Abstr.)
6. Ball, G.D., M.L. Leibffied, R.W. Lenz, R.L. Ax, B.D. Bavister, and N.L. First. 1983. Factors affecting successful in vitro fertilization of bovine follicular oocytes. Biol. Reprod. 28:717-725.
7. Ball, G.D., M.L. Leibfried, R.L. Ax, and N.L. First. 1984 Maturation and fertilization of bovine oocytes in vitro. J. Dairy Sci. 67:2775-2785.
8. Bavister, B.D. 1988. Role of oviductal secretions in embryonic growth in vivo or in vitro. Theriogenology 29:143-154.
9. Berg, U., and G. Brem. 1990. Developmental rates of in vitro produced IVM- IVF bovine oocytes in different cell culture systems. Theriogenology 33:195.(Abstr.)
10. Biggers, J.D., R.B.L. Gwatkin, and R.L. Brinster. 1962. Development of mouse embryos in organ cultures of fallopian tubes on a cehmically defined medium. Nature 194:747-749.
11. Blakewood, E.G., and R.A. Godke. 1989. A method using the chick embryo amnion for mammalian embryo culture. J. Tissue Cult. Meth. 12:73-76.
119
120
12. Blake wood, E.G., J.M. Jaynes, and R.A. Godke. 1988. Culture of pronuclear mammalian embryos using domestic chicken eggs. Theriogenology 29:226. (Abstr.)
13. Blakewood, E.G., J.M. Jaynes, W.A. Johnson, and R.A. Godke. 1989. Using the amniotic cavity of the developing chick embryo for the in vitro culture of early-stage mammalian embryos. Poultry Sci. 68:1695-1702.
14. Blakewood, E.G., K.E. Wiemer, and R.A. Godke. 1989. Post-thaw viability of bovine embryos cultured in domestic chicken eggs or on epithelial monolayers prior to freezing in liquid nitrogen (LN2). Theriogenology 31:177.(Abstr.)
15. Blakewood, E.G., S.H. Pool, K.E. Wiemer, and R.A. Godke. 1989. Culturing early stage bovine morulae using domestic chicken eggs. Theriogenology 31:176. (Abstr.)
16. Blakewood, E.G., S.H. Pool, J.F. Prichard, and R.A. Godke. 1990. Culturing two- to eight-cell caprine embryos using domestic chicken eggs. Mol. Reprod. Develop. 27:288-294.
17. Boccart, C., P. Mermillod, C. Delecoeuillerie, and F. Dessy. 1991. Bovine oviduct cell monolayers for supporting the blastocyst formation of bovine embryos. Theriogenology 35:187. (Abstr.)
18. Boland, M.P. 1984. Use of the oviduct as a screening tool for the viability of mammalian embryos. Theriogenology 21:126-137.
19. Bondioli, K.R., and R.W. Wright. 1983 In vitro fertilization of bovine oocytes by spermatozoa capacitated in vitro. J. Anim. Sci. 57:1001-1005.
20. Bongso, T.A., S.C. Ng, S.S. Ratnam, and A.H. Sathananthan. 1990. Human oviductal/endometrial cell lines: Co-culture and assisted reproduction. Assist. Reprod. Technol. Androl. 1:39-53.
21. Boomsma, R.A., and H.G. Varhage. 1982. The uterine progestional response in cats: ultrastructural changes during chronic administration of progesterone to estradiol-primed and non primed animals. Amer. J. Anat. 164:243-254.
22. Boomsma, R.A., R.C. Jaffe, and H.G. Varhage. 1982. The uterine progestional response in cats: changes in morphology and progestional receptors during chronic administration of progesterone to estradiol-primed and non primed animals. Biol. Reprod. 26:511-521.
23. Brackett, B.G., and G. Oliphant. 1975. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12:260-274.
121
24. Brackett, B.G., D. Bousquet, M.L. Boice, M.J. Donawick, J.F. Evans, and M.A. Dressel. 1982 Normal development following in vitro fertilization in the cow. Biol. Reprod. 27:147-158.
25. Breener, R.M. 1969. Renewal of oviduct cilia during menstrual cycle of the rhesus monkey. Fertil. Steril. 20:599-611.
26. Bunch, T.D., W.C. Foote, J.W. Call, R.W. Wright, J.P. Selgrath, and W.D. Foote. 1987. Long term culture of two to eight-cell ovine embryos in various co-culture systems. Encyclia 64:66-72.
27. Camous, S., Y. Heyman, W. Meziou, and Y. Menezo. 1984. Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J. Reprod. Fertil. 72:479-485.
28. Chaminadas, G., A.Y. Propper, M. Royez, O. Prost, J.P. Remy-Martin, and G.L. Adessi. 1986. Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-173 on the epithelial cells. J. Reprod. Fertil. 77:547-558.
29. Chaminadas, G., M. Alkhalaf, J.P. Remy-Martin, A.Y Propper, and G.L. Adessi. 1989. Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium. J. Endocrinol. 123:233-241.
30. Chaminadas, G., J.P. Remy-Martin, M. Alkhalaf, A.Y. Propper, and G.L. Adessi. 1989. Effect of progesterone on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium. Cell Tissue Res. 257:129-136.
31. Chen, L., H.R. Linder, and M. Lancet. 1973. Mitogenic action of oestradiol- 173 on human myometrial and endometrial cells in long-term tissue cultures. J. Endocrinol. 59:87-97.
32. Cole, R.J., and J. Paul. 1965. Properties of cultured preimplantation mouse and rabbit embryos and cell strains developed from them. Pages 82-155 in Preimplantation stages of pregnancy. G.E.W. Wolstenhoumeand M. O'Conner, ed. Little, Brown and Co., Boston, MA
33. Conti, C.J., I.B. Gimenez-Conti, E.A. Conner, J.M. Lehman, and L.E. Gerschenson. 1984. Estrogen and progesterone regulation of proliferation, migration and loss in different target cells of rabbit uterine epithelium. Endocrinol. 114:345-351.
34. Critser, E.S., and N.L. First. 1986. Use of a fluorescent stain for visualization of nuclear material in living oocytes and early embryos. Stain Technol. 61:1-5.
35. Dallenbach-Hellweg, G. 1975. Histopathology of the endometrium. 2nd ed. Springer-Verlag, New York, NY
122
36. Dorman, B.H., V.A. Varma, J.M. Siegfried, S.A. Melin, T.A. Adamec, C.R. Norton, and D.G. Kaufman. 1982. Morphology and growth potential of stromal cell cultures derived from human endometrium. In Vitro 18:919-928.
37. Ellington, J.E., P.B. Farrell, M.E. Simkin, R.H. FoOte, E.E. Goldman, and A.B. McGrath. 1989. Development and survival after transfer of cow embryos cultured from 1-2 cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells. J. Reprod. Fertil. 89:293- 299.
38. Ellington, J.E., E.W. Carney, P.B. Farrell, M.E. Simkin, and R.H. Foote.1990. Bovine 1-2-cell development using a simple medium in three oviduct epithelial cell co-culture systems. Biol. Reprod. 43:97-104.
39. Ellington, J.E., P.B. Farrell, and R.H. Foote. 1990. Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow. Theriogenology 34:837-844.
40. Eyestone, W.H., and N.L. First. 1988. Co-culture of bovine embryos with oviductal tissue. Proc. 11th Intern. Cong. Anim. Reprod. Artific. Insem. 4:471.
41. Eyestone, W.H., and N.L. First. 1989. Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. 85:715-720.
42. Eyestone, W.H., and N.L. First. 1991. Characterization of developmental arrest in early bovine embryos cultured in vitro. Theriogenology 35:613-624.
43. Eyestone, W.H. , J. Vignier, and N.L. First. 1987. Co-culture of early bovine embryos wth oviductal epithelium. Theriogenology 27:228.(Abstr.)
44. Eyestone, W.H., J.M. Jones, and N.L. First. 1991. Some factors affecting the efficacy of oviduct tissue-conditioned medium for the culture of early bovine embryos. J. Reprod. Fertil. 92:59-64.
45. Eyestone, W.H., M.L. Leibfried-Rutledge, D.L. Northey, B.G. Gilligan, and N.L. First. 1987. Culture of one- and two-cell bovine embryos to the blastocyst stage in the ovine oviduct. Theriogenology 28:1-7.
46. Faundez, R., I. Spohr, and Z. Boryczko. 1988. Effect of follicle cells on maturation and in vitro fertilization of cattle oocytes. Proc. 11th Intern. Cong. Anim. Reprod. Artif. Insem. 5:325.(Abstr.)
47. Figge, D.C. 1960. Growth characteristics of human endometrium in tissue culture. Obstet. Gynecol. 16:269-277.
48. First, N.L., and J.J. Parrish. 1987. In vitro fertilization of ruminants. J. Reprod. Fertil. 34(Suppl.): 151-165.
123
49. Fleming, H., and £. Guipide. 1982. Growth characteristics of primary culture of stromal cells from human endometrium. J. Steroid Biochem. 16:717.
50. Fredricsson, B. 1959. Proliferation of rabbit oviduct epithelium after estrogenic stimulation, with reference to the relationship between ciliated and secretory cells. Acta Moiphol. Neerl. Scand. 2:193-202.
51. Freshney, R.I. 1987. Culture of animal cells: a manual of basic technique. 2nd ed. John Wiley and Sons, New York, NY
52. Fukui, Y. 1989. Effects of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-cultured with bovine oviduct epithelial cells. J. Anim. Sci. 67:1318-1323.
53. Fukui, Y., and H. Ono. 1988. In vitro development to blastocysts of in vitro matured and fertilized bovine oocytes. Vet. Rec. 122:282.
54. Fukui, Y., M. Fukushima, Y. Terawaki, and H. Ono. 1982. Effect of gonadotropins, steroids and culture media on bovine oocyte maturation in vitro. Theriogenology 18:161-175.
55. Fukuda, Y., M. Ichikawa, K. Maito, and Y. Toyoda. 1990. Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage. Biol. Reprod. 42:114-119.
56. Fukui, Y., L.T. McGowan, R.W. James, P.A. Pugh, and H.R. Tervit. 1991. Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J. Reprod. Fertil. 92:125-131.
57. Gandolfi, F., and R.M. Moor. 1987. Stimulation of early embryonic development in the sheep by co-culture with oviductal epithelial cells. J. Reprod. Fertil. 81:23-28.
58. Gandolfi, F., T.A.L. Brevini, and R.M. Moor. 1989. Effect of oviduct environment on embryonic development. J. Reprod. Fertil. 38(Suppl.): 107-115.
59. Gandolfi, F., T.A.L. Brevini, L. Richardson, C.R. Brown, and R.M. Moor. 1989. Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development. Develop. 106:303-312.
60. Gill, J.L., and H.D. Hafs. 1971. Analysis of repeated measurements of animals. J. Anim. Sci. 33:331-336.
61. Glass, R.H., A.I. Spindle, and R.A. Pederson. 1979. Mouse embryos attachment to substratum and interaction of trophoblast with cultured cells. J. Exp. Zool. 208:327-335.
124
62. Glasser, S.R., and S.A. McCormack. 1981. Separated cell types as analytical tools in the study of decidualization and implantation. Pages 217-239 in Cellular and molecular aspects of implantation. S.R. Glasser and D.W. Bullock, ed. Plenum Press, New York, NY
63. Godke, R.A., and R.W. Rorie. 1988. Methods and applications of embryo bisection for cattle. Proc. 8th Amer. Embryo Transf. Conf. p. 66-84.
64. Goodeaux, L.L., C.A. Anzalone, J.K. Thibodeaux, Y. Menezo, J.D. Roussel, and S.A. Voelkel. 1990. Successful nonsurgical collection of Macaco, mulatto embryos. Theriogenology 34:1159-1167.
65. Goodeaux, L.L., J.K. Thibodeaux, S.A. Voelkel, C.A. Anzalone, J.D. Roussel, J.C. Cohen, and Y. Menezo. 1990. Collection, co-culture and transfer of rhesus pre-implantation embryos. Assist. Reprod. Technol. Androl. 1:370- 379.
66. Goto, K., Y. Kajihara, S. Kosaka, M. Koba, Y. Nakanishi, and K. Ogawa. 1988. Pregnancies after in vitro fertilization of cow follicular oocytes, incubation in vitro an their transfer to the cow uterus. Theriogenology 29: 251.(Abstr.)
67. Goto, K., Y. Kajihara, S. Kosaka, M. Koba, Y. Nakanishi, and K. Ogawa. 1988. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in vitro fertilization of in vitro matured follicular oocytes. J. Reprod. Fertil. 83:753-758.
68. Griffiths, J.B., and P. A. Riley. 1985 Cell biology: basic concepts. Pages 17-48 in Animal cell biotechnology. R.E. Spier and J.B. Griffiths, ed. Academic Press, New York, NY
69. Hammer, R.E., V.G. Pursel, C.E. Rexroad, R.J. Wall, K.J. Bolt, K.M. Ebert, R.D. Palmiter, and R.L. Brinster. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315:680-683.
70. Henriksen, T., T. Tanbo, Th. Abyholm, B.R. Oppedal, O.P. Claussen, and T. Hovig. 1990. Epithelial cells from human fallopian tube in culture. Human Reprod. 5:25-31.
71. Heyman, Y., and Y. Menezo. 1987. Interaction of trophoblastic vesicles with bovine embryos developing in vitro. Pages 175-191 in The mammalian preimplantation embryo. B.D. Bavister, ed. Plenum Press, New York, NY
72. Heyman, Y., S. Camous, J. Fevre, W. Meziou, and J. Martal. 1984. Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and sheep. J. Reprod. Fertil. 70:533-540.
125
73. Heyman, Y., Y. Menezo, P. Chesne, S. Camous, and V. Gamier. 1987. In vitro cleavage of bovine and ovine early embryos:Improved development using co-culture with trophoblastic vesicles. Theriogenology 27:59-68.
74. Hishinuma, M., Y. Takahashi, and H. Kanagawa. 1989. Isolation and monolayer culture of bovine oviduct epithelial cells. Jpn. J. Vet. Sci. 51:1201- 1208.
75. Hu, Y., S.A. Voelkel, and R.A. Godke. 1989. One-cell murine embryos cultured on rat liver cell monolayers and mouse oviduct cells. Proc. 1st CAASS Conf. p. 83.(Abstr.)
76. Iwasaki, S., N. Yoshiba, H. Ushijima, S. Watanabe, and T. Nakahara. 1990. Moiphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. J. Reprod. Fertil. 90:279-284.
77. Jiang, H.S., W.L. Wang, K.H. Lu, I. Gordon, and C. Polge. 1991. Roles of different cell monolayers in the co-culture of IVF bovine embryos. Theriogenology 35:216.(Abstr.)
78. Joshi, M.S. 1988. Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow. J. Reprod. Fertil. 83:249-261.
79. Kim, H.N., Y.X. Hu, J.D. Roussel, and R.A. Godke. 1989. Culturing murine embryos on bovine fetal spleen cell fibroblast and chick embryo fibroblast monolayers. Theriogneology 21:211.(Abstr.)
80. Kim, H.N., J.D. Roussel, G.F. Amborski, Y.X. Hu, and R.A. Godke. 1989. Monolayers of bovine fetal spleen cells and chick embryo fibroblasts for co- culture of bovine embryos. TTieriogenology 31:212.(Abstr.)
81. Kim, H.N., L. Zhang, J.D. Roussel, and R.A. Godke. 1991. Development of in vitro fertilized (IVF)-bovine oocytes using fetal spleen cell and chick embryo fibroblast monolayers. Biol. Reprod. 44(Suppl. l):73.(Abstr.)
82. Kinis, A., £ . Vergos, I. Gordon, A. Gordon, and M. Gallagher. 1990. Studies in the production of chimeric cattle embryos by aggregation of blastomeres from embryos derived from oocytes matured and fertilized in vitro. Theriogenology 33:268.(Abstr.)
83. Kirk, D., R.J.B. King, J. Heyes, L. Peachey, P.J. Hirsch, and R.W.T. Taylor. 1978. Normal human endometrium in cell culture. I. Separation and characterization of qpithelial and stromal components in vitro. In Vitro 14:651-662.
84. Kitiyanant, Y., C. Thonabulsombat, C. Tocharaus, B. Sanituongse, and K. Pavasuthipaisit. 1989. Co-culture of bovine embryos from oocytes matured and fertilized in vitro to the blastocyst stage with oviductal tissue. J. Sci. Soc. Thailand 15:251-260.
126
85. Kuzan, F.B., and R.W. Wright. 1981. Attachment of porcine blastocyst to fibroblast monolayers in vitro. Theriogenology 16:651-658.
86. Kuzan, F.B., and R.W. Wright. 1982. Observations on the development of bovine morulae on various cellular and noncellular substrata. J. Anim. Sci. 54:811-816.
87. Kuzan, F.B., and R.W. Wright. 1982. Blastocyst expansion, hatching and attachment of porcine embryos co-cultured with bovine fibroblast in vitro. Anim. Reprod. Sci. 5:57-63.
88. Lakhanpal, S., N.J. Gonchoroff, J.A. Katzmann, and B.S. Handwerger. 1987. A flow cytofluorometric double staining technique for simultaneous determination of human mononuclear cell surface phenotype and cell cycle phase. J. Immunol. Meth. 96:35-40.
89. Lavranos, T.C., and R.F. Seamark. 1989. Enhanced in vitro survival of mouse embryos cocultured with uterine cells in the presence of steroids. Reprod. Fertil. Dev. 1:41-46.
90. Leibfned, L., and N.L. First. 1979. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci. 48:76-86.
91. Leibfried-Rutledge, M.L., E.S. Crister, J.J. Parrish, and N.L. First. 1989. In vitro maturation and fertilization of bovine oocytes. Theriogenology 31:61-74.
92. Lenz, R.W., G.D. Ball, M.L. Leibfned, R.L. Ax, and N.L. First. 1983. In vitro maturation and fertilization of bovine oocytes are temperature-dependent processes. Biol. Reprod. 29:173-179.
93. Liszczak, T.M., G.S. Richardson, D.T. McLaughlin, and P.L. Komblith. 1977. Ultrastructure of human endometrial epithelium in monolayer culture with and without steriod hormones. In Vitro 13:344-356.
94. Lu, K.H., I. Gordon, M. Gallagher, and H. McGovern. 1987. Pregnancy established in cattle by transfer of embryos derived from in vitro. Vet Rec. 121:259-260.
95. Lu, K.H., I. Gordon, H.B. Chen, M. Gallagher, and H. McGovern. 1988. Birth of twins after transfer of cattle embryos produced by in vitro techniques. Vet. Rec. 122:539-540.
96. Maier, D.B., R.R. Newbold, and J.A. McLachlan. 1985. Prenatal diethyl- stilbesterol exposure alters murine uterine responses to prepubertal estrogen stimulation. Endocrinol. 116:1878-1886.
97. McCaffery, C., T.G. McEvoy, M.G. Diskin, F.C. Gwazdauskas, M.T. Kane, and J.M. Sreenan. 1991. Successful co-culture of 1-4 cell cattle ova to the morula or blastocyst stage. J. Reprod. Fertil. 91:119-124.
127
98. McDaniel, J.W., H. Scalzi, and D.L. Black. 1968. Influence of ovarian hormones on histology and histochemistry of the bovine oviduct. J. Dairy Sci. 51:754-761.
99. Merchant, D.J. 1990. Terminally differentiating epithelial tissues in primary explant culture: a model of growth and development. In Vitro 26:543-553.
100. Milgrom, E., M. Perrot, M. Atger, and E.E. Baulieu. 1972. Progesterone in uterus and plasma: V. An assay of the progesterone cytosol receptor of the guinea-pig uterus. Endocrinol. 90:1064-1070.
101. Milgrom, E., M. Perrot, M. Atger, and E.E. Baulieu. 1972. Progesterone in uterus and plasma: VI. Uterine progesterone receptors during the estrous cycle and implantation in guinea-pig. Endocrinol. 90:1071-1078.
102. Milgrom, E., T. Luu, M. Atger, and E.E. Baulieu. 1973. Mechanisms regulating the concentration and the conformation or progesterone receptor(s) in the uterus. J. Biol. Chem. 248:6366-6374.
103. Moore, N.W., B.G. Miller, and M.N. Trappl. 1983. Transport and development of embryos transferred to the oviducts and uteri of entire and ovariectomized ewes. J. Reprod. Fertil. 68:129-135.
104. Moutaouakkil, M., O. Prost, N. Dahan, and G.L. Adessi. 1984. Estrone and dehydroepiandrosterone sulfatase activities in guinea-pig uterus and liver: estrogenic effect of estrone sulfate. J. Steroid Biochem. 21:321-328.
105. Mukku, V.R., and G.M. Stancel. 1985. Receptors for epidermal growth factor in the rat uterus. Endocrinol. 117:149-154.
106. Mukku, V.R., and G.M. Stancel. 1985. Regulation of epidermal growth factor receptor by estrogen. J. Biol. Chem. 260:9820.
107. Mulhollard, J., E. Winterhager, and H.M. Beier. 1988. Changes in proteins synthesized by rabbit endometrial epithelial cells following primary culture. Cell Tissue Res. 252:123-132.
108. Munson, L., S.K. Chandler, and D.H. Schlafer. 1988. Cultivation of bovine fetal and adult endometrial epithelial cells. J. Tissue Culture Meth. 11:129-133.
109. Nakao, H., and N. Nakatsuji. 1990. Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos. Theriogenology 33:591-600.
110. Odor, D.L., P. Gaddum-Rosse, and R.E Rumery. 1983. Secretory cells of the oviduct of die pig-tailed monkey, Macaco nemestrina, during the menstrual cycle and after estrogen treatment. Amer. J. Anat. 166:149-172.
128
111. Oki, T., K. Takeuchi, S. Yamamoto, A. Mori, S. Fukumoto, T. Douchi, and Y. Nagata. 1990. Effect of aging on human oviductal epithelia in culture. Assist. Reprod. Technol. Androl. 1:69-75.
112. Ouhibi, N., Y. Menezo, G. Benet, and B. Nicollet. 1989. Culture of epithelial cells derived from the oviduct of different species. Human Reprod. 4:229-235.
113. Ouhibi, N., J. Hamidi, J.. Guillavd, and Y. Menezo. 1990 Co-culture of 1-cell mouse embryos on different cell supports. Human Reprod. 5:737-743.
114. Overskei, T.L., and A.H. Cincotta. 1987. A new approach to embryo coculture. Theriogenology 27:266.(Abstr.)
115. Parrish, J.J., J.L. Susko-Parrish, M.L. Leibfried-Rutledge, E.S. Crister, W.H. Eyestone, and N.L. First. 1986. Bovine in vitro fertilization with frozen-thawed semen. TTheriogenology 25:591-600.
116. Pollard, J.W., K.P. Xu, R. Rorie, W.A. King, and K.J. Betteridge. 1989. Influence of various oviductal epithelial cell culture systems on the development of early cleavage stage bovine embryos in vitro. Theriogenology 31:239. (Abstr.)
117. Pollard, J.W., J.M. Scodras, L. Plante, W.A. King, and K.J. Betteridge. 1990. Partial characterization of the embryotrophic nature of bovine endosalpingeal epithelial cell conditioned media. Biol. Reprod. 42(Suppl. 1): 168.(Abstr.)
118. Pool, S.H., K.E. Wiemer, R.W. Rorie, and R.A. Godke. 1988. The use of trophoblastic vesicles and fetal uterine fibroblast cells for the culture of precompaction-stage bovine embryos. Proc. 11th Intern. Congr. Anim. Reprod. Artif. Insem. 4:479.
119. Pool, S.H., R.W. Rorie, R.J. Pendleton, A.R. Menino, and R.A. Godke. 1988. Culture of early-stage bovine embryos inside day-13 and day-14 precultured trophoblastic vesicles. Ann. N.Y. Acad. Sci. 541:407-418.
120. Prather, R.S., F.L. Bames, M.M. Sims, J.M. Robl, W.H. Eyestone, and N.L. First. 1990. Nuclear transplantation in die bovine embryo assessment of donor nuclei and recipient oocyte. Biol. Reprod. 37:859-866.
121. Prichard, J.F., S.H. Pool, E.G. Blakewood, and R.A. Godke. 1990. Culture of early-stage caprine embryos using goat oviductal and uterine cell monolayers. Theriogenology 33:300. (Abstr.)
122. Prichard, J.F., S.H. Pool, E.G. Blakewood, Y. Menezo, and R.A. Godke. 1991. Culture of early-stage caprine embryos using goat oviductal cell monolayers. Theriogenology 35:259.(Abstr.)
123. Quarmby, V.E., and K.S. Korach. 1984. The influence of 17fi-estradiol on patterns of cell division in the uterus. Endocrinol. 114:694-702.
129
124. Rexroad, C.E. 1989. Co-culture of domestic animal embryos. Theriogenology 31:105-114.
125. Rexroad, C.E., and A.M. Powell. 1986. Co-culture of sheep ova and cells from sheep oviduct. Theriogenology 37:859-866.
126. Rexroad, C.E., and A.M. Powell. 1988. Co-culture of ovine ova with oviductal tissue in medium 199. J. Anim. Sci. 66:947-953.
127. Rexroad, C.E., and A.M. Powell. 1988. Co-culture of ovine eggs with oviductal cells and trophoblastic vesicles. Theriogenology 29:387-397.
128. Rexroad, C.E., and A.M. Powell. 1991. Effect of serum-free co-culture and synchrony of recipients on development of cultured sheep embryos to fetuses. J. Anim. Sci. 2066-2072.
129. Ricketts, A.P., M. Hagensee, and D.W. Bullock. 1983. Characterization in primary monolayer culture of separated cell types from rabbit endometrium. J. Reprod. Fertil. 67:151-160.
130. Robl, J.M., R. Prather, F. Barnes, W. Eyestone, D. Northy, B. Gilligan, and N.L. First. 1987. Nuclear transplantation in bovine embryos. J. Anim. Sci. 64:642-647.
131. Rodriguez, H.F., K.E. Wiemer, R.S. Dennis ton, and R.A Godke. 1990. A bilayered fetal-cell co-culture system for culturing bovine embryos. Theriogenology 33:309.(Abstr.)
132. Rodriguez, H.F., R.S. Denniston, and R.A. Godke. 1991. Co-culture of bovine embryos using a bilayer of bovine oviductal and granulosa cells. Theriogenology 35:264.(Abstr.)
133. Rorie, R.W., K.P. Xu, and K.J. Betteridge. 1990. Effects of culture on the post-thaw viability of cryopreserved in vitro fertilized bovine embryos. Theriogenology 33:331.(Abstr.)
134. Saeki, K., M. Hoshi, M.L. Leibfried-Rutledge, and N.L. First. 1991. In vitro fertilization and development of bovine oocytes matured in serum-free medium. Biol. Reprod. 44:256-260.
135. Sakkas, D., P.A. Batt, and A.W.N. Cameron. 1989. Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro. J. Reprod. Fertil. 87:359-365.
136. Salamonsen, L.A., W. Sum, B. Doughton, and J.K. Findlay. 1985. The effects of estrogen and progesterone in vivo on protein synthesis and secretion by cultured epithelial cells from sheep endometrium. Endocrinol. 117:2148-2159.
130
137. Salamonsen, L.A., B. Doughton, and J.K. Findlay. 1986. The effects of preimplantation blastocyst in vivo and in vitro on protein synthesis and secretion by cultured epithelial cells from sheep endometrium. Endocrinol. 119:622-628.
138. Scodras, J.M., J.W. Pollard, L. Plante, and K.J. Betteridge. 1990. Establishment of a bovine trophoblast cell line supporting the development of in vitro matured and fertilized oocytes. Biol. Reprod. 42(Suppl. 1): 167.(Abstr.)
139. Sirard, M.A., and R.D. Lambert. 1986. Birth of calves after in vitro fertilization using laparoscopy and rabbit oviducts incubation of zygotes. Vet. Rec. 119:167-169.
140. Sirard, M.A., J.J. Parrish, C.B. Ware, M.L. Leibfried-Rutledge, and N.L. First. 1988. The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. 39:546-552.
141. Smith, J.A., L. Martin, R.J.B. King, and M. Vertes. 1970. Effects of oestradiol-17£ and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17fl. Biochem. J. 119:773.
142. Sumida, C., C. Geliy, and J.R. Peasqualini. 1987. Modulation of the progesterone receptor in the fetal uterus of the progesterone primed guinea-pig in vivo and in organ culture. Life Sci. 40:1185.
143. Sumida, C., F. Lecerf, and J.R. Peasqualini. 1988. Control of progesterone receptors in fetal uterine cells in culture: effects of estradiol, progestins, antiestrogens, and growth factors. Endocrinol. 112:3-11.
144. Sutton, R., C.D. Nancarrow, A.L.C. Wallace, and N.W. Rigby. 1984. Identification of an oestrus-associated glycoprotein in oviductal fluid of the sheep. J. Reprod. Fertil. 72:415-422.
145. Thiard, M.C., M. Nicollier, A. Mahfoudi, and G.L. Adessi. 1989. Effects of oestrone sulphate, oestradiol and progesterone on protein sulphation in the guinea-pig uterus. J. Reprod. Fertil. 87:687-697.
146. Thibodeaux, J.K., L.L. Goodeaux, J.D. Roussel, Y. Menezo, G.F. Amborski, J.D. Moreau, and R.A. Godke. 1991. Effects of stage of the bovine estrous cycle on in vitro characteristics of uterine and oviductal epithelial cells. Human Reprod. 6:(in press).
147. Thibodeaux, J.K., J.D. Roussel, L.L. Goodeaux, Y. Menezo, M.A. Dietrich, and R.A. Godke. 1991. The effect of culture media on in vitro growth and proliferation of bovine uterine epithelial cells. Theriogenology 35:283.(Abstr.)
148. Thibodeaux, J.K., J.D. Roussel, Y. Menezo, R.A. Godke, and L.L. Goodeaux.1991. A method for in vitro cell culture of superficial bovine uterine endometrium epithelium. J. Tissue Culture Meth. 13:(in press).
131
149. Thibodeaux, J.K., J.R. Broussard, L.L. Goodeaux, J.D. Roussel, Y. Menezo, and R.A. Godke. 1991. The effect of incubator temperature on in vitro growth and proliferation of oviductal epithelial cells. J. Dairy Sci. 74(Suppl. l):298.(Abstr.)
150. Thompson, D.L., R.A. Godke, and T.M. Nett. 1983. Effects of melatonin and thyrotropin releasing hormone on mares during the nonbreeding season. J. Anim. Sci. 56:668-677.
151. Tomooka, Y., R.P. Diaugusdne, and J.A. McLaughlin. 1986. Proliferation of mouse uterine epithelial cells in vitro. Endocrinol. 118:1011-1018.
152. Trent, J.M., J.R. Davis, and C.M. Payne. 1980. The establishment and morphologic characterization of finite cell lines from normal human endometrium. Am. J. Obstet. Gynecol. 136:352-362.
153. van de Sandt, J.J.M., A.C. Schroder, and J.J. Eppig. 1990. Culture media for mouse oocyte maturation affect subsequent embryonic development. Mol. Reprod. Dev. 25:164-171.
154. Varma, V.A., S.A. Melin, T.A. Adamec, B.H. Dorman, J.M. Siegfried, L.A. Walton, C.N. Carney, C.R. Norton, and D.G. Kaufman. 1982. Monolayer culture of human endometrium: Methods of culture and identification of cell types. In Vitro 18:911-918.
155. Voelkel, S.A., G.F. Amborski, K.G. Hill, and R.A. Godke. 1985. Use of uterine-cell monolayer culture system for micromanipulated bovine embryos. Theriogenology 24:271-281.
156. White, K.L., K. Hehnke, L.F. Rickords, L.L. Southern, D.L. Thompson, and T.C. Woods. 1989. Early embryonic development in vitro by coculture with oviductal epithelial cells in pigs. Biol. Reprod. 41:425-430.
157. Whittingham, D.G. 1968. Development of zygotes in cultured mouse oviducts.I. The effect of varying oviductal conditions. J. Exp. Zool. 169:391-398.
158. Wiemer, K.E., G.F. Amborski, R.S. Denniston, K.L. White, and R.A. Godke. 1987. Use of a hormone-treated fetal uterine fibroblast monolayer system for in vitro culture of bovine embryos. Theriogenology 27:294.(Abstr.)
159. Wiemer, K.E., R.S. Denniston, G.F. Amborski, K.L. White, and R.A. Godke. 1987. A fetal fibroblast monolayer system of in vitro culture of bovine embryos. J. Anim. Sci. 65(Suppl. 1): 122.(Abstr.)
160. Wiemer, K.E., P.L. Casey, and R.A. Godke. 1988. Short term storage of equine embryos on a fetal bovine uterine monolayer followed by transfer to recipients. Proc. 11th Intern. Cong. Anim. Reprod. Artif. Insem. 2:198.
132
161. Wiemer, K.E., J. Cohen, G.F. Amborski, S. Wiker, G. Wright, L. Munya- kazi, and R. A. Godke. 1989. In vitro development and implantation of human embryos following culture on fetal bovine uterine fibroblast cells. Human Reprod. 4:595-600.
162. Wiemer, K.E., J. Cohen, S.R. Wiker, H.E. Malter, G. Wright, and R.A. Godke. 1989. Coculture of human zygotes on fetal bovine uterine fibroblast: embryonic morphology and implantation. Fertil. Steril. 52:503-508.
163. Wiemer, K.E., P.L. Casey, P.S. Mitchell, and R.A. Godke. 1989. Pregnancies following 24-hour co-culture of equine embryos on foetal bovine uterine monolayer cells. Equine Vet. J. 8(Suppl.): 117-122.
164. Willadsen, S.M. 1982. Micromanipulation of embryos of the large domestic species. Pages 185-210 in Mammailian egg transfer. C.E. Adams, ed. CRC Press, Boca Raton, FL
165. Willadsen, S.M. 1986. Nuclear transplantation in sheep embryos. Nature 320:63-65.
166. Witkowska, E. 1979. Reactivity of the epithelial cells of the bovine oviduct in vitro on the exogenic gonadotropic and steroid hormones. Part I: The effect of gonadotropic and steroid hormones on the amount of lipids and activity of dehydrogenases. Folia Histochem. 17:225-238.
167. Wright, R.W., and K.R. Bondioli. 1981. Apsects of in vitro fertilization and embryo culture in domestic animals. J. Anim. Sci. 53:702-729.
168. Wynn, R.M. 1977. Histology and ultrastructure of the human endometrium. Pages 341-376 in Biology of the uterus. R.M. Wynn, ed. Plenum Press, New York, NY
169. Xu, K.P., T. Greve, H. Callesen, and P. Hyttel. 1987. Pregnancy resulting from cattle oocytes matured and fertilized in vitro. J. Reprod. Fertil. 81:501- 504.
170. Younis, A.I., and B.G. Brackett. 1990. In vitro development of bovine oocytes into morulae and blastocysts. Theriogenology 33:355.(Abstr.)
171. Zhang, L., R.S. Denniston, and R.A. Godke. 1991. The effect of insulin on the development of in vz'rro-fertilized porcine oocytes. J. Anim. Sci. 64:(Suppl. l):49.(Abstr.)
172. Zhang, L., R.S. Denniston, and R.A. Godke. 1991. A simple method for in vitro maturation, in vitro fertilization and co-culture of bovine oocytes. J. Tissue Cult. Metii. (in press).
V IT A
John Kevin Thibodeaux was bom on November 9, 1963 in Lafayette,
Louisiana, the son of Herbert and Peggy Thibodeaux. He was raised on a small farm
located near Church Point, Louisiana and later graduated from Church Point High
School in May of 1981. Following high school graduation he attended the University
of Southwestern Louisiana. He majored in Animal Science and received his Bachelor
of Science degree in December of 1986. Following graduation he worked at the
University of Southwestern Louisiana Beef Farm as manager.
In August of 1987, he enrolled as a graduate student in the Department of
Dairy Science at Louisiana State University and completed the requirements for a
Master of Science degree in August of 1989. On March 20, 1991, his wife Holly S.
Thibodeaux gave birth to a beautiful baby girl named Sydney Paige Thibodeaux.
Currently, he is a candidate for the degree of Doctor of Philosophy in
Reproductive Physiology in the Department of Dairy Science under the direction of
Dr. Joseph D. Roussel.
133
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate: John Kevin Thibodeaux
Major Field: Dairy Science - Reproductive Physiology
Title of Dissertation: In V itro C u ltu re o f Bovine U te r in e and O viduct E p i th e l i a lC e lls
Approved:
M ajor P rofessor and C hairm an
D ean of the G raduate School
EXAM INING CO M M ITTEE:
Date of Examination:
August 26, 1991