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J. Embryol. exp. Morph. Vol. 31, 3, pp. 621-634, 1974 6 2 1Printed in Great Britain
Artificial insemination
of deermice (Peromyscus maniculatus) withsperm from other rodent species
By MICHAEL B. MADDOCK1 AND WALLACE D. DAWSONFrom the Department of Biology, University of South Carolina
SUMMARYDeermice (Peromyscus maniculatus) were hormonally induced to ovulate and artificially
inseminated with sperm of 13 other rodent species varying in taxonomic distinction fromspecies to family level. These were compared with inter se and sham inseminated controls.Embryos were recovered at various stages from 5 h to 10 days. Hybrid embryos failed todevelop fully in all crosses except those within the same species group. Closely allied inter-species group crosses produced implantation embryos, but intersubgeneric and inter-generic crosses did not develop beyond early cleavage. Interfamilial hybrid embryos couldnot be obtained.
INTRODUCTION
The cricetid rodent genus Peromyscus (deermice and allies) affords a parti-cularly valuable model for species hybrid studies because of its many formswhich exhibit varying, and often subtle, degrees of relationship, and becauseof the relative ease with which they can be maintained in the laboratory.Additionally, hormone-induced ovulation and implantation, and artificialinsemination methods developed with common laboratory animals (rats, mice,hamsters) can be employed. These techniques make possible hybridizationswhich could not be accomplished through natural mating, and permit thestudy of developmental interplay between genotypes from divergent sources.
The rationale of the present study was to inseminate female deermice (P.maniculatus) with sperm of progressively more distantly related species. It wasanticipated that the extent of ontogenetic development would be negativelycorrelated with phylogenetic divergence, and that given a specific degree oftaxonomic distinction, developmental end points could be predicted. Addition-ally, sensitive ontogenetic stages should be recognized, and some evolutionaryimplications could be drawn.
1 Author's address: Department of Biology, University of South Carolina, Columbia,S.C., 29208, U.S.A.
622 M. B. MADDOCK AND W. D. DAWSON
MATERIALS AND METHODS
Young (40- to 90-day-old) virgin female deermice were induced to ovulatewith intraperitoneal injections of 15i.u. pregnant mare serum gonadotrophin(PMS) followed two days later by lOi.u. of human chorionic gonadotrophin(HCG). Ovulation occurred about 13 h after the second injection.
Epididymal sperm from rodents killed one hour previously were suspendedin reconstituted 9 % skim milk at concentrations in excess of 106/mm3 (Dziuk& Runner, 1960). Sperm counts and motility were confirmed visually. Artificialinsemination was accomplished through the cervix using a syringe with ablunted needle. Insemination was timed to coincide with ovulation.
Mice from each cross were autopsied at intervals from 5 to 96 h afterinsemination. Fallopian tubes and uterine horns were flushed with physiologicalsaline, and ova and/or pre-implantation embryos recovered. In crosses wheredevelopment proceeded to the blastocyst stage, mice were examined for im-plantation sites between the 7th and 10th day, where day 0 was the day ofinsemination.
One mg medroxyprogesterone acetate (MPA), as DepoProvera (Upjohn),was injected subcutaneously at both 5 and 24 h post-insemination to facilitateegg transport. Additional daily injections of 1 mg MPA were given to somemice together with 0-025 mg estradiol in 0-025 cm3 sesame oil on day 5 post-insemination, to induce implantation of blastocysts.
Freshly recovered ova and embryos were examined and photographed underphase microscopy at x 100. Representative specimens were fixed, stained andmounted for further study.
Sperm donors represented 13 other rodent species differentiated from P.maniculatus at species to family level, with six intermediate categories (Table 1).P. maniculatus, P. polionotus, Mesocricetus auratus, Meriones unguiculatus,Rattusnorvegicus and Mus musculus were from domesticated or semi-domesticatedstocks, while wild captured animals of the other species were used.
RESULTS
In the P. maniculatus inter se inseminated controls, 49 (83 %) of 59 miceinseminated and 140 (81 %) of 172 ova recovered were fertilized. Thecriteria for fertilization in each instance were: (1) sperm penetration of ova;(2) cleavage; or (3) blastocyst development and implantation (Fig. 1). Noneof the 40 ova recovered from the milk sham inseminated control showedgynogenesis at 48 h. These and numerous additional observations indicatethat spontaneous cleavage (fragmentation) is not a common occurrence inunfertilized deermouse ova during the first two days. Therefore any cleavageobserved at 48 h was assumed to result from fertilization rather than gyno-genesis.
Artificial insemination of deermice 623
Table 1. Comparisons o/Peromyscus maniculatusand sperm donor species
Sperm donor species
Level of Diploid Usualtaxonomic chromosome adult
differentiation number wt (g)
Estimated time ofmost recent common
ancestry (108 yrs)
P. maniculatus(deermouse)
P. polionotus(oldfield mouse)
P. leucopus \(woodmouse) I
P. gossypinus j(cottonmouse)J
P. truei(pinyon mouse)
P. floridanus(Florida mouse)
Reithrodontomys humulis(harvest mouse)
Ochrotomys nuttalli(golden mouse)
Sigmodon hispidus -\(cotton rat) I
Oryzomys palustris j(rice rat) J
Mesoericetus auratus(golden hamster)
Meriones unguieulatus(Mongolian gerbil)
Rattus norvegicus N
(laboratory Norwayrat)
Mus museulus(laboratory housemouse) ,
Species
Species group
Species group
Subgenus
Genus
Genus
Geographic line
Tribe
Subfamily
Family
48
48
48
48
48
48
42
52
52
56
44
44
f42
40 25-35
002(Pleistocene-Wisconsin)
0-4(Pleistocene-Sangamon)
10(Mid-Pleistocene)
20(Pleistocene-Nebraskan)
40(Pliocene)
70(Pliocene)
20(Miocene)
20-30
20-30
20-30
Inseminations with sperm from other species are compared with the controlsin Tables 2 and 3. Each type of insemination within the genus Peromyscusproduced some examples of development through first cleavage, but develop-ment to blastocyst and beyond was restricted to crosses within the maniculatusand leucopus species groups. Inseminations with P. polionotus sperm produceda high proportion of fertilized ova (62 %) and all embryos examined hadnormal morphology (Fig. 2). This was expected and consistent with the occur-rence of viable, fertile hybrids in laboratory matings of these species (Dice,1933; Dawson, 1965).
P. leucopus and P. gossypinus are closely related species of the leucopusspecies group, and are interfertile in laboratory crosses (Dice, 1937). Hybridsof either of these two leucopus group species with P. maniculatus were expected
V) EMB 31
624 M. B. MADDDOCK AND W. D. DAWSON
Fig. 1. Early embryonic development of P. maniciilatus. (A) Fertilized ovum withpronuclei and sperm tail. (B-D) Cleavage. (E) Morula. (F) Blastocyst in zona.(G) Section of uterus showing implanted blastocyst. (H) Uterus with fourconceptuses.
Artificial insemination of deermice 625
Table 2. Fertility of deermice artificially inseminatedwith sperm from other rodents
Sperm source
P. maniculatusP. polionotusP. leucopusP. gossypinusP. trueiP. floridamisReithrodontomysOchrotomysSigmodonOryzomysMesocricetusMen'onesMusRattus
* Total number
Percentage ofinseminated mice
successfullyfertilized
8383172930101708
3311000
of eggs in sample given in
Percentageof eggs
fertilized5-48 h
62 (85)*58 (48)15(46)87(15)52 (46)13(30)8(12)0(26)3(25)
15(27)9(40)0(9)0(29)0(15)
parentheses.
to show similar development. Earlier attempts at laboratory matings of P.maniculatus x P. leucopus produced no progeny (Dice, 1933), although thereare unconfirmed reports of adult hybrids in nature. Chang, Pickworth &McGaughey (1969) obtained pre-implantation embryos by artificial insemi-nation, and, in one instance, observed an 11-day-old implanted embryo.
Insemination with P. leucopus sperm gave a lower frequency of fertilizedova than expected (Table 2). However, some representatives of various stagesfrom sperm penetration to implantation were observed. These had normalmorphology. Sperm quality (motility and concentration) was not good in thelimited number of P. leucopus we had available, and this probably contributedto the reduced proportion of fertilized ova. Some P. maniculatus x P. gossypinusembryos showed normal cleavage, but in other instances irregular shapedblastocysts were observed (Fig. 3). A single implantation site was seen in onemouse, indicating that hybrids of P. gossypinus as well as P. leucopus are capableof initiating post-implantation development. Another study (Dawson, Mintz,Maddock & Lewin, 1972) has shown that occasional P. maniculatus-leucopushybrids produced by artificial insemination may be live-born but non-viable;however, development is usually interrupted before 15 days post-insemination.
P. truei also differs from P. maniculatus at the species group level, but thetruei group is usually considered more remotely related to P. maniculatus thanthe leucopus group (Hooper & Musser, 1964). Moreover, P. truei are sub-stantially larger than P. maniculatus. Six experimental matings attempted by
39-2
OS
Tab
le 3
. Em
bryo
nic
deve
lopm
ent
in a
rtif
icia
lly
inse
min
ated
dee
rmic
e
Sper
m s
ourc
e
P. m
anic
ulat
usP
. pol
iono
tus
P.
leuc
opus
P. g
ossy
pinu
sP
. tru
eiP
. fl
orid
anus
Rei
thro
don
tom
ysO
chro
tom
ysSi
gmod
onO
ryzo
mys
Mes
ocri
cetu
sM
erio
nes
Mus
Rat
tus
Milk
sha
m
Nn
mh
pr
mic
ein
sem
inat
ed
59 12 30 24 30 20 6 7 12 6 18 6 5 5 6
Nnm
hpr
mic
efe
rtile
49 10 5 7 9 2 1 0 1 2 2 0 0 0 —
1
Sper
mpe
netr
atio
n
18(2
6)8(
16)
3(27
)0(
1)6(
14)
1(25
)1(
2)0(
20)
0(25
)0(
9)1(
5)0 0(
4)0(
4) —
Dev
elop
men
t ob
serv
ed
Cle
avag
e(2
-8 c
ell)
32 (
53)
16(2
4)4(
19)
12(1
3)18
(31
)3(
4)0(
10)
0(6)
1 (1
0)4(
18)
2(35
)0(
9)0(
25)
0(11
)0(
40)
A
Mor
ula
3(6)
4(8)
0 KD
0(1)
0(1)
0 0 0 0 — — — — —
Impl
anta
tion
Bla
stoc
yst
site
s
43 (4
3)
44
8 (1
0)
—1(
7)
1*
0 1
0 —
0 —
—
——
—
—
——
—
—
——
—
—
——
—
—
—
Num
ber
AV
Q\J
V
C*.
and
embr
yos
172 58 54 16 46 30 12 26 35 27 40 9 29 15 40
Tot
alfp
rtil
p
ova
orem
bryo
s
140 36 9 14 24 4 1 0 1 4 3 0 0 0 —
x •fcd X d 0 o z b p d o z*
Doe
s no
t in
clud
e 16
im
plan
tatio
ns r
epor
ted
else
whe
re (
Daw
son
et a
l. 19
72).
Num
bers
in
pare
nthe
ses
repr
esen
t to
tal
eggs
obs
erve
d at
equ
ival
ent
time.
Artificial insemination of deer mice 627
Fig. 2. Early embryonic development of P. mamculatus-polionotus F1 hybridsproduced by artificial insemination. (A). Fertilized ovum with pronuclei. (B-D).Cleavage. (E). Morula. (F). Blastocyst in zona.
Dice (1933) between P. mcmiculatus and P. truei were sterile. About half ofthe ova which we recovered from inseminations with P. truei sperm had beenfertilized (Table 2), and the first cleavage appeared normal. However, thesecond cleavage was abortive (Fig. 4A-D), and no development beyond thefour-cell stage was observed.
Inseminations with P. floridanus sperm gave a small number of fertile ova.The first cleavage was normal, but irregular cleavage followed (Fig. 4E-F).
628 M. B. MADDOCK AND W. D. DAWSON
Fig. 3. (A) Fertilized ovum of P. maniculatus inseminated with P. leucopus spermshowing sperm penetration and pronuclei. (B) Two-cell hybrid embryo of P.maniculatus inseminated with P. gossypinus. (C-D) Embryos of P. maniculatus-gossypinus hybrids showing irregular cleavage. (E) Morula of P. maniculatus-gossypinus hybrid. (F) Single conceptus of P. maniciilatus-gossypinus hybrid.
One 6-cell embryo was observed. No previous experimental hybridization hasbeen attempted with this species, but Dice (1933) had demonstrated that otherintersubgeneric crosses in Peromyscus do not yield progeny.
Reithrodontomys (harvest mice) and Peromyscus are immediately alliedgenera within the Peromyscine line (Hooper & Musser, 1964). In a limitednumber of observations, one P. maniculatus ovum had been penetrated bya R. humulis sperm, but no subsequent cleavage was seen (Table 2).
Artificial insemination ofdeer mice 629
Fig. 4. (A-C) Sperm penetration and cleavage of P. maniculatus-truei hybridembryos. (D-F) Sperm penetration and cleavage of P. maniculatus-florhkinushybrid embryos. Note excess nucleoli and irregular cleavage in F.
Ochrotomys nuttalli (golden mouse) formerly was classified under Peromyscusas a monotypic subgenus, but its chromosome number and phallic morphologyevidence its distinctness from Peromyscus (Blair, 1942; Hooper, 1958; Patton& Hsu, 1967). The generic status of Ochrotomys is further supported in ourstudy by the failure of any of 26 P. maniculatus ova recovered to be fertilizedby O. nuttalli sperm. Golden mouse sperm differs markedly from other NewWorld cricetids in both morphology and motility (Fig. 5E), as was noted alsoby Hirth (1960).
630 M. B. MADDOCK AND W. D. DAWSON
Fig. 5.(A). Reithrodontomys humulis sperm penetration of P. manicitlatus. (B-D).Abortive and apparently normal first cleavage of P. maniculatus-Oryzomys palustrisembryos. (E). Ochrotomys nuttalli sperm. (F). P. maniculatus sperm.
Although North American species of Sigmodon (cotton rats) and Oryzomys(rice rats) occur, these genera represent a distinct cricetid line with neotropicalaffinities. Insemination of deermice with sperm from these rat-size rodentsproduced some abortive first cleavage, and two regular two-cell embryos wereobserved - one with Sigmodon and one with Oryzomys (Fig. 5).
Insemination with hamster {Mesocricetus) sperm gave two instances of firstcleavage. In one case a regular two-cell stage was observed, and each blastomere
Artificial insemination of deermice 631
indicated regular organization of a mitotic apparatus for the second division.An irregular two-cell embryo was observed in the same mouse. Probable spermpenetration was observed in an ovum from another mouse.
Inseminations of deermice with sperm from other Old World myomorphs(Meriones, Rattus, and Mus) gave entirely negative results (Table 2).
DISCUSSION
There are numerous reports (Gray, 1953) of viable mammalian specieshybrids, including classic cases such as mule and cattalo, but few examplesof prenatally interrupted hybrid development have been investigated. Theseexamples were reviewed by Chang & Hancock (1967). They are limited togoat x sheep, mink x ferret, and artificial crosses among various species ofrabbits and hares. Goats (Copra hircus) inseminated by sheep (Ovis aries)customarily give rise to conceptuses which may survive to midgestation, butdevelopment in the reciprocal cross is limited to early cleavage.
Various crosses among domestic rabbits, European and American hares,and cottontails by artificial insemination do not progress beyond the blastocyststage (Chang & Hancock, 1967). Mink (Mustela visori) sperm may fertilizeferret (Mustela furo) eggs which undergo cleavage and occasionally developto blastocyst and implant abnormally (Chang, 1965).
These studies imply that taxonomic relationship per se is not a reliableindicator of the extent of embryonic development in species hybrids since(1) an intergeneric hybrid (goat x sheep) may develop further than an intra-generic one (ferret x mink), and (2) reciprocal crosses differ significantly.However, within a restricted taxonomic grouping (Leporidae), crosses betweenmore closely related genera (Oryctolagus x Lepus) apparently give betterpercentages of fertilization and greater development than those between themore distinctly related Oryctolagus and Syhilagus (Chang & Hancock, 1967).It is noteworthy that in most instances development fails at or before im-plantation. Only in goat-sheep hybrids were fetuses formed.
The interspecific inseminations of Peromyscus gave a spectrum ranging fromcompletely viable hybrids among species of the same species group, to failureof sperm penetration at the subfamily level (Fig. 6). As in lagomorph andmustelid hybridizations, when reproductive failure occurs, it was more likelyto affect preimplantation embryos. Only in P. maniculatus x P. leucopus (orP. gossypinus) did limited fetal organization occur, analogous to goat x sheep.It is reasonable to generalize that if inseminations between mammalian speciesof the same or a closely related genus fail to produce living offspring, thereproductive block most likely occurs between first cleavage and implantation.The probability of normal morphogenesis diminishes rapidly with increasingphylogenetic separation.
Crosses between Peromyscus subgenera or closely related genera occasionally
632 M. B. MADDOCK AND W. D. DAWSON
0 5
1 'I-Pcromy.scu.smamvulutits
(female)
X
(sperm donor)
X X X
Spermpenetration
Cleavane,2-cell
Clcnvaye.4-cell
8-eell
Morula
Blastocyst
Implantation
Fetaldevelopment
Live birth
Viability
Fig. 6. Maximum development of embryos of P. maniculatus artificiallyinseminated with sperm from various species.
initiated cleavage, but this often was abnormal and rarely progressed beyonda few divisions. The concept of Dice (1940) that Peromyscus subgeneric crosseswill not produce viable hybrids is reinforced.
A wide variety of agents can induce sperm capacitation (Yanagimachi, 1970).Species specificity of capacitation was not indicated by our investigation. Ifcapacitation is requisite to egg penetration in these rodents, conditions of theP. maniculatus genital tract apparently are sufficient to induce it.
The inseminations in this study were all in vivo with zona pellucida intact.
Artificial insemination of deermice 633
Observation that some deermouse eggs were penetrated by sperm of othercricetid genera indicates that neither the zona nor vitelline block is particularlyspecific here. Yanagimachi (1972) proposed that the vitelline surface in zona-free murid (rat and mouse) eggs is strongly species specific, whereas the zona-free hamster egg, which can be penetrated by guinea-pig spermatozoa, is not.However, Hanada & Chang (1972) reported that a low percentage of zona-free mouse eggs were penetrated by rat or hamster sperm, and that rat eggsare fertilized by hamster sperm. Additionally, they obtained penetration ofzona-free hamster eggs by rat sperm in 8-26 % of the attempts, and highpercentages of zona-free rat and hamster eggs penetrated by mouse sperm. Thepossibility exists that both the zona and vitelline blocks in cricetids (hamstersand deermice) show less specificity than in murids, particularly mouse.
Wild deermice reproduce at the rate of approximately two generations peryear. Various lines of evidence indicate that the leucopus and maniculatusspecies groups became distinct in the Sangamon interglacial (ca. 5xl05yrsago). From this it may be deduced that a million generations of separationfrom a common ancestral gene pool is sufficient to evolve strong geneticisolating mechanisms.
The authors gratefully acknowledge the generous assistance of Mr Read Lewin. Portionsof this research were supported by grant HD 06754 from the Institute of Child Health andHuman Development, U.S. Public Health Service.
REFERENCES
BLAIR, W. F. (1942). Systematic relationships of Peromyscus and several related generaas shown by the baculum. J. Mammal. 23, 196-204.
CHANG, M. C. (1965). Implantation of ferret ova fertilized by mink sperm. /. exp. Zool.160, 67-80.
CHANG, M. C. & HANCOCK, J. L. (1967). Experimental hybridization. In ComparativeAspects of Reproductive Failure (ed. K. Benirschke), pp. 206-217. New York: SpringerVerlag.
CHANG, M. C, PICKWORTH, S. & MCGAUGHEY, R. W. (1969). Experimental hybridizationand chromosomes of hybrids. In Comparative Mammalian Cytogenetics (ed. K. Benirschke),pp. 132-145. New York: Springer Verlag.
DAWSON, W. D. (1965). Fertility and size inheritance in a Peromyscus species cross. Evolution19, 44-55.
DAWSON, W. D., MINTZ, J., MADDOCK, M. B. & LEWIN, A. R. (1972). On hybridizationbetween deermice (Peromyscus maniculatus) and woodmice (P. leucopus). ASB Bulletin19, 64 (Abstract).
DICE, L. R. (1933). Fertility relationships between some of the species and subspecies ofmice in the genus Peromyscus. J. Mammal. 14, 298-305.
DICE, L. R. (1937). Fertility relations in the Peromyscus leucopus group of mice. Contr.Lab. vertebr. Genet Univ. Mich. 4, 1-3.
DICE, L. R. (1940). Speciation in Peromyscus. Am. Nat. 74, 289-298.DZIUK, P. J. & RUNNER, M. N. (1960). Recovery of blastocysts and induction of implantation
following artificial insemination of immature mice. /. Reprod. Fert. 1, 321-331.GRAY, A. P. (1953). Mammalian Hybrids. Farnham Royal: Technical Communication of
the Commonwealth Agricultural Bureau.HANADA, A. & CHANG, M. C. (1972). Penetration of zona-free eggs by spermatozoa of
different species. Biol. Reprod. 6, 300-309.
634 M. B. MADDOCK AND W. D. DAWSON
HIRTH, H. F. (I960). Spermatozoa of some North American bats and rodents. /. Morph.106, 77-83.
HOOPER, E. T. (1958). The male phallus in mice of the genus Peromyscus. Misc. Publs. Mus.Zool. Univ. Mich. 105, 1-24.
HOOPER, E. T. & MUSSER, G. G. (1964). Notes on classification of the rodent genus Peromyscus.Occ. Pap. Mus. Zool. Univ. Mich. 635, 1-13.
PATTON, J. L. & Hsu, T. C. (1967). Chromosomes of the golden mouse Peromyscus(Ochrotomys) nuttalli (Harlan). /. Mammal. 48, 637-639.
YANAGIMACHI, R. (1970). The movement of golden hamster spermatozoa before and aftercapacitation. /. Reprod. Fert. 23, 193-196.
YANAGIMACHI, R. (1972). Penetration of guinea-pig spermatozoa into hamster eggs in vitro.J. Reprod. Fert. 28, 477-480.
{Received 5 October 1973)
