Evaluation of the pregnancy immunotrophism hypothesisby assessment of the reproductive performance of young

adult mice of genotype scid/scid.bg/bgB. A. Croy and C. Chapeau

Department of Biomedicai Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2W1

Summary. The hypothesis that enhancement of pregnancy success results from immunerecognition of the conceptus was evaluated by studying reproductive performance in a

new line of mice deficient in NK cells and lacking B cells and T cells. Doubly mutantmice of genotype scid/scid.bg/bg are both viable and fertile. The numbers of offspringborn to pairs of this genotype were not different from numbers born to heterozygouspairs. Differences in prenatal loss could not be found between genotypes by counts ofeither fetal resorption sites or corpora lutea. The timing of developmental stages andthe differentiation of trophoblast, placenta, decidua and metrial gland in scid/scid.bg/bg mice appeared normal. These results suggest either that lymphokine influences on

trophoblast cells in vivo do not contribute, in a major way, to pregnancy success or thatthe important cytokines are derived from uterine cell populations that are not classical,mature B cells, T cells or NK cells.

Keywords: mouse; severe combined immunodeficiency (SCID); beige mutant; pregnancy; trophoblast;cytokines

IntroductionThe roles of uterine lymphocytes during mammalian pregnancy are incompletely defined. Since thefetus expresses paternal and unique fetal antigens that can be recognized by the mother it has oftenbeen suggested that uterine lymphocytes harm the fetus unless blocked (Medawar, 1954; Beer &Billingham, 1976; Clark & Croy, 1986; Gill & Wegmann, 1987; Clark et ai, 1988; Parhar et ai,1988). This model has failed to explain the immunological enigma of viviparous reproduction(Goodfellow, 1983). Data have recently been collected suggesting that T-cell lymphokine productscan act on a wide variety of cell types (Dedhar et ai, 1988) and can enhance trophoblast cellfunction (Athanassakis et ai, 1987; Armstrong & Chaouat, 1989) and promote pregnancy success

in vivo (Wegmann et ai, 1989).Immunohistochemical studies have been used for in-situ demonstration of known immuno-

competent cell populations within the pregnant mouse uterus (Lala et ai, 1983; Parr & Parr, 1985;Redline & Lu, 1989) but few functional effector cell populations have been demonstrated (Gambelet ai, 1985). Antigen-processing cells (Matthews & Searle, 1988) and antibody production (Bell& Billington, 1986) have been found but macrophages and cells appear to be located at theperiphery of the implantation site, associated with the myometrium rather than with the conceptus(Redline & Lu, 1989; Stewart & Mitchell, 1989). Transient NK-cell activity appears in deciduaearly after implantation but declines rapidly. Since NK cells appear unable to lyse embryonic cells,including trophoblast, unless artificially activated in vitro it has been postulated that theirfunctional role in the uterus in early pregnancy is cytokine production (Gambel et ai, 1985; Croy &Rossant, 1987; Croy et ai, 1988; Zuckermann & Head, 1988). suppressor cells (Chaouat et ai,1982; Ribbing et ai, 1988) and non-T, non-B suppressor lymphocytes (Clark et ai, 1988) have alsobeen isolated from pregnant uteri.

The requirement for lymphocyte function during pregnancy, however, must be questioned sinceantibody-mediated depletion of T- or NK-cell subsets does not usually lead to abortion of pregnantrodents (Croy et ai, 1985; Athanassakis et ai, 1987; Gendron & Baines, 1988; Mattsson &Holmdahl, 1987; Sulila et ai, 1988). Furthermore, female and male mice of genotypes nu/nu (T-celldeficient), scid/scid (combined B-cell and T-cell deficient) and of the small thymic strain SL/Nireproduce (Croy & Osoba, 1973; Clark et ai, 1989; Michael et ai, 1988), although nu/nu femaleshave reduced fertility because of follicular atresia (Kindred, 1979) and SL/Ni mice experienceovarian and reproductive abnormalities after 1 year of age (Michael et ai, 1988).

To address the question of whether an essential role exists for mature lymphocytes and cells in promotion of fetal success, we reasoned that lymphocyte depletion could be more success¬

fully accomplished in mice by genetic mutation than by short-term antibody administration. Thewell defined mutation seid was selected for depletion of mature lymphocytes due to the absence ofany pleiotrophic effects on non-lymphoid tissue (Lieber et ai, 1988; Malynn et ai, 1988) and themutation bg was selected for its major depletion of NK-cell function (Roder & Duwe, 1979) onimmunodeficient backgrounds (Clark et ai, 1981; Andriole et ai, 1985, Hioki et ai, 1987; Shultz& Sidman, 1987). A line of doubly mutant scid/scid.bg/bg mice was established and studied forreproductive competence.

Materials and Methods

Mice. Three female C57BL/6J bg/bg mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) andmated to three C.B-17 scid/scid males, kindly provided by Dr G. Fulop and Dr R. A. Phillips (Hospital for SickChildren, Toronto, Canada). Offspring from these matings were intercrossed to produce litters containing immuno¬deficient animals. The mice were maintained in a sterile environment using microisolation cages (Lab Products Inc.,Maywood, NJ, USA) and housed in rooms illuminated from 07:00 to 17:00 h each day. Weaned mice were bled forgenotyping at 4 weeks of age and new breeding pairs were established by random mating of individual males to oneor more females having the same immune deficiency. Mated, female random-bred CD1 mice (Charles River Labora¬tories, St Constant, Quebec, Canada) from a conventional animal colony were used for some comparisons.

Identification of the scid/scid genotype. Mice homozygous for the gene seid have no detectable serum immuno¬globulins (Ig). To assess the presence of Igs, 50 pi heparinized blood were collected from the tail vein and centrifuged(at a relative centrifugal force of 828, 4°C, 10 min) to obtain plasma. Immunodiffusion was conducted in 1 % gels ofNoble Agar (Difco, Detroit, MI, USA) in phosphate-buffered saline pH 7-4 (PBS) on a glass microscope slide. Wellswere cut symmetrically in a hexagonal pattern around the centre well and 15 pi rabbit anti-mouse immunoglobulins(DAKOJZ109 diluted 1:1 with PBS) were placed in the central well. Test plasma (15 pi) was placed in each surround¬ing well and the slides were incubated overnight in a humid atmosphere at 37°C Slides were scored for the presence( + /+ or +/scid) or absence (scid/scid) of a precipitin line by using light box illumination. Mice which displayed Igswere designated + /scid(?) since the hétérozygotes ( + /seid) were not studied further to distinguish them from mice ofwild genotype ( + / + ).

Identification ofthe bg/bg genotype. Beige mice are most readily identified by staining blood films and detecting thepresence of giant sudanophilic granules in 100% of their blood neutrophils (Bennett et ai, 1969) and by coat colourdilution, in non-white animals. Smears of blood were prepared from all animals by touching a clean glass slide to theincised tail vein of each animal then spreading the drop using a second glass slide. Slides were air dried then fixed inparaformaldehyde vapour for 10 min and washed in running tap water for 10 min. Next, slides were immersed in a

filtered staining solution made by mixing 60 ml of a 0-3% (w/v) solution of Sudan black (Sigma) in 95% ethanolwith 40 ml of a buffered solution made of 53% (w/v) crystalline phenol in 95% ethanol mixed at 1:3 with a 0-3% w/vaqueous solution of Na2HP04.12H20 for 60 min. The slides were then dipped 5 times, for 10 sec each dip, in 50%ethanol, washed for 5 min in running tap water and blown dry. The slides were counterstained with Gill's haema¬toxylin for 3 min, blown dry, and examined using a light microscope at 400 or 1000 oil magnification. At least 5neutrophils were examined from each animal and scored for granule size. Mice with blood smears displaying giantgranules were designated bg/bg, and this coincided with coat colour dilution in all of the non-white mice. Mice withblood films displaying neutrophil granules of normal size were designated non-beige, and may have been +/bg or+ / +

.

The non-beige animals have been designated + /bg(?).Timed matings, embryo collection and evaluation ofcorpora lutea. To obtain timed pregnancies paired females were

checked daily for copulation plugs. The morning of plug detection was designated Day 0-5 of pregnancy. Pregnantfemales were killed by cervical dislocation and the ovaries and uterus removed. Uteri with preimplantation stageswere flushed as previously described (Croy et ai, 1982) and some embryos were passed through 1 % sodium citrate for3 min and mounted on slides using 1:1 methanol-acetic acid followed by 3:1 methanol-acetic acid and stained using

4% Giemsa for assessment of cell number by enumeration of stained, dispersed nuclei. Uteri containing post-implantation stages were fixed in PBS-buffered formalin, and individual implantation sites or placentae wereembedded in glycol methacrylate, sectioned at 2 pm, mounted and stained using eosin and haematoxylin or periodicacid-Shiffs reagent (Fisher Scientific) for histological assessment.

Statistics. A paired / test was used for data comparisons. Significance was accepted at 5%.

Results

Development of the seid/scid.bg¡bg line and study of its litter sizes

Matings of C51BL/63-bg/bg females to C.B-17 scid/scid males provided 3 litters of offspringhaving the genotype +/scid. + /bg. These mice were intercrossed to obtain a generation containingrecombinant mice of the genotypes of interest. All offspring were genotyped and males and femaleshaving the same immunodeficiency were paired, to create four lines, seid/scid.bg/bg, scid/scid. + /bg(?), +/scid(?).bg/bg and +/scid(?). + /bg/(?). All lines, except the non-scid non-bg, which was

terminated, have been maintained under identical housing conditions for several generations byrandom mating. It was observed that male and female mice of genotype scid/scid.bg/bg were bothviable and fertile and that they were produced in approximately equal numbers. The sex ratio atweaning was 50-93% for males and 4907% for females. Of 81 males studied all except one were

fertile, as assessed by production of offspring within 4 weeks of pairing. The numbers of pairsstudied at each generation are presented in Table 1. Mean litter size at each generation is reportedin Table 2, by litter in sequence of production. The mean litter sizes of the foundation lines are

reported to be 4-6 for BALB/c and 6-7 for C57BL/6J mice (Heininger & Dorey, 1980). The littersizes observed for the immunodeficient mice therefore equalled or exceeded that reported for thefoundation stocks. There are no statistically different values in Table 2. Litter sizes ranged from 1to 13 and the type of immune deficiency had no bearing on number of offspring born. In many pairshomozygous for seid, dystocia or maternal mortality before weaning of the 4th litter terminated thestudy.

Table 1. Males siring litters within 4 weeks of pairing

Genotype

No. ofmalestested

No. oflitters

produced

Intercross generation

scid/scid.bg/bg 38 80 0 6 7 8 6 11scid/scid. + /bg(?) 21 52 0 14* 2 5 0 0+ /scid(?).bg/bg 13 61 063400+ /scid(?). + /bg(?) 8 13 800000Total 80 206 8 26 12 17 6 11

*1 of 15 males tested did not produce offspring when paired with 3 different proven females.

Evaluation of pregnancy failure in seid/scid.bg/bg mice

To define whether the sizes of the litters born accurately measured reproductive performancesome pregnant females were killed for counts of conceptuses and corpora lutea. The number ofcorpora lutea corresponded well to the number of conceptuses (Table 3), indicating that littlepreimplantation loss had occurred. The ratio of dead:viable blastocysts and post-implantationconceptuses was about 1:10 for each of the various immunodeficiences (Table 3).

Table 2. Litter size by genotype and order of litter

MeanNo. of No. of Total offspring/

Generation Genotype females litters offspring litter

Mean offspring by sequenceof litter (range)

1st 2nd 3rd 4th

Gl

G2

G3

G4

G 5

+ /scid. + /bg

scid/scid.bg/bg

scid/scid. + /bg+ /scid.bg/bg+ /scid. + /bg

scid/scid.bg/bg

scid/scid. + /bg+ 1scid.bg/bg

scid/scid.bg/bg

scid/scid. + ¡bg+ /scid.bg/bg

scid/scid.bg/bg

10

4

6

13

9

13

9

6

9

13

5

4

25

6

12

25

13

34

14

14

22

26

22

7

177

49

87

201

111

233

90

89

145

150

142

56

7-4

8-2

7-3

8 0

8-5

6-9

6-4

6-4

6-6

7-5

6-5

80

7-7(3-10)

80(7-9)

6-7(5-9)

7-5(5-11)

8-6(6-11)

7-3(2-10)

6-8(4-9)

5-7(5-6)

7-2(4-12)

7-3(4-13)

6-8(4-13)

8-3(6-10)

6-5(2-11)

8-5(8-9)

7-3(4-9)

8-7(4-11)

8-5(5-12)

5-9(3-9)

5-8(2-7)

7-3(6-9)

6-8(5-9)

7-8(4-12)

5-8(2-9)

8-0(6-10)

70(5-8)

90(9)8-5

(5-12)

6-9(5-9)

60(5-7)

7-0(3-9)

60(4-8)

7-2(3-10)

7

6-7(5-9)

6-8(6-7)

70(5-9)

4-3(2-7)

8-3(5-11)

6-4(1-12)

Table 3. Conceptus success compared to numbers of corpora lutea

Genotype ofpregnant female

Before implantation

Conceptus

After implantation

ConceptusDead Viable CL Dead Viable

CL(litters)

scid/scid.bg/bgscid/scid. + ¡bg(?)+ /scid(?).bg/bg

9*12+9*

1013

1306937

137(18)74 (7)35 (4)

*Day 3-5 of gestation, 1 pregnancy per genotype.tDay 3-5 of gestation, 2 pregnancies.

Timing of embryonic differentiation in scid/scid.bg/bg mice

If cytokines released from maternal lymphocytes (Athanassakis et ai, 1987) or NK cells(Croy & Rossant, 1987) influence trophoblast cell function, the absence of immune recognition andcytokine release might result in slower development of embryos. Therefore the stage of develop¬ment of embryos resulting from matings of scid/scid.bg/bg and scid/scid.+ /bg(?) mice was

examined. From females of both genotypes morulae were obtained from the oviduct on Day 2-5 ofgestation and expanded blastocysts were flushed from uteri on Day 3-5 of gestation. Both stageswere enclosed in zonae pellucidae. From a single scid/scid.bg/bg animal studied later on Day 3 (i.e.3-75) 5 corpora lutea were detected and 5 hatched embryos were recovered that had elongated in

preparation for implantation, suggesting that implantation in the scid/scid.bg/bg uterus occurs lateon Day 3 to early Day 4. The number of cells present in zona-enclosed blastocysts was estimated bycounting nuclei. The mean cell number per blastocyst recovered from scid/scid.bg/bg females was62 (range 58-64). From a single scid/scid.+ /bg(?) female 8 late, elongated blastocytes wererecovered with a mean cell number of 72 (range 55-85). One to three metaphase plates were

observed per embryo. Dissection of 6-5-day implantation sites from scid/scid.bg/bg and scid/scid. +/bg(?) mice revealed a decidua-enclosed conceptus comprised of ectoplacental cone and earlyprimitive streak. Similar dissections on Day 7-5 of gestation revealed no differences in stage from7-5-day conceptuses from random-bred immunocompetent CD1 mice previously studied in our

laboratory (Crepeau et ai, 1989).

Morphology of implantation sites in scid/scid.bg/bg mice

The biological role of many cytokines appears to be the regulation of cellular differentiation(Williams et ai, 1988; Pollard et ai, 1987; Dedhar et ai, 1988; Bussolino et ai, 1989). If immunecytokines promote trophoblast cell differentiation rather than cell proliferation, the absence of animmune response could lead to anomalous differentiation of trophoblast and/or decidua, ratherthan to loss of conceptuses. A time course study of implantation sites in scid/scid.bg/bg andscid/scid.+ /bg(?) mice was therefore undertaken. Implantation sites were studied on Days 5-16of gestation. Progressive differentiation of a mature normal placenta was observed, includingtrophoblast giant cells, spongiotrophoblast and labyrinthine trophoblast (Fig. 1). Decidua andmetrial gland development occurred but these tissues appeared to lack normal cellularity; differen¬tiated granulated metrial gland (GMG) cells were prominent (Fig. lc, Id). Surprisingly, the granulesin the GMG cells of scid/scid.bg/bg mice observed by light microscopy did not appear of 'giant'size, as might be expected for mice carrying the bg/bg genotype in which neutrophils all containedvery large sudanophilic granules. This absence of giant granules has been confirmed by electronmicroscopy (Yamashiro et ai, 1989). Placentae of all mice examined seemed small, suggestingthat germ-free mice, rather than immunodeficient mice, might have small placentae. This was notsupported, however, by weighing separate implantation sites (Table 4).

Discussion

Mice of genotype scid/scid.bg/bg were bred to address the question of whether there is an essentialrequirement for immune recognition of the fetus during mammalian pregnancy. It was observedthat these doubly mutant animals were both viable and fertile, although they lacked serum

immunoglobulins, failed to respond to B-cell or T-cell mitogens, had giant neutrophil granules andcould not differentiate lymphokine-activated killer cells when stimulated by human recombinantinterleukin-2 in vitro (unpublished data). Male fertility was excellent and that of females exceededthe fertility reported for BALB/c mice, the congenie partner of C.B-17, and for C.B-17 scid/scidfemales raised in the parent colony (Clark et ai, 1989) and matched or exceeded the fertility knownfor C57BL/6J mice (Heininger & Dorey, 1980) and observed in the + /scid(?). + /bg(?) females. Noevidence was thus obtained to support the hypothesis that lymphocyte-deficient scid/scid.bg/bgfemales would have small litters. Furthermore, maternal immunocompetence would not haveenhanced reproductive performance since the sizes of litters born corresponded to ovulation rates,the frequency of résorptions was low and mature placentae from scid/scid.bg/bg females were notsmall when compared to those from matched timed pregnancies in immunocompetent mice.

Studies were therefore undertaken to address more subtle effects that an immunodeficiencymight have on either the timing of development or the differentiaton of placental cell types fromDays 2-16 of gestation. Both preimplantation and postimplantation stages developed at the rateexpected in immunocompetent animals (Hogan et ai, 1986). The number of cells present at the

Fig. 1. Photomicrographs of regions of a 14-5-day placenta, from a scid/scid.bg/bg female,stained with H + E; (a) labyrinthine trophoblast region showing sinusoids containing maternalblood (arrows), 340; (b) trophoblast giant cells (arrows) in a lateral placental area, 340; (c)decidua basalts, showing sparse cellularity but a prominent population of granulated metrialgland (GMG) cells, 136; (d) region of junction between the decidua basalis and metrial glandwith smooth muscle fibres (S) and characteristic large GMG cells (arrow), 340.

Table 4. Weight of conceptus sites, placentae and embryosMean weight (g)

Day of Implantationgestation Genotype No. site Placenta Embryo12 scid/scid.bg/bg 3 0-350 0-090 0-093

scid/scid. + /bg(?) 2 0-347 0-093 0-114CD1 3 0-419 0083 0120

14 scid/scid.bg/bg 3 0-670 0176 0-290scid/scid. + ¡bg(?) 3 0-730 0130 0-360CD1 3 0-813 0140 0-380

blastocyst stage was similar to that found in immunocompetent mice. In addition the cell popu¬lations expected to be present in the normal placenta were found, including giant cell trophoblast,spongiotrophoblast, labyrinthine trophoblast, decidua, metrial gland and GMG cells. Theacellularity of decidua left a meshwork appearance not unlike that identified as prominent stromalelements in the spleen, thymus and lymph nodes of C.B-17'-seid/seid mice (Custer et ai, 1985;Deschryver-Kecskemeti et ai, 1988). These morphological studies again indicate that a level ofplacental function at least sufficient to ensure pregnancy success occurs in the absence of a mature,functional immune system. The results of this study suggest either that the pregnancy immuno-trophism hypothesis is invalid or, more likely, that its framework is too narrow for considering therole of cytokines at the feto-maternal interface.

Maternally and fetally derived cytokines are present in the uterus and have been shown to bereleased from non-lymphoid cells including uterine epithelium and trophoblast (Pollard et ai,1987; Imakawa et ai, 1987). Further, there are several populations of bone marrow-derived cellspresent in the pregnant uterus that are believed to be immunocompetent but are not classicallydefined cells or NK cells. These include decidual cells (Lala et ai, 1983), macrophages (Redline &Lu, 1989) and GMG cells (Peel et ai, 1983), all of which are potential sources of cytokine growthfactors. Characterization of the cytokine production abilities of these cells and their regulation are

essential before consideration can be given to rejection of the pregnancy immunotrophism hypo¬thesis. We find GMG cells of particular interest. These bone marrow-derived cells differentiatefrom lymphoid-like precursor cells under the influence of progesterone and oestrogen (Peel et ai,1983), are readily detected in the murine uterus at Days 8-15 of gestation and release a complexcytokine mixture that includes colony stimulating factor-1, interleukin-1, leukaemia inhibitoryfactor (Williams et ai, 1988) and at least one other activity presently under characterization (Croyet ai, 1989). Studies of pregnancy in scid/scid.bg/bg mice will provide a unique comparison fordefining the activities of GMG cells and of other cell subsets in the pregnant uterus in an environ¬ment free from the regulatory influences of mature lymphocytes.

To date, the pregnancy immunotrophism hypothesis has focussed upon the effects of embryo-stimulated maternal lymphokines on trophoblast cells. It is important to consider that cytokinesmay also originate from the embryo (Imakawa et ai, 1987) and, regardless of their origin, haveimportant maternal effects. Studies with human umbilical cord endothelial cell cultures demon¬strated that both recombinant human G- and GM-CSF induced endothelial cell migration anddivision (Bussolino et ai, 1989). These activities would seem essential during differentiation of themature placenta and may represent the important and overlapping cytokine effects at the feto-maternal interface that have not been eliminated in the scid/scid.bg/bg mice.

This work was supported by awards from the Natural Sciences and Engineering Council ofCanada, the Ontario Ministry of Agriculture and Food and The University of Guelph. We thankMiss Nancy Reed for technical assistance; Mr Tony Cengija and Mr Will Wistowsky for dedicated

care in handling of the immunodeficient mice; Mrs Jean Claxton and Dr T. Lumsden for advice onsudan black staining; Dr A. King on enumeration of blastocyst nuclei; and Dr S. Yamashiro forhelp with preparation of the photomicrographs.

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Received 2 May 1989