Induction of embryonic major histocompatibility complex antigen expression by γ-IFN

Journal of Reproductive Immunology, 24 (1993) 111-121

111

Elsevier Scientific Publishers Ireland Ltd.

JRI 00825

Induction of embryonic major histocompatibility complex antigen expression by 7-IFN C a r o l M. W a r n e r , C a t h a r i n e D. A l m q u i s t , M o h a m e d H. T o u l i m a t a n d Yuanxin Xu Department of Biology, 414 Mugar Hall, Northeastern University, Boston, MA 02115 (USA) (Accepted for publication 9 March 1993)

Summary Preimplantation mouse embryos were incubated in vitro with mouse recombinant 7-interferon (IFN). The effect of the 7-IFN on major histocompatibility complex (MHC) class I antigen expression was tested using an ELISA procedure. It was found that there is a doubling of D b antigens and a tripling of Qa-2 antigens on C57BL/6 mouse embryos cultured from the 8-cell stage for 24 h in the presence of 105 units/ml 7-IFN. The effect of 7-IFN on the rate of preimplantation embryonic development was tested by culturing 2-cell embryos for 48 h and 8-cell embryos for •24 h in the presence of varying concentrations of 7-IFN up to 10 6 units/ml. Two methods were used to assess the cell number per embryo after the culture period: incorporation of [3H]thymidine into DNA, and direct counting of nuclei in fixed and stained embryos. Both methods showed that treatment with 3,-IFN increases the rate of development of preimplantation mouse embryos. Since rate of preimplantation embryonic development is genetically controlled by the Ped gene, it is suggested that 7-IFN has a direct effect on the Ped gene phenotype of preimplantation mouse embryos.

Key words: mouse major histocompatibility complex; 7-interferon; Ped gene

Correspondence to: Carol M. Warner, Department of Biology, 414 Mugar Hall, Northeastern University, Boston, MA 02115, USA. 0165-0378/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Introduction

Since the discovery of the major histocompatibility complex (MHC) and transplant rejection, immunologists have been puzzled by the fact that fetal tissue evades this phenonmenon. For years it was not clear whether or not preimplantation embryos express MHC antigens. Many studies have now shown that preimplantation mouse embryos do indeed express both classical (K,D,L) and nonclassical (Qa-2) class I MHC antigens (reviewed in Warner et al., 1988). Techniques used for MHC antigen detection have included electron microscopy (Searle et al., 1976; Warner and Spannaus, 1984), [125I]lactoperoxidase labeling (Webb et al., 1977); complement dependent cytotoxicity (Krco and Goldberg, 1977; Cozad and Warner, 1982) cell mediated cytotoxicity (Ewoldsen et al., 1987), and an enzyme linked immunosorbent assay (ELISA) procedure (Goldbard et al., 1984; Warner et al., 1987). Recently, using both slot-blot hybridization and the reverse-transcription polymerase chain reaction (RT-PCR), mRNA for class I MHC antigens has been detected in preimplantation mouse embryos (Arcellana-Panlilio and Schultz, 1991; Jin et al., 1992). Thus, class I MHC genes are actively transcribed and translated in preimplantation mouse embryos. Lindahl et al. (1973) were the first to observe the induction of MHC proteins by interferon (IFN). Cells which express class I MHC antigens have the amount of antigen increased by treatment with 7-IFN if they express the IFN receptor and if the MHC genes contain an IFN response sequence (IRS) (Singer and Maguire, 1990). The enhancement of MHC proteins by interferons has been found on virtually every cell type, both marrow-derived and non-marrow derived (reviewed in Halloran et al. 1986). Marrow-derived cells which have been shown to increase expression of MHC proteins in this manner include T cells, B cells, macrophages, monocytes, Langerhans cells, and mast cells. Non-marrow derived cells which have been shown to increase expression of MHC proteins in response to interferons in culture include those derived from heart, kidney, colon, pancreas, bladder, lung, ovary, brain, thyroid, and many melanomas. Little is known regarding the effects of 7-IFN on embryo development. Drasner et al. (1979) found no effects on development or tritiated thymidine ([SH]TdR) incorporation by 2-cell and 8-cell mouse embryos maintained in vitro in the presence of 10 3 units/ml of IFN. Carthew et al. (1986) confirmed these results for 2-cell mouse embryos cultured for 72 h in the presence of 102 units/ml of IFN. Neither of these studies utilized recombinant IFN or a range of IFN concentrations. Hill et al. (1987) did use recombinant IFN at varying concentrations but it was of human and rat, not mouse, origin. They found that 10 6 units/ml of human or rat IFN was toxic to mouse embryos. However, it is generally accepted that 3,-IFN does not bind to IFN receptors across species, so the observed toxic effects must be non-specific.

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The present studies were undertaken to test the effects of varying concentrations of mouse recombinant 3,-IFN on class I MHC antigen expression by preimplantation mouse embryos. In addition, the effect of mouse recombinant 3,-IFN on the rate of embryonic development was testedl Materials and Methods

Mice and embryos C57BL/6J mice (H-2 b, Qa-2 positive, Pedfast) were originally purchased from the Jackson Laboratory, Bar Harbor, ME and bred in our laboratory. The mice were housed in a day-night cycled room (lights on 0400-1800 h standard time) with food and water ad libitum. Female mice were superovulated with 5 I.U. eCG (Sigma, St. Louis, MO), injected at the 9th hour of the light cycle, followed 48 h later by 10 I.U. hCG (ICN BioMedicals, Inc., Costa Mesa, CA). One female was placed with a single male immediately following hCG injection and vaginal plugs checked the next morning. Plug positive mice were sacrificed 41-47 h post-hCG injection for the 2-cell stage and 65-69 h post-hCG injection for the 8-cell stage.

Culture of embryos The embryos were collected in Whitten and Biggers (1968) medium, washed, and prepared for culture in the same medium. Embryos were cultured for 48 h from the 2-cell stage or for 24 h from the 8-cell stage either by placing them in microdrops under oil or by placing them in flat-bottomed microtiter plates. All incubations were at 37°C in 5-7% CO2 in air. To obtain the desired concentration of 3,-IFN, double strength 3'-IFN was mixed with an equal volume of double strength Whitten and Biggers medium.

Cell lines and antibodies The WEHI-3 cell line (H-2 d) was purchased from the ATCC (American Type Culture Collection, Rockville, MD) and used as a positive control since this cell line is known to increase the amount of cell surface class I MHC antigen in response to 3,-IFN (McNicholas et al., 1983). The monoclonal antibody (McAb) producing cell line used to detect H-2 antigens on the WEHI-3 cells and on the C57BL/6 embryos was the mouse McAb 27-11-13 (anti-H-2Db/d), provided by Dr. David Sachs (Ozato and Sachs, 1981). As a negative control a mouse McAb, TIB 109 (purchased from the ATCC), to sheep red blood cells (SRBC), an antigen known not to be expressed on either WEHI-3 cells or C57BL/6 embryos, was used. A mouse McAb specific for Qa-2 antigen, 141.16.6, was provided by Dr. Ian McKenzie (Hogarth et al., 1982).

"r-Interferon Mouse recombinant 3,-IFN was obtained from two sources. The first

114

source was a gift from Dr. Michael Shepherd, Genentech, Inc., San Francisco CA. It was at a concentration of 2.3 × 10 7 units/ml. The second source was commercially available material from Amgen, Thousand Oaks, CA, at a concentration of _ 107units/ml.

Fluorescence assay for the detection of cell surface MHC antigens on cells WEHI-3 cells were analyzed for expression of Class I MHC molecules by a procedure modified from that described by McNicholas et al. (1983). Cells were cultured for 72 h in the absence or presence of 25-100 units/ml of murine recombinant ~,-IFN. Cells were washed three times in R P M I 1640 medium (GIBCO/BRL) supplemented with 0.1% sodium azide and then placed in a 96-well V-bottom microtiter plate at a concentration of 2.5 × 105 cells/well in a volume of 50 tA. Then 50/~1 of appropriate McAb diluted in RPMI 1640 were added to each well as the first antibody and the plate incubated at room temperature for 30 min. After the incubation the plate was washed three times with RPMI 1640 and the cells resuspended in 50 t~l of fresh medium. Then 50 t~l of the appropriate dilution of the second antibody, FITC-conjugated goat antimouse IgG antiserum (ICN), were added to each well and the plate incubated at room temperature for 30 min. The plate was washed as before. The cells were analyzed on a flow cytometer (either EPICS 752 or FACScan). A standard curve was created by analyzing FITCconjugated beads of known fluorescence intensity on a logarithmic scale.

ELISA procedure for the detection of cell surface MHC antigens on embryos Mouse embryos were subjected to the ELISA procedure, as previously described (Goldbard et al., 1984; Tian et al., 1992), to detect cell surface MHC antigens in the absense or presence of 3,-IFN. At the end of the incubation period the embryos were washed three times in a modified PBS (10% heat inactivated FCS and 0.1% sodium azide) and placed in 200 #1 of a 1:8 dilution of the appropriate first McAb diluted in the modified PBS. The embryos were incubated for 2 h at 37°C in 7% CO2 in air and then washed five times in the modified PBS. Embryos were then incubated with the second antibody, sheep anti-mouse IgG F(ab')2 fragments conjugated to /3galactosidase (BRL, Gaithersburg, MD) diluted 1:25 in the modified PBS. After a 1-h incubation the embryos were again washed five times as described above. The embryos were then placed, at least 15 per well, in 100 #1 of freshly prepared substrate solution [4 mg/ml p-nitrophenyl /3-D-galactopyranoside (Sigma), 7 t~l/3-mercaptoethanol, in assay buffer] in a 96 well Immulon I microtitration plate (Dynatech, Alexandria, VA). The plates were incubated 3-4 h at 37°C. The reaction was stopped by the addition of 1.0 M Na2CO3. The absorbance at 410 nm of each well was read on an ELISA plate reader (Dynatech).

115

Determination of embryonic cell number Two methods were used to determine the number of cells per embryo after culturing the embryos with 7-IFN. In the first, incorporation of [3H]TdR into cultured mouse embryos was used as a direct measure of the number of cells per embryo (Cozad et al., 1981). A working stock of [3H]TdR was made by diluting methyl-labeled [3H]TdR (20 Ci/mmol, Dupont/New England Nuclear) with an equal volume of double strength Whitten and Biggers medium. This was further diluted with Whitten and Biggers medium to give a concentration of 8/zCi/ml. Fifty microliters of Whitten and Biggers medium were added to the wells of a 96-well flat-bottomed microtiter plate, followed by 5-20 embryos in as small a volume of medium as possible (< 1 t~l). Then 50 #1 of the 8/~Ci/ml [3H]TdR were added to give a final concentration of 4 #Ci/ml. The plates were incubated at 37°C for 3-5 h. The reaction was stopped by placing the plates on ice and the contents of the wells were harvested onto filter paper using a Titertek cell harvestor. The filters were dried in a 65°C drying oven, placed in scintillation vials with toluene fluor, and counted in a liquid scintillation counter. The second method used to assess the number of cells per embryo after culturing with or without 7-IFN was direct counting of the nuclei after fixing and staining (Goldbard et al., 1982).

Statistical analysis of data The Student's t-test was used to compare the means of the different sample populations. All the experimental groups (presence of 7-IFN) were compared to the controls (absence of T-IFN). The degrees of freedom were based on the number of experiments for the ELISA results and the [3H]TdR incorporation assays. For the nuclei staining assays the degrees of freedom were based on the number of embryos scored. Results and Discussion

Mouse recombinant 3,-IFN increases the expression of class I MHC antigens on preimplantation mouse embryos and also increases their rate of development. In the first set of experiments the efficacy of the 3,-IFN was tested on WEHI-3 cells. The results (Fig. 1) show that both the Genentech and Amgen 7-IFNs gave similar results. At 50 units/ml, the approximately 20-fold increase in the amount of class I MHC antigen on the WEHI-3 cells was similar to that reported by others (McNicholas et al., 1983). Next, the effect of the Genentech 7-IFN on the expression of class I MHC antigens on mouse embryos was tested. First, embryo development-was assessed in the presence of varying concentrations of 7-IFN up to 10 6 units/ml. There were no apparent toxic effects at any of these concentrations

116

200 (,.) C o u)

175

0

rr e¢-

150

0 e-

!

125

N 100

2

3

y-IFN Treatment

y-IFN (U/ml)

MCF

0

125

1

[]

None

2

~

Genentech

50

170

3

[]

Amgen

25

117

4

~

Amgen

50

159

5

[]

Amgen

100

190

Fig. 1. Effect of T-IFN on MHC class I expression by WEHI-3 cells. Mean Channel Fluorescence (MCF) is on a logarithmic scale.

so a concentration of 105 units/ml was chosen to test the effect of the 3,-IFN on MHC class I antigen expression. The results (Table 1) show that incubation of 8-cell embryos for 24 h with 3,-IFN causes a doubling of D b antigen expression and a tripling of Qa-2 antigen expression. The implication is that preimplantation mouse embryos express receptors for 3,-IFN and that the embryonic MHC genes contain the IRS element. These results are particularly interesting in light of a recent report which shows that mice repeatedly injected in vivo with 3,-IFN show a high rate of resorptions at day 12.5 of gestation (Mattsson et al., 1992). Moreover, the same phenomenon may be found in humans, based on a recent study which suggests that 3,-IFN may cause recurrent spontaneous abortion in women (Hill et al., 1992). The results showing an increase in class I MHC antigen expression prompted us to test whether this increase would be accompanied by an increase

117

TABLE 1 Effect of 3,-IFN on M H C class I antigen expression by preimplantation mouse embryos, a Concentration of 3'-IFN (units/ml)

McAb

No. embryos

No. expts.

Mean A4I 0 nm ± S.D.

0 (Control) 105 0 (Control) 105 0 (Control) 105 0 (Control) 105

cx-SRBC ot-SRBC c~-Db t~-D b c~-SRBC a-SRBC ct-Qa-2 c~-Qa-2

45 45 45 45 30 30 30 30

3 3 3 3 2 2 2 2

0.11 0.10 0.21 0.37 0.04 0.06 0.54 1.55

± ± + ± ± ± ± ±

P

0.01 0.01 0.00 0.02 0.01 0.02 0.04 0.10

N.S. < 0.05 N.S. < 0.01

aEight-cell mouse embryos were incubated with or without (control) ~/-IFN for 24 h and cell surface M H C antigen expression assessed by the ELISA procedure, as described in the text.

in the rate of development of the embryos. In the first set of experiments 8cell embryos were cultured for 24 h with varying concentrations of the Genentech 3,-IFN. The number of cells per embryo was assessed both by incorporation of [3H]TdR (Table 2) and direct counting of stained nuclei (Table 3). The results from both methods show a statisically significant increase in the rate of development of the embryos in the presence of 105 and 10 6 units/ml of 3,-IFN. In the second set of experiments, the embryos were collected at the 2-cell stage and incubated for 48 h with the Genentech 3,-IFN. Again (Tables 4 and 5) there was a significant increase in the rate of development with 105 and 10 6 units/ml of 3,-IFN.

TABLE 2 Effect of Genentech ~-IFN on rate of development (counts/min/embryo per hour) of 8-cell embryos cultured for 24 h. a Concentration of )'-IFN (units/ml)

No. of embryos

No. of experiments

count/min/embryo per h ± S.D.

0 (Control) 103 104 105 106

15 15 15 15 15

3 3 3 3 3

131 135 141 147 151

± ± ± ± ±

6 2 4 2 1

P

N.S. N.S. < 0.05 < 0.01

aRate of development was assessed by incorporation of [ 3H]thymidine, as described in the text. P values from Student's t-test.

118 TABLE 3 Effect of Genentech 3,-IFN on rate of development (cell number per embryo) of 8-cell embryos cultured for 24 h. a Concentration of 3,-IFN (units/ml)

No. embryos

Cell number per embryo (S.E.M.)

0 (Control) 103 104 105 106

17 16 15 15 15

31.4 30.8 33.3 36.2 36.1

P

(1.4) (1.6) (1.5) (1.3) (0.8)

N.S. N.S. <0.05 <0.05

aRate of development was assessed by direct counting of cell nuclei, as described in the text. P values from Student's t-test.

TABLE 4 Effect of Genentech "t-IFN on rate of development (counts/min/embryo per hour) of 2-cell embryos cultured for 48 h.a Concentration of 3,-IFN (units/ml)

No. embryos

No. experiments

counts/min/embryo per h ± S.D.

0 (Control) 103 104 105 106

15 15 15 15 15

3 3 3 3 3

84 80 92 105 125

± ± ± ± ±

11 11 9 2 3

P

N.S. N.S. <0.05 <0.01

aRate of development was assessed by incorporation of [3H]thymidine, as described in the text. P values from Student's t-test.

TABLE 5 Effect of Genentech "r-IFN on rate of development (cell number per embryo) of 2-cell embryos cultured for 48 h.a Concentration of ~'-IFN (units/ml)

Number of embryos

Cell number per embryo (S.E.M.)

0 (Control) 103 104 105 l06

17 18 16 15 17

26.3 25.8 26.4 30.2 3",7

(1.4) (1.2) (1.4) (1.2) (1.2)

P

N.S. N.S. <0.05 <0.01

aRate of development was assessed by direct counting of cell nuclei, as described in the text. P values from Student's t-test.

119 TABLE 6 Effect of Amgen -¢-IFN on rate of development (counts/min/embryo per hour) of 2-cell embryos cultured for 48 h. a Concentration of ~,-IFN (units/ml)

No. embryos

No. experiments

counts/min/embryo per h ± S.D.

0 (Control) 101 102 103 104

56 60 25 56 30

6 6 2 6 2

42 47 75 63 66

± ± ± ± ±

12 7 2 9 7

P

N.S. <0.01 <0.01 <0.05

aRate of development was assessed by incorporation of [3H]thymidine, as described in the text. P values from Student's t-test.

Finally, a third set of experiments was performed with the Amgen 3,-IFN. Preliminary experiments showed that the Amgen ~,-IFN was toxic to the embryos at 105 units/ml. The most reasonable explanation is that the different buffer in the Amgen IFN compared to the Genentech IFN caused the toxicity. Therefore, two-cell embryos were incubated for 48 h with the Amgen 3,-IFN only at concentrations up to 104 units/ml. The results are shown in Tables 6 and 7. These experiments showed a statistically significant increase in cell number after incubation with the Amgen 3,-IFN. Although the increase is noted at lower concentrations with the Amgen 3,-IFN than with the Genentech 3,-IFN, it should be noted (Tables 2-5) that there is a trend in the direction of an increase with the Genentech material even though the results are not statistically significant at the lower concentrations. Overall, the results with both interferons show that 3~-IFN causes an increase in the rate of development of preimplantation mouse embryos.

TABLE 7 Effect of Amgen 3,-IFN on rate of development (cell number/embryo) of 2-cell embryos cultured for 48 h. a Concentration of ~,-IFN (units/ml)

No. embryos

Cell number per embryo (S.E.M.)

0 (Control) 103 104

27 42 29

16.6 (1.1) 17.8 (0.8) 21.2 (1.3)

P

N.S. <0.01

aRate of development was assessed by direct counting of cell nuclei, as described in the text. P values from Student's t-test.

120

The large increase in Qa-2 antigen expression in response to 7-IFN merits further discussion. It has been hypothesized that Qa-2 antigen is the product of the Ped (Preimplantation embryo development) gene (reviewed in Warner et al., 1988). The Ped gene, which maps to the Q region of the mouse MHC on chromosome 17, controls the rate of preimplantation embryonic cleavage division and subsequent embryo survival (Warner et al., 1991). We have recently demonstrated that removal of Qa-2 antigen from the embryonic cell surface slows down the rate of cleavage division (Tian et al., 1992). Therefore, it seemed logical that increasing the amount of Qa-2 antigen might increase the rate of preimplantation embryonic development. The experiments reported in this paper show that indeed this is the case. Qa-2 antigen is encoded by four different Q region genes: Q6, Q7, Q8, and Q9. It remains to be determined which of these genes is responsible for the increase in embryonic Qa-2 antigen expression in response to 7-IFN. The mechanisms by which the increase in the levels of cell surface MHC proteins might influence the rate of cleavage division of preimplantation embryos remain to be determined. Class I MHC antigens have a groove to which peptide binds (Matsumura et al., 1992). It would be interesting if the binding of a particular peptide to embryonic cell surface class I MHC antigens could modulate the rate of cell division.

Acknowledgements This work was supported by NIH grant HD 13748.

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