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Immunological studies of mouse decidual cells. II. Studies of cells in artificially induced decidua
Journal of Reproductive Immunology, 3 (1981) 41-48 Elsevier/North-Holland Biomedical Press
41
IMMUNOLOGICAL STUDIES OF MOUSE DECIDUAL CELLS. II. STUDIES OF CELLS IN A R T I F I C I A L L Y INDUCED DECIDUA
FLORE RACHMAN 1, OLIVIER BERNARD 1, MARGRIT P. SCHEID 2 and DOROTHEA BENNETT 2 1 Clinique de Pddiatrie and Unitd de Recherche d 'Hdpatologie Infantile [INSERM U 56], Hdpital d'Enfants, 94270, Kremlin Bicdtre, France, 2 Memorial Sloan-Kettering Cancer Center, New York, N Y 10021, U.S.A.
(Received 26 July 1980; revised 15 September; accepted 10 October)
Cells from artificially induced decidual tissue (deciduoma) in the mouse were examined for Thy-1 surface antigen and receptors for the Fc portion of immunoglobulin G (FcR) and compared with cells of the normal decidua from day 6 to day 9 of pregnancy. It was shown that (1) Thy-1 antigen is present on the same proportion of cells in decidua and deciduoma on day 6 and day 7, (2) FcR-bearing cells can be detected in similar numbers on day 6 and day 7 but this does not increase on day 8 in deciduoma as it does in decidua, and (3) progesterone treatment after induction of decidualization allowed further increase of FcR-bearing cells in deciduoma. These results present further evidence of the similarity between deciduoma and decidua in the mouse. They indicate that these two membrane markers are present in the early decidua, regardless of the presence of an embryo, and suggest that progesterone may play a part in the increase of FcR-bearing cells in the decidua during pregnancy.
INTRODUCTION In mammals, the close relationships occurring after implantation between the embryo and the maternal uterine tissues raise several questions concerning (1) how the embryo is not rejected as an allograft by the mother (Beer and Billingham, 1976), and ( 2 ) h o w the mother prevents excessive invasion o f the uterus b y trophoblastic cells that possess considerable invasive and phagocytic properties at that time (Kirby, 1963). The decidual tissue developing in the uterus at the time of implantation may play a protective role in both respects (Kirby et al., 1966; Kirby and Cowell, 1968; Beer and BiUingham, 1974). Although it is clear that maternal immunoglobulins, decidual and trophoblastic cells are in close association from the time o f implantation onwards (Bernard et al., 1977), little is known of the mechanisms controlling, in the decidua, the discrete balance between excessive invasion by the trophoblast and rejection o f the embryo by the mother. Previous studies o f cells o f the mouse decidua in the days after implantation have shown that no significant numbers o f B and T lymphocytes can be detected, although a majority of cells express Thy-1 antigen, presumably reflecting their fibroblastic origin. In addition, an increasing number o f cells bearing surface receptors for IgG (FcR) are present from day 6 to day 8 o f gestation (Bernard et al., 1978). In mice and rat uteri, a decidual reaction (deciduoma) can be artificially induced in the 0165-0378/81/0000-0000/$02.50 © Elsevier/North-HoUand Biomedical Press
42 absence of an embryo and has proved a useful tool in analyzing the differentiation of the decidua and its hormonal control (for review, see Finn and Porter, 1975). In this paper, we report the results of an immunological study of the cells present in mouse deciduoma as compared with the cells in the decidua during a normal pregnancy. The results indicate that both Thy-l-bearing cells and FcR-bearing cells are also present in deciduoma, and suggest tha.t progesterone may play a part in controlling the increasing amount of FcRbearing cells in the decidua as pregnancy proceeds. MATERIALS AND METHODS
Mice Inbred C57B1/6(B6) and AKR/J mice, and random-bred CF1 and ICR albino mice were obtained as previously described (Bernard et al., 1978). Inbred and outbred matings were set up with normal or vasectomized males, and the day a vaginal plug was found was considered day 0 of pregnancy or pseudo-pregnancy. Induction of deciduoma Virgin female mice ( 8 - 1 0 weeks old) were placed overnight with vasectomized males. On the third day of pseudo-pregnancy, females were anaesthetized with tribromoethanol (Avertin) and 4 - 0 silk threads were placed at three different levels transversally through each uterine horn. From day 6 of pseudo-pregnancy, deciduomata appear as hypertrophic areas spread along the uterine horns, and centered by the threads left in situ. The lifespan of the mouse deciduoma is limited and by day 9 it is undergoing necrosis. Regression of deciduoma can, however, be delayed for a few days by treatment with progesterone (Atkinson, 1944). To study the possible influence of progesterone on FcR bearing-cells in deciduoma, 0.2 ml of a 7.5 mg/ml progesterone solution in sesame off were injected daffy subcutaneously from the day of trauma onwards. Control mice, in which decidualization also had been induced, received the same amount of sesame oil without progesterone. Preparation of cells from decidua and deciduoma Females pregnant for the first time, or females in which deciduomata had been artificially induced, were killed by cervical dislocation at 6, 7, 8 and 9 days of pregnancy or pseudo-pregnancy. Decidual capsules were dissected out of the myometrium in phosphate-buffered saline (PBS) and the embryo and its immediate surrounding tissues (including the chorioallantoic placenta on day 9) were carefully removed. Deciduomata were dissected similarly and the remnants of uterine epithelium were removed; absence of an embryo was checked in each case. Cell suspensions were obtained from decidua or deciduoma using a slight modification of the procedure already described (Bernard et al., 1978). Briefly, pieces of tissue were chopped in medium and incubated in 1 mg/ml collagenase type II (Sigma Chemical Co., St. Louis, Mo.), then washed twice in Ca 2+- and Mg2*-f~eeDulbecco's PBS (Grand Island Biological Co., Grand Island, N.Y.) with 0.3% fructose and 0.3% dextrose, and incubated for 4 periods of 5 min each in 1 mg/ml trypsin (1 : 250, Difco Laboratories, Detroit, Mich.) in the same Ca 2+- and Mg2÷-free medium. Cells from each trypsin incubation were
43 recovered after pipetting, treated as described previously, and their viability assessed by trypan blue exclusion. All four fractions contained over 90% viable cells and were pooled for further studies. However, on day 9 of pseudo-pregnancy the viability of cells prepared from deciduomata undergoing necrosis was so poor (averaging 15-20%) that no study could be carried out at that stage.
Other procedures Anti-Thy-l.2 serum, complement-mediated cytotoxicity, detection of surface immunoglobulins and E rosette (with non-sensitized sheep red cells) and EA rosette (7S antibody-coated sheep red cells) studies were carried out as previously described (Bernard et al., 1978). Each test was performed using cells prepared from pooling all decidual or deciduomal capsules from one mouse. Student's t-test was used for statistical analysis. RESULTS
Cell suspensions from deciduas and deciduomata Mean cell yields per decidual or deciduomal capsule are indicated in Fig. 1. Mean cell yield per capsule was 8.7 _+0.4 X l0 s on day 9 of pregnancy, but could not be stated precisely at that stage in the deciduoma from mice treated with progesterone since capsules were very large and sometimes fused. Red blood cell (RBC) counts were always less than two RBC per nucleated cell, making contamination by blood nucleated cells quite minimal.
12
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E=4
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8
Days of pregnancy or pseudopregnancy Fig. 1. Number of ceils recovered per decidual or deciduomal capsule from day 6 to day 8 of pregnancy or pseudo-pregnancy in outbred albino female mice. Mean number of cells X10 5 and standard deviations. ~, decidua; ~, deciduoma; l , deciduoma with progesterone.
44
Thy-1 antigen Anti-Thy-1.2 antiserum was used in complement-dependent cytotoxicity tests on cells from deciduas and deciduomata from B6 (Thy-1.2) and AKR/J (Thy-l.1) females on day 6 and day 7 of pregnancy or pseudo-pregnancy. In B6 mice, Thy-l.2-bearing cells can be found in similar amounts in both decidua and deciduoma. Fig. 2 shows the results of tests carried out on day 6 where the cytotoxic index is 0.6 at 1 : 100 dilution in both cases. No significant activity could be detected against AKR/J deciduoma cells. In B6 mice, the morphology of cells killed by anti-Thy-1.2 serum was the same in decidua and deciduoma, made up mostly of medium size or large, granular, often bi- or multi-nucleated cells. Surface immunoglobulins [sIg) In cell suspensions from deciduas and deciduomata from outbred albino mice, no slgbearing cells could be detected, under the same conditions where 40% of spleen cells and less than 1% of thymocytes are labelled by sheep Fab anti-mouse Ig coupled with peroxidase. Previous data showed that the enzymatic treatment we used did not alter the detection of sIg-bearing cells in the spleen (Bernard et al., 1978). Fc receptors on cells in deciduoma Studies of EA rosette-forming cells were carried out from day 6 to day 9 in randombred albino mice, on decidua, deciduoma and deciduoma from mice treated with progesterone. At least three separate experiments were done at each stage. Fig. 3 confirms that the mean percentage of EA rosette-forming cells increases significantly in the course of a normal pregnancy from 6% on day 6 to 10% on day 7 (P < 0.05) and from 10% on day 7 to 17.8 and 17% on day 8 (P < 0.05) and day 9 (P < 0.05) respectively. In deciduoma, FcR-bearing cells can be detected in amounts similar to those in decidua on day 6 and
O.7 O...--- O
06
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0.3
~ N
S 02
o~-.-° 0.1
o~
~o~
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50
\
\
...~..~.0~ \A
100 250 500 1000 2000 1 / Dilution of Antiserum
Fig. 2. Titration of anti-Thy-t.2 serum: on 6-day B6 deciduomal cells (-), on 6-day AKR/J deci-
duomal cells (o), and on 6-day B6 decidual ceils (A).
45
30 25
!
8 20 E
T
6
7
it 8
9
Days of pregnancy or pseudopregnancy
Fig. 3. Percentage EA rosette-forming cells in outbred mouse decidua and deciduoma from day 6 to day 9 of pregnancy or pseudo-pregnancy(mean figures and standard deviations). Controls using unsensitized SRBC (E rosettes)were all negative. ~, decidua;r~, deciduoma; m, deciduomawith progesterone. Mean figures correspond to the mean of the percentages of EA rosette-forming cells in at least three tests for each stage.
day 7, but on day 8 the amount of EA rosette-forming cells does not increase further and remains equal to that on day 7. On day 9 no study could be performed in the deciduoma undergoing necrosis. Daily treatment with 1.5 mg of progesterone from the day of trauma onwards alters somewhat the amount of FcR-bearing cells in deciduoma. While on day 6 and day 7 the mean level of EA rosette-forming cells remains stable, it significantly increases thereafter, reaching 11.3 and 23% of deciduoma cells on day 8 ( P < 0.05) and day 9 ( P < 0.05), respectively. FcR-bearing cells in mice treated with sesame oil instead of progesterone followed a pattern similar to that in untreated mice (data not shown). EA rosettes in deciduomata were similar in size and appearance to those in deciduas. DISCUSSION The results presented here indicate that in mouse deciduoma, as in mouse decidua, Thy-1 antigen can be detected on a large fraction of cells on day 6 and day 7 of pseudopregnancy, and that FcR-bearing cells are present in similar amounts at the same time. However, in deciduoma the amount of FcR-bearing cells does not increase after day 7 as in decidua, unless mice are treated with progesterone from the day of trauma onwards. These findings can be discussed from three viewpoints: (1) they provide further data to support the analogies between decidua and deciduoma, (2) they stress that the presence of the two cell surface markers studied on cells in the decidua is actually independent of
46 the presence of an embryo, and (3) they suggest a hormonal control for the level of FcRbearing cells in the deciduoma and in the decidua. Further analogies between deciduoma and decidua Several studies have shown that early decidua and deciduoma, in rats and mice, share several features. Morphological, histochemical and autoradiographic observations show that early decidual and deciduomal cells derive mostly from uterine stromal cells and their evolution from this stage to the differentiated, often bi- or multi-nucleated decidual cells has been documented in both normal and pseudo-pregnancy (Krehbiel, 1937; Finn and Hinchliffe, 1964; Galassi, 1968). The present data provide further evidence regarding this analogy. In both tissues, Thy-1 antigen is present on a comparable fraction of cells on day 6 and day 7 of pregnancy or pseudo-pregnancy. As previously discussed (Bernard et al., 1978), Thy-1 antigen probably reflects the fibroblastic origin of the majority of cells in decidua and deciduoma (Stern, 1973). Similarly, FcR-bearing cells are present in the same proportion in the early (6 and 7 day) decidua as well as in deciduoma. Although the origin and role of these cells are unknown, their presence indicates that similarities between early decidua and deciduoma exist not only on morphological grounds but also from a functional viewpoint. Cell surface markers detected on cells in the decidua are independent o f the presence of a n embryo The presence of an increasing number of FoR-bearing cells in the early decidua during a normal pregnancy could be accounted for by contamination with cells of embryonic origin: most early trophoblastic cells contain maternal immunoglobulin (Bernard et al., 1977) and the presence of FoR on trophoblastic cells has been reported at later stages of pregnancy in mice and humans (Elson et al., 1975; Jenkinson et al., 1976). It could thus be argued that cells described as bearing FcR in the decidua are, at least partly, embryonic in origin. Data presented here rule out this possibility on day 6 and day 7 since the same amounts of FcR-bearing cells are present in decidua and deciduoma, the latter in the absence of any embryo. As far as day 8 and day 9 are concerned, it is also unlikely that the increase in FcR-bearing cells in decidua is due to embryonic cells. Thus, lack of increase of FcR-bearing cells in deciduoma on day 8 rather corresponds to an early sign of regression of deciduoma, while the temporary 'rescue' of deciduoma in progesteronetreated mice is in fact paralleled by an increase in FcR-bearing cells. It is quite possible that, in untreated mice, the absence of an embryo and/or of the corresponding luteotrophic placental factors (see McCormack and Greenwald, 1974), or even the lack of production of progesterone by the placenta, as in the rat (Sanyal, 1978), is actually responsible for both the regression of deciduoma and the lack of increase in FoR-bearing cells. The results presented here also show that the expression of Thy-1 antigen on decidual cells does not depend on the presence of an embryo. It is, however, still not known whether Thy-1 antigen on decidual cells results from the persistence of a membrane antigen already present on uterine stromal cells before decidualization or if it is the result of a particular step in the differentiation of decidual cells. Studies of Thy-1 antigen expression during in vitro decidualization (Vladimirsky et al., 1977; Sananes et al., 1978)
47 should make it possible to answer this question and also to study the regulation o f Thy-1 expression under various hormonal conditions.
Possible endocrine control o f FcR-bearing cells in the decidua The present results show that an increase in FcR-bearing cells in 8- and 9-day deciduoma can be obtained when regression of deciduoma is prevented by progesterone treatment. In pregnant rats, progesterone is necessary to maintain normal development of the decidua in the days after implantation (Deanesley, 1973). It is therefore tempting to suggest that in the pregnant mouse the presence of FcR-bearing cells in the decidua is under the control of progesterone. In the mouse, plasma progesterone levels are known to remain fairly high from day 5 to day 10 and to grow even higher in the second half of pregnancy (McCormack and Greenwald, 1974; Murr et al., 1974). FcR-bearing cells are in fact present, in even greater amounts, at later stages in the rat metrial gland (Bray et al., 1978), and it may be that at least some of these cells fulfdl the same role and are under the same hormonal control as those present in the mouse decidua at earlier stages. However, it is still not clear whether FcR-bearing cells are differentiating within the uterus as a part of the decidua or deciduoma, possibly also from fibroblasts (Bourgois et al., 1977), or if they are migrating into the decidua and the deciduoma in the postimplantation period. Nor can the data we report confirm that FcR-bearing cells in decidua and deciduoma are identical. Finally, it must be stressed that the role of FcR-bearing cells is still not understood, although several hypotheses have been proposed (Bernard et al., 1978; Bray et al., 1978), such as clearing of immune complexes around the embryo, prevention of excessive invasion by the trophoblast or simply provision of nutritive proteins for the rapidly growing decidua. Further investigation of the origin and role of FcR-bearing cells in the decidua must await purification of this cell population; this is currently in progress. ACKNOWLEDGEMENTS We are grateful to Dr. B. Peitz for helpful discussion. The work was supported by INSERM CRL 78.5.022.4, UER Kremlin-Bicetre, and NIH Grants CA08748, HD 10668, and HD 08415, AI 00270. REFERENCES Atkinson, W.B. (1944) The persistence of deciduomata in the mouse. Anat. Rec. 88,271-283. Beer, A.E. and BiUingham, R.E. (1974) Host responses to intrauterine tissue, cellular and fetal allografts. J. Reprod. Fertil. Suppl. 21,59~88. Beer, A.E. and BiUingham, R.E. (1976) The immunobiology of mammalian reproduction. Prentice Hall, Englewoods Cliffs, N.J. Bernard, O., Ripoehe, M.A. and Bennett, D. (1977) Distribution of maternal immunoglobulins in the mouse uterus and embryo in the days after implantation. J. Exp. Med. 145, 58-75. Bernard, O., Scheid, M., Ripoche, M.A. and Bennett, D. (1978) Immunological studies of mouse decidual cells. I - Membrane markers of decidual cells in the days after implantation. J. Exp. Med. 148,580-591. Bourgois, A., Abney, E.R. and Parkhouse, RJVI.E. (1977) Structure of mouse Fc receptor. Eur. J. Immunol. 7,691-695.
48 Bray, J., Stewart, I. and Craggs, R. (1978) The demonstration of cells bearing Fc receptors in the metrial gland of the pregnant rat uterus. Cell Tiss. Res. 192, 8 9 - 9 6 . Deanesley, R. (1973) Termination of early pregnancy in rats after ovariectomy is due to immediate collapse of the progesterone-dependent decidua. J. Reprod. Fertil. 3 5 , 1 8 3 - 1 8 6 . Elson, J., Jenkinson, E.J. and Billington, W.D. (1975) Fc receptors on mouse placenta and yolk sac cells. Nature (London) 2 5 5 , 4 1 2 - 4 1 4 . Finn, C.A. and Hinchliffe, J.R. (1964) Reaction of the mouse uterus during implantation and deciduoma form~ition as demonstrated by changes in the distribution of alkaline phosphatase. J. Reprod. Fertil. 8 , 3 3 1 - 3 3 8 . Finn, C.A. and Porter, D.G. (1975) The Uterus. Elek Science, London. Galassi, L. (1968) Autoradiographic study of the decidual cell reaction in the rat. Dev. Biol. 17, 7 5 84. Jenkinson, E.J., Billington, W.D. and Elson, J. (1976) Detection of receptors for immunoglobulin on human placenta by EA rosette formation. Clin. Exp. Immunol. 2 3 , 4 5 6 - 4 6 1 . Kirby, D.R.S. (1963) Development of the mouse blastocyst transplanted to the spleen. J. Reprod. Fertil. 5, 1-12. Kirby, D.R.S. and CoweU, T.P. (1968) Trophoblast-Host interactions. In Epithelial-Mesenchymal Interactions, 18th Hahnemann Symposium (Fleischmajer, R. and Billingham, R.E., eds.), pp. 6 4 77. Williams and Wilkins Co., Baltimore. Kirby, D.R.S., Billington, W.D. and James, D.A. (1966) Transplantation of eggs to the kidney and uterus of immunised mice. Transplantation, 4 , 7 1 3 - 7 1 8 . McCormack, J.T. and Greenwald, G.S. (1974) Progesterone and oestradiol-1713 concentrations in the peripheral plasma during pregnancy in the mouse. J. Endocrinol. 6 2 , 1 0 1 - 1 0 7 . Murr, S.M., Stabenfelt, G.H., Bradford, G.E. and Geschwind, I.I. (1974) Plasma progesterone during pregnancy in the mouse. Endocrinology 94, 1209-1211. Sananes, N.E., Weiller, S., Baulieu, E.E. and Le Goascogne, C. (1978) In vitro decidualization of rat endometrial cells. Endocrinology 103, 8 6 - 9 5 . Sanyal, M.K. (1978) Secretion of progesterone during gestation in the rat. J. Endocrinol. 79, 1 7 9 190. Stern, P.L. (1973) 0 alloantigen on mouse and rat fibroblasts. Nat. New Biol. 246, 7 6 - 7 8 . Vladimirsky, F., Chen, L., Amsterdam, A., Zor, U. and Lindner, H.R. (1977) Differentiation of decidual cell in cultures of rat endometrium. J. Reprod. Fertil. 49, 6 1 - 6 8 .
41
IMMUNOLOGICAL STUDIES OF MOUSE DECIDUAL CELLS. II. STUDIES OF CELLS IN A R T I F I C I A L L Y INDUCED DECIDUA
FLORE RACHMAN 1, OLIVIER BERNARD 1, MARGRIT P. SCHEID 2 and DOROTHEA BENNETT 2 1 Clinique de Pddiatrie and Unitd de Recherche d 'Hdpatologie Infantile [INSERM U 56], Hdpital d'Enfants, 94270, Kremlin Bicdtre, France, 2 Memorial Sloan-Kettering Cancer Center, New York, N Y 10021, U.S.A.
(Received 26 July 1980; revised 15 September; accepted 10 October)
Cells from artificially induced decidual tissue (deciduoma) in the mouse were examined for Thy-1 surface antigen and receptors for the Fc portion of immunoglobulin G (FcR) and compared with cells of the normal decidua from day 6 to day 9 of pregnancy. It was shown that (1) Thy-1 antigen is present on the same proportion of cells in decidua and deciduoma on day 6 and day 7, (2) FcR-bearing cells can be detected in similar numbers on day 6 and day 7 but this does not increase on day 8 in deciduoma as it does in decidua, and (3) progesterone treatment after induction of decidualization allowed further increase of FcR-bearing cells in deciduoma. These results present further evidence of the similarity between deciduoma and decidua in the mouse. They indicate that these two membrane markers are present in the early decidua, regardless of the presence of an embryo, and suggest that progesterone may play a part in the increase of FcR-bearing cells in the decidua during pregnancy.
INTRODUCTION In mammals, the close relationships occurring after implantation between the embryo and the maternal uterine tissues raise several questions concerning (1) how the embryo is not rejected as an allograft by the mother (Beer and Billingham, 1976), and ( 2 ) h o w the mother prevents excessive invasion o f the uterus b y trophoblastic cells that possess considerable invasive and phagocytic properties at that time (Kirby, 1963). The decidual tissue developing in the uterus at the time of implantation may play a protective role in both respects (Kirby et al., 1966; Kirby and Cowell, 1968; Beer and BiUingham, 1974). Although it is clear that maternal immunoglobulins, decidual and trophoblastic cells are in close association from the time o f implantation onwards (Bernard et al., 1977), little is known of the mechanisms controlling, in the decidua, the discrete balance between excessive invasion by the trophoblast and rejection o f the embryo by the mother. Previous studies o f cells o f the mouse decidua in the days after implantation have shown that no significant numbers o f B and T lymphocytes can be detected, although a majority of cells express Thy-1 antigen, presumably reflecting their fibroblastic origin. In addition, an increasing number o f cells bearing surface receptors for IgG (FcR) are present from day 6 to day 8 o f gestation (Bernard et al., 1978). In mice and rat uteri, a decidual reaction (deciduoma) can be artificially induced in the 0165-0378/81/0000-0000/$02.50 © Elsevier/North-HoUand Biomedical Press
42 absence of an embryo and has proved a useful tool in analyzing the differentiation of the decidua and its hormonal control (for review, see Finn and Porter, 1975). In this paper, we report the results of an immunological study of the cells present in mouse deciduoma as compared with the cells in the decidua during a normal pregnancy. The results indicate that both Thy-l-bearing cells and FcR-bearing cells are also present in deciduoma, and suggest tha.t progesterone may play a part in controlling the increasing amount of FcRbearing cells in the decidua as pregnancy proceeds. MATERIALS AND METHODS
Mice Inbred C57B1/6(B6) and AKR/J mice, and random-bred CF1 and ICR albino mice were obtained as previously described (Bernard et al., 1978). Inbred and outbred matings were set up with normal or vasectomized males, and the day a vaginal plug was found was considered day 0 of pregnancy or pseudo-pregnancy. Induction of deciduoma Virgin female mice ( 8 - 1 0 weeks old) were placed overnight with vasectomized males. On the third day of pseudo-pregnancy, females were anaesthetized with tribromoethanol (Avertin) and 4 - 0 silk threads were placed at three different levels transversally through each uterine horn. From day 6 of pseudo-pregnancy, deciduomata appear as hypertrophic areas spread along the uterine horns, and centered by the threads left in situ. The lifespan of the mouse deciduoma is limited and by day 9 it is undergoing necrosis. Regression of deciduoma can, however, be delayed for a few days by treatment with progesterone (Atkinson, 1944). To study the possible influence of progesterone on FcR bearing-cells in deciduoma, 0.2 ml of a 7.5 mg/ml progesterone solution in sesame off were injected daffy subcutaneously from the day of trauma onwards. Control mice, in which decidualization also had been induced, received the same amount of sesame oil without progesterone. Preparation of cells from decidua and deciduoma Females pregnant for the first time, or females in which deciduomata had been artificially induced, were killed by cervical dislocation at 6, 7, 8 and 9 days of pregnancy or pseudo-pregnancy. Decidual capsules were dissected out of the myometrium in phosphate-buffered saline (PBS) and the embryo and its immediate surrounding tissues (including the chorioallantoic placenta on day 9) were carefully removed. Deciduomata were dissected similarly and the remnants of uterine epithelium were removed; absence of an embryo was checked in each case. Cell suspensions were obtained from decidua or deciduoma using a slight modification of the procedure already described (Bernard et al., 1978). Briefly, pieces of tissue were chopped in medium and incubated in 1 mg/ml collagenase type II (Sigma Chemical Co., St. Louis, Mo.), then washed twice in Ca 2+- and Mg2*-f~eeDulbecco's PBS (Grand Island Biological Co., Grand Island, N.Y.) with 0.3% fructose and 0.3% dextrose, and incubated for 4 periods of 5 min each in 1 mg/ml trypsin (1 : 250, Difco Laboratories, Detroit, Mich.) in the same Ca 2+- and Mg2÷-free medium. Cells from each trypsin incubation were
43 recovered after pipetting, treated as described previously, and their viability assessed by trypan blue exclusion. All four fractions contained over 90% viable cells and were pooled for further studies. However, on day 9 of pseudo-pregnancy the viability of cells prepared from deciduomata undergoing necrosis was so poor (averaging 15-20%) that no study could be carried out at that stage.
Other procedures Anti-Thy-l.2 serum, complement-mediated cytotoxicity, detection of surface immunoglobulins and E rosette (with non-sensitized sheep red cells) and EA rosette (7S antibody-coated sheep red cells) studies were carried out as previously described (Bernard et al., 1978). Each test was performed using cells prepared from pooling all decidual or deciduomal capsules from one mouse. Student's t-test was used for statistical analysis. RESULTS
Cell suspensions from deciduas and deciduomata Mean cell yields per decidual or deciduomal capsule are indicated in Fig. 1. Mean cell yield per capsule was 8.7 _+0.4 X l0 s on day 9 of pregnancy, but could not be stated precisely at that stage in the deciduoma from mice treated with progesterone since capsules were very large and sometimes fused. Red blood cell (RBC) counts were always less than two RBC per nucleated cell, making contamination by blood nucleated cells quite minimal.
12
~8 o)
"56 t-~
E=4
Y.,~
.:l
6
Illlg
YZJ
7
8
Days of pregnancy or pseudopregnancy Fig. 1. Number of ceils recovered per decidual or deciduomal capsule from day 6 to day 8 of pregnancy or pseudo-pregnancy in outbred albino female mice. Mean number of cells X10 5 and standard deviations. ~, decidua; ~, deciduoma; l , deciduoma with progesterone.
44
Thy-1 antigen Anti-Thy-1.2 antiserum was used in complement-dependent cytotoxicity tests on cells from deciduas and deciduomata from B6 (Thy-1.2) and AKR/J (Thy-l.1) females on day 6 and day 7 of pregnancy or pseudo-pregnancy. In B6 mice, Thy-l.2-bearing cells can be found in similar amounts in both decidua and deciduoma. Fig. 2 shows the results of tests carried out on day 6 where the cytotoxic index is 0.6 at 1 : 100 dilution in both cases. No significant activity could be detected against AKR/J deciduoma cells. In B6 mice, the morphology of cells killed by anti-Thy-1.2 serum was the same in decidua and deciduoma, made up mostly of medium size or large, granular, often bi- or multi-nucleated cells. Surface immunoglobulins [sIg) In cell suspensions from deciduas and deciduomata from outbred albino mice, no slgbearing cells could be detected, under the same conditions where 40% of spleen cells and less than 1% of thymocytes are labelled by sheep Fab anti-mouse Ig coupled with peroxidase. Previous data showed that the enzymatic treatment we used did not alter the detection of sIg-bearing cells in the spleen (Bernard et al., 1978). Fc receptors on cells in deciduoma Studies of EA rosette-forming cells were carried out from day 6 to day 9 in randombred albino mice, on decidua, deciduoma and deciduoma from mice treated with progesterone. At least three separate experiments were done at each stage. Fig. 3 confirms that the mean percentage of EA rosette-forming cells increases significantly in the course of a normal pregnancy from 6% on day 6 to 10% on day 7 (P < 0.05) and from 10% on day 7 to 17.8 and 17% on day 8 (P < 0.05) and day 9 (P < 0.05) respectively. In deciduoma, FcR-bearing cells can be detected in amounts similar to those in decidua on day 6 and
O.7 O...--- O
06
%~
0.5 :~ 0.4
0.3
~ N
S 02
o~-.-° 0.1
o~
~o~
"~.0~" 0
50
\
\
...~..~.0~ \A
100 250 500 1000 2000 1 / Dilution of Antiserum
Fig. 2. Titration of anti-Thy-t.2 serum: on 6-day B6 deciduomal cells (-), on 6-day AKR/J deci-
duomal cells (o), and on 6-day B6 decidual ceils (A).
45
30 25
!
8 20 E
T
6
7
it 8
9
Days of pregnancy or pseudopregnancy
Fig. 3. Percentage EA rosette-forming cells in outbred mouse decidua and deciduoma from day 6 to day 9 of pregnancy or pseudo-pregnancy(mean figures and standard deviations). Controls using unsensitized SRBC (E rosettes)were all negative. ~, decidua;r~, deciduoma; m, deciduomawith progesterone. Mean figures correspond to the mean of the percentages of EA rosette-forming cells in at least three tests for each stage.
day 7, but on day 8 the amount of EA rosette-forming cells does not increase further and remains equal to that on day 7. On day 9 no study could be performed in the deciduoma undergoing necrosis. Daily treatment with 1.5 mg of progesterone from the day of trauma onwards alters somewhat the amount of FcR-bearing cells in deciduoma. While on day 6 and day 7 the mean level of EA rosette-forming cells remains stable, it significantly increases thereafter, reaching 11.3 and 23% of deciduoma cells on day 8 ( P < 0.05) and day 9 ( P < 0.05), respectively. FcR-bearing cells in mice treated with sesame oil instead of progesterone followed a pattern similar to that in untreated mice (data not shown). EA rosettes in deciduomata were similar in size and appearance to those in deciduas. DISCUSSION The results presented here indicate that in mouse deciduoma, as in mouse decidua, Thy-1 antigen can be detected on a large fraction of cells on day 6 and day 7 of pseudopregnancy, and that FcR-bearing cells are present in similar amounts at the same time. However, in deciduoma the amount of FcR-bearing cells does not increase after day 7 as in decidua, unless mice are treated with progesterone from the day of trauma onwards. These findings can be discussed from three viewpoints: (1) they provide further data to support the analogies between decidua and deciduoma, (2) they stress that the presence of the two cell surface markers studied on cells in the decidua is actually independent of
46 the presence of an embryo, and (3) they suggest a hormonal control for the level of FcRbearing cells in the deciduoma and in the decidua. Further analogies between deciduoma and decidua Several studies have shown that early decidua and deciduoma, in rats and mice, share several features. Morphological, histochemical and autoradiographic observations show that early decidual and deciduomal cells derive mostly from uterine stromal cells and their evolution from this stage to the differentiated, often bi- or multi-nucleated decidual cells has been documented in both normal and pseudo-pregnancy (Krehbiel, 1937; Finn and Hinchliffe, 1964; Galassi, 1968). The present data provide further evidence regarding this analogy. In both tissues, Thy-1 antigen is present on a comparable fraction of cells on day 6 and day 7 of pregnancy or pseudo-pregnancy. As previously discussed (Bernard et al., 1978), Thy-1 antigen probably reflects the fibroblastic origin of the majority of cells in decidua and deciduoma (Stern, 1973). Similarly, FcR-bearing cells are present in the same proportion in the early (6 and 7 day) decidua as well as in deciduoma. Although the origin and role of these cells are unknown, their presence indicates that similarities between early decidua and deciduoma exist not only on morphological grounds but also from a functional viewpoint. Cell surface markers detected on cells in the decidua are independent o f the presence of a n embryo The presence of an increasing number of FoR-bearing cells in the early decidua during a normal pregnancy could be accounted for by contamination with cells of embryonic origin: most early trophoblastic cells contain maternal immunoglobulin (Bernard et al., 1977) and the presence of FoR on trophoblastic cells has been reported at later stages of pregnancy in mice and humans (Elson et al., 1975; Jenkinson et al., 1976). It could thus be argued that cells described as bearing FcR in the decidua are, at least partly, embryonic in origin. Data presented here rule out this possibility on day 6 and day 7 since the same amounts of FcR-bearing cells are present in decidua and deciduoma, the latter in the absence of any embryo. As far as day 8 and day 9 are concerned, it is also unlikely that the increase in FcR-bearing cells in decidua is due to embryonic cells. Thus, lack of increase of FcR-bearing cells in deciduoma on day 8 rather corresponds to an early sign of regression of deciduoma, while the temporary 'rescue' of deciduoma in progesteronetreated mice is in fact paralleled by an increase in FcR-bearing cells. It is quite possible that, in untreated mice, the absence of an embryo and/or of the corresponding luteotrophic placental factors (see McCormack and Greenwald, 1974), or even the lack of production of progesterone by the placenta, as in the rat (Sanyal, 1978), is actually responsible for both the regression of deciduoma and the lack of increase in FoR-bearing cells. The results presented here also show that the expression of Thy-1 antigen on decidual cells does not depend on the presence of an embryo. It is, however, still not known whether Thy-1 antigen on decidual cells results from the persistence of a membrane antigen already present on uterine stromal cells before decidualization or if it is the result of a particular step in the differentiation of decidual cells. Studies of Thy-1 antigen expression during in vitro decidualization (Vladimirsky et al., 1977; Sananes et al., 1978)
47 should make it possible to answer this question and also to study the regulation o f Thy-1 expression under various hormonal conditions.
Possible endocrine control o f FcR-bearing cells in the decidua The present results show that an increase in FcR-bearing cells in 8- and 9-day deciduoma can be obtained when regression of deciduoma is prevented by progesterone treatment. In pregnant rats, progesterone is necessary to maintain normal development of the decidua in the days after implantation (Deanesley, 1973). It is therefore tempting to suggest that in the pregnant mouse the presence of FcR-bearing cells in the decidua is under the control of progesterone. In the mouse, plasma progesterone levels are known to remain fairly high from day 5 to day 10 and to grow even higher in the second half of pregnancy (McCormack and Greenwald, 1974; Murr et al., 1974). FcR-bearing cells are in fact present, in even greater amounts, at later stages in the rat metrial gland (Bray et al., 1978), and it may be that at least some of these cells fulfdl the same role and are under the same hormonal control as those present in the mouse decidua at earlier stages. However, it is still not clear whether FcR-bearing cells are differentiating within the uterus as a part of the decidua or deciduoma, possibly also from fibroblasts (Bourgois et al., 1977), or if they are migrating into the decidua and the deciduoma in the postimplantation period. Nor can the data we report confirm that FcR-bearing cells in decidua and deciduoma are identical. Finally, it must be stressed that the role of FcR-bearing cells is still not understood, although several hypotheses have been proposed (Bernard et al., 1978; Bray et al., 1978), such as clearing of immune complexes around the embryo, prevention of excessive invasion by the trophoblast or simply provision of nutritive proteins for the rapidly growing decidua. Further investigation of the origin and role of FcR-bearing cells in the decidua must await purification of this cell population; this is currently in progress. ACKNOWLEDGEMENTS We are grateful to Dr. B. Peitz for helpful discussion. The work was supported by INSERM CRL 78.5.022.4, UER Kremlin-Bicetre, and NIH Grants CA08748, HD 10668, and HD 08415, AI 00270. REFERENCES Atkinson, W.B. (1944) The persistence of deciduomata in the mouse. Anat. Rec. 88,271-283. Beer, A.E. and BiUingham, R.E. (1974) Host responses to intrauterine tissue, cellular and fetal allografts. J. Reprod. Fertil. Suppl. 21,59~88. Beer, A.E. and BiUingham, R.E. (1976) The immunobiology of mammalian reproduction. Prentice Hall, Englewoods Cliffs, N.J. Bernard, O., Ripoehe, M.A. and Bennett, D. (1977) Distribution of maternal immunoglobulins in the mouse uterus and embryo in the days after implantation. J. Exp. Med. 145, 58-75. Bernard, O., Scheid, M., Ripoche, M.A. and Bennett, D. (1978) Immunological studies of mouse decidual cells. I - Membrane markers of decidual cells in the days after implantation. J. Exp. Med. 148,580-591. Bourgois, A., Abney, E.R. and Parkhouse, RJVI.E. (1977) Structure of mouse Fc receptor. Eur. J. Immunol. 7,691-695.
48 Bray, J., Stewart, I. and Craggs, R. (1978) The demonstration of cells bearing Fc receptors in the metrial gland of the pregnant rat uterus. Cell Tiss. Res. 192, 8 9 - 9 6 . Deanesley, R. (1973) Termination of early pregnancy in rats after ovariectomy is due to immediate collapse of the progesterone-dependent decidua. J. Reprod. Fertil. 3 5 , 1 8 3 - 1 8 6 . Elson, J., Jenkinson, E.J. and Billington, W.D. (1975) Fc receptors on mouse placenta and yolk sac cells. Nature (London) 2 5 5 , 4 1 2 - 4 1 4 . Finn, C.A. and Hinchliffe, J.R. (1964) Reaction of the mouse uterus during implantation and deciduoma form~ition as demonstrated by changes in the distribution of alkaline phosphatase. J. Reprod. Fertil. 8 , 3 3 1 - 3 3 8 . Finn, C.A. and Porter, D.G. (1975) The Uterus. Elek Science, London. Galassi, L. (1968) Autoradiographic study of the decidual cell reaction in the rat. Dev. Biol. 17, 7 5 84. Jenkinson, E.J., Billington, W.D. and Elson, J. (1976) Detection of receptors for immunoglobulin on human placenta by EA rosette formation. Clin. Exp. Immunol. 2 3 , 4 5 6 - 4 6 1 . Kirby, D.R.S. (1963) Development of the mouse blastocyst transplanted to the spleen. J. Reprod. Fertil. 5, 1-12. Kirby, D.R.S. and CoweU, T.P. (1968) Trophoblast-Host interactions. In Epithelial-Mesenchymal Interactions, 18th Hahnemann Symposium (Fleischmajer, R. and Billingham, R.E., eds.), pp. 6 4 77. Williams and Wilkins Co., Baltimore. Kirby, D.R.S., Billington, W.D. and James, D.A. (1966) Transplantation of eggs to the kidney and uterus of immunised mice. Transplantation, 4 , 7 1 3 - 7 1 8 . McCormack, J.T. and Greenwald, G.S. (1974) Progesterone and oestradiol-1713 concentrations in the peripheral plasma during pregnancy in the mouse. J. Endocrinol. 6 2 , 1 0 1 - 1 0 7 . Murr, S.M., Stabenfelt, G.H., Bradford, G.E. and Geschwind, I.I. (1974) Plasma progesterone during pregnancy in the mouse. Endocrinology 94, 1209-1211. Sananes, N.E., Weiller, S., Baulieu, E.E. and Le Goascogne, C. (1978) In vitro decidualization of rat endometrial cells. Endocrinology 103, 8 6 - 9 5 . Sanyal, M.K. (1978) Secretion of progesterone during gestation in the rat. J. Endocrinol. 79, 1 7 9 190. Stern, P.L. (1973) 0 alloantigen on mouse and rat fibroblasts. Nat. New Biol. 246, 7 6 - 7 8 . Vladimirsky, F., Chen, L., Amsterdam, A., Zor, U. and Lindner, H.R. (1977) Differentiation of decidual cell in cultures of rat endometrium. J. Reprod. Fertil. 49, 6 1 - 6 8 .