Cell Type-Specific Expression of Transforming Growth Factor-/? 1 in the Mouse Uterus during the Periimplantation Period
Hiromichi Tamada*, Michael T. McMasterj-, Kathleen C. Flanders, Glen K. Andrews, and Sudhansu K. Dey Departments of Obstetrics-Gynecology and Physiology (H.T., S.K.D.) Department of Biochemistry and Molecular Biology (M.T.M., G.K.A.) University of Kansas Medical Center Ralph L. Smith Research Center Kansas City, Kansas 66103 Laboratory of Chemoprevention National Cancer Institute National Institutes of Health (K.C.F.) Bethesda, Maryland 20892
Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-/? 1 (TGF/31) in the mouse uterus during the periimplantation period. The polyclonal antisera antiLC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF/J1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF/91 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF/J1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-01 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-/31 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4. On day 5 (after initiation of implantation on the evening of day 4), while the intracellular staining was restricted to the luminal epithelium and primary decidual zone (PDZ), intense extracellular staining was noted in the decidualizing stroma
around the PDZ. On days 6 and 7, the PDZ was still positive for intracellular staining, but the extracellular staining was limited to the secondary decidual zone (SDZ) on day 6 and to the decidua capsularis on day 7. The extracellular staining persisted in the decidua capsularis on day 8, while the intracellular staining was diffuse in the regressing decidua. On days 7 and 8, decidua at the mesometrial pole also showed intense extracellular staining. TGF/91 mRNA was diffusely distributed throughout the decidua on days 5-8; however, the presence of intracellular staining in the PDZ provides evidence that this zone is the primary site of TGF/31 synthesis, while extracellular immunostaining in the SDZ and decidua capsularis is indicative of the sites of accumulation. The colocalization of mRNA with intracellular staining of the protein in the implanting embryo indicates that the embryo also synthesizes this growth factor. Northern blot hybridization of total RNA confirmed the presence of 2.4-kilobase TGF/31 mRNA in uteri and decidua during the periimplantation period. The results suggest that TGF/? has a role in regulating interactions between the epithelium and surrounding stroma in the uterus during the preimplantation period, and between the PDZ and SDZ in the deciduum during the postimplantation period. Furthermore, because of TGF/3's role in proliferation and differentiation as well as in the formation of extracellular matrix and cell surface molecules, this TGF is likely to participate in various events of blastocyst implantation, decidualization, placentation, and embryogenesis. (Molecular Endocrinology 4: 965-972, 1990)
0888-8809/90/0965-0972$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
The implantation process involves complex interactions between embryonic and uterine cells. The major events
INTRODUCTION
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of the implantation process are 1) synchronized development of the preimplantation embryo into a blastocyst and establishment of the receptive uterus (1); 2) escape of the embryo from immunological responses of the mother (2); 3) increased endometrial capillary permeability at the site of the blastocyst apposition (1); 4) localized decidualization of the endometrial stroma immediately after blastocyst attachment (1, 3); and 5) controlled uterine invasion by trophoblasts (4). These events are a conglomerate of temporally and spatially regulated proliferation, differentiation, migration, and remodelling of heterogeneous cell types of both embryonic and uterine tissues. Although these critical events are primarily dependent upon temporal and cell typespecific interactions mediated by progesterone (P4) and estrogen (E), the molecular and cellular mechanisms involved in these P4/E-regulated processes are not clearly understood. Regulation of expression of epidermal growth factor, insulin-like growth factor-l, and their receptors in the uterus by ovarian steroids and during the periimplantation period provides circumstantial evidence that P4/E actions in the uterus are at least partially mediated in an autocrine/paracrine fashion by growth factors (5-12). However, an important multifunctional growth factor, transforming growth factor-/? (TGF/3), has not yet been studied in the uterus. TGF/3 belongs to a large gene family, and multiple forms of this growth factor have been identified (reviewed in Ref. 13). Because of its role in cell growth, differentiation, and migration as well as in the formation of extracellular matrix and cell surface molecules (13-15), this family of growth factors is likely to play important roles in various events of the implantation process. Therefore, in the present investigation cell type-specific expression of TGF/31 was studied in the mouse uterus during the periimplantation period.
RESULTS In Situ Hybridization Analysis of TGF01 mRNA The cell type-specific expression of TGF/31 mRNA was examined by in situ hybridization. Autoradiographic signals of TGF/31 hybrids on days 1-8 are shown in Figs. 1-3 as brightfield and darkfield photomicrographs (A and B, respectively). On days 1-4 (preimplantation period), TGF/31 mRNA was localized primarily in the luminal and glandular epithelia. However, on days 3 and 4, stromal cells close to the luminal epithelium also contained TGF/31 mRNA. These observations suggest that during the preimplantation period, epithelial cells are the major sites of TGF/31 synthesis. During the postimplantation period (days 5-8), however, TGFj31 hybrids showed a diffuse distribution throughout the deciduum (Figs. 2 and 3). Positive hybrids were also detected in implanting embryos on these days of pregnancy.
Immunohistochemistry Uterine sections in which endogenous peroxidase was blocked showed no staining after incubation with normal rabbit immunoglobulin G (IgG), antibody preabsorbed with TGF/31 resin, or antibody neutralized with excess antigenic peptide (data not shown). The staining pattern of immunoreactive TGF/31 seen with anti-LC was intracellular, and that with anti-CC was extracellular, as reported previously (16, 17). The staining with anti-LC (intracellular) and anti-CC (extracellular) for days 1-8 are represented by C and D, respectively, in Figs. 1-3. On day 1 of pregnancy, while intense intracellular staining was seen in the luminal and glandular epithelia, extracellular staining was evident in the stroma (Fig. 1). Little extracellular staining of the stroma was noted on day 2, in spite of the presence of intracellular staining in the luminal epithelium (Fig. 1). On day 3, intracellular staining persisted in the luminal and some glandular epithelia, and moderately intense extracellular staining reappeared in the stroma (Fig. 1). The intensity of both the intracellular staining in the epithelia and the extracellular staining in the stroma showed a further increase on day 4 (Fig. 1). After blastocyst attachment on day 5, intracellular staining was limited to the luminal epithelium and the primary decidual zone (PDZ), while extracellular staining was intense in the decidualizing stroma surrounding the PDZ (Fig. 2). On day 6, the staining patterns with these two antibodies became clearly distinct; the intracellular staining persisted in the PDZ, while the extracellular staining was restricted to the secondary decidual zone (SDZ; Fig. 2). On day 7, although the PDZ still showed intracellular staining, extracellular staining was only evident at the periphery of the SDZ (decidua capsularis; Fig. 3). The decidua capsularis on day 8 still showed extracellular staining, but the regressing decidua proper showed diffuse intracellular staining. On days 7 and 8, decidua at the mesometrial pole surrounding the blood islands showed intense extracellular staining (Fig. 3). Furthermore, while intracellular staining was present in the embryo on all days examined, extracellular staining was clearly evident on day 8 only (Figs. 2 and 3). During the periimplantation period, TGFj81 mRNA colocalized with immunostaining for intracellular TGF/31 (anti-LC), but not with extracellular immunostaining for this protein (antiCC). This suggests that anti-LC recognizes the sites of synthesis of TGF/31. Northern Blot Hybridization of TGF/31 mRNA To confirm our findings of TGF/31 mRNA in the uterus and deciduum, as determined by in situ hybridization, uterine and decidual RNAs were analyzed by Northern blotting. Adult female mouse adrenal gland RNA served as a positive control for the 2.4-kilobase (kb) TGF/31 transcript (17). As shown in Fig. 4, a 2.4-kb TGF-/31 transcript was detected in total RNA from the uterus (days 1 -4) and the deciduum (days 7-11). There were no remarkable changes in the steady state levels of
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Fig, 1. In Situ Hybridization of TGF/31 mRNA and Immunohistochemical Localization of TGF/31 in the Mouse Uterus on Days 1-4 of Pregnancy In situ hybridization: Mice on the indicated days of pregnancy were anesthetized, and uteri were perfusion fixed with 4% paraformaldehyde in PBS. Paraffin-embedded samples were sectioned at 7 ^M. TGF/31 mRNA was hybridized in situ for 5 h at 42 C with a 35S-labeled human TGF/31 cRNA probe, and RNase-A-resistant hybrids were detected after 4 days of autoradiography using Kodak NTB-2 liquid emulsion. Slides were poststained lightly in hematoxylin. Brightfield (A) and darkfield (B) photomicrographs (x40) of uterine sections on specific days of pregnancy are shown. Uterine sections from each day of pregnancy were hybridized onto the same microscope slides. LE, Luminal epithelium; GE, glandular epithelium; S, stroma; LM, longitudinal muscle; CM, circular muscle. Immunohistochemistry: Bouin's-fixed paraffin-embedded uterine sections (7 HM) from specific days of pregnancy were mounted onto the same slide. After deparaffinization and hydration, sections were incubated in primary antibodies at a concentration of 20 ^g/ml for 24 h at 4 C. Immunostaining was performed by employing the avidin-biotin-peroxidase complex technique. Photomicrographs (x40) of immunostaining by anti-LC (intracellular staining) and anti-CC (extracellular staining) are represented by C and D, respectively, for each day of pregnancy.
uterine or decidual TGF/?1 transcripts on various days of pregnancy. This is probably a reflection of expression of this gene in a subpopulation of cells in these tissues, as evident from in situ hybridization.
DISCUSSION
The present study shows, for the first time, that expression of TGF/31 occurs in the uterus and deciduum during the periimplantation period. Expression of this gene is
temporally and spatially regulated. Sequential steroid hormone interplays before ovulation and after conception initiate a series of changes that involve temporal and cell type-specific proliferation and differentiation of the uterus (18). In the adult ovariectomized mouse, proliferation of luminal and glandular epithelial cells occurs in response to E, whereas maximal stromal cell proliferation requires the presence of both P4 and E (18). A similar pattern of P4/E-regulated cell type-specific proliferation and differentiation occurs in the mouse uterus during the periimplantation period (18). Preovulatory ovarian E secretion results in intense proliferation
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Fig. 2. In Situ Hybridization of TGFj81 mRNA and Histochemical Localization of TGF/31 in Sections of Implantation Sites on Days 5 and 6 of Pregnancy In situ hybridization: Brightfield (A) and darkfield (B) photomicrographs (x40) of sections of implantation sites on the indicated days of pregnancy are shown. Autoradiographic exposure was for 8 days. Immunohistochemistry: C and D represent photomicrographs (x40) of immunostaining of TGFj81 in sections of implantation sites on the indicated days of pregnancy by anti-LC (intracellular staining) and anti-CC (extracellular staining), respectively. Procedures for in situ hybridization and immunohistochemistry were described in Fig. 1. E, Embryo. The left and right sides of each photomicrograph represent mesometrial and antimesometrial poles, respectively.
of the luminal epithelium early on day 1, and that of the glandular epithelium on day 2 of pregnancy. Rising P4 levels from day 3 onward result in proliferation of the stromal cells and affects differentiation of the luminal epithelium. Stromal cell proliferation is enhanced by preimplantation E secretion early on day 4. After the process of implantation is initiated on the evening of day 4, stromal cells begin to differentiate into decidual cells, a process requiring both preimplantation ovarian steroid hormone secretion and the presence of a blastocyst (1, 18). Decidualization is first initiated at antimesometrial sites where blastocysts are implanting. In
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Fig. 3. In Situ Hybridization of TGFj81 mRNA and Histochemical Localization of TGF/31 in Sections of Implantation Sites on Days 7 and 8 of Pregnancy In situ hybridization: Brightfield (A) and darkfield (B) photomicrographs (x40) of sections of implantation sites on the indicated days of pregnancy are shown. Autoradiographic exposure was for 8 days. Immunohistochemistry: C and D represent photomicrographs (x40) of immunostaining of TGF/31 in sections of implantation sites on the indicated days of pregnancy by anti-LC (intracellular staining) and anti-CC (extracellular staining), respectively. Procedures for in situ and immunohistochemistry were described in Fig. 1. E, Embryo; DC, decidua capsularis. The orientations of mesometrial and antimesometrial poles are the same as in Fig. 2. Sections of implantation sites on days 5-8 of pregnancy were mounted onto the same slides in each experiment for comparison.
the mouse, proliferating and differentiating stromal cells surrounding the blastocyst initially form the PDZ on day 5. By day 6, the SDZ is formed around the PDZ, and the PDZ progressively degenerates. After day 8, the growing placenta and embryo slowly displace the SDZ, which is reduced to a thin layer of cells termed the decidua capsularis. The mesometrial decidual cells ultimately form the decidua basalis, a part of the definitive placenta (19). The colocalization of mRNA with intracellular protein in uterine epithelial cells on days 1-4 provides strong evidence for the synthesis of TGF/31 in these cells during the preimplantation period. On the other hand, the presence of extracellular TGF/31 in the stroma in the absence of mRNA in these cells suggests that this
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Fig. 4. Northern Blot Detection of Steady State Levels of TGF/31 mRNA in the Mouse Uterus and Deciduum on the Indicated Days of Pregnancy Total RNA (6 ng) samples were separated by formaldehyde agarose gel electrophoresis and blotted to nylon filters. The filters were hybridized with 32P-labeled cRNA probe for human TGF/31 under highly stringent conditions (67 C in 40% fomamide), as described in Materials and Methods. Adrenal gland (AD) RNA was used as a positive control for its TGF/31 mRNA trancript of 2.4 kb. Decidual tissues were isolated by microdissection. A 2.4-kb transcript of TGF/31 mRNA was present in the uterus and deciduum during the periimplantation period.
growth factor, after being synthesized in the epithelium, is released and binds to the extracellular matrix of the stroma, as has been shown to occur in various other tissues (16,17). This finding suggests that TGF/31 plays an important role in controlling epithelial-mesenchymal interactions in the uterus and could be involved in epithelial and stromal cell proliferation and differentiation during the preimplantation period. TGF/3 has been found to have both positive and negative effects on cell growth, depending on the cell type. Inhibition of proliferation by TGF/3 is often associated with terminal differentiation of cells (reviewed in Refs. 12 and 20). The diffuse distribution of mRNA throughout the deciduum from day 5 onward indicates that expression of TGFjSI gene is not restricted to a particular zone of the deciduum. However, the presence of intracellular immunostaining primarily in the PDZ suggests that these cells are the major site of synthesis of TGF/31. The presence of immunostaining in the extracellular matrix of the SDZ again shows that the sites of synthesis and accumulation of TGF/31 in the deciduum are different. The discordant distribution of the mRNA and protein is not uncommon for TGF/31 (17). This finding suggests that TGF/31 may play a role in mediating interactions between the PDZ and SDZ. However, a caveat to interpreting these data is the recent identification of multiple members of the TGF-/3 gene family which share amino acid sequence homology (13). This raises the possibility that antibodies to TGF/31 may also cross-react with other isoforms of TGF/3. Despite this possibility, the colocalization of TGF-/31 mRNA and intracellular staining for TGF/31 coupled with the fact that anti-CC has been shown to react with TGF/31, but not with TGF/32
(16), suggest that TGF/31 was the primary immunoreactive protein in these studies. Although P4 and E are critical for uterine proliferation and differentiation, the mechanism by which they regulate these processes is not known. Epidermal growth factor, insulin-like growth factor-l, and their receptors are expressed in specific uterine cell types during early pregnancy and under E/P4 stimulation, which has led to the suggestion that these proteins are involved in uterine growth and differentiation (5-12). Because of its roles in the regulation of formation of extracellular matrix and cell surface molecules, its involvement in regulation of cell proliferation and differentiation (1315), and its potent immunoregulatory effects, the selective synthesis and accumulation of TGF/31 in the uterus and deciduum suggest that this growth factor plays critical roles during the periimplantation period. The changing endocrine state of the female during the reproductive cycle as well as during pregnancy causes the uterus to undergo extensive remodelling (tissue formation and repair) that is associated with changes in basement-membrane components (21, 22). For example, the various basement membrane components (collagens, laminin, fibronectin, and proteoglycans) in the human uterus undergo changes throughout the menstrual cycle and during pregnancy (21). Likewise, mouse stromal cells undergoing decidualization also synthesize these extracellular components (22). TGF/3 is known to cause accumulation of matrix proteins not only by enhancing their synthesis, but also by decreasing matrix degradation by down-regulating the secretion of proteases and up-regulating the synthesis of protease inhibitors (13-15). Furthermore, this TGF has recently been shown to control a superfamily of cell surface molecules called integrins (14,15). The integrins mediate cell-cell and cell-matrix adhesions (14,15). The apposition and adhesion of the blastocyst trophectoderm with the uterine luminal epithelium as well as the later invasion of the endometrium by the trophoblast cells are dependent upon cell-cell interactions and cell migration, which could be mediated in an autocrine/ paracrine manner by TGF/3. Because TGF/3 down-regulates a specific laminin-binding integrin and because cell migration depends upon laminin binding (14, 15), we postulate that this growth factor participates in controlled invasion of the uterus by the trophoblast cells. Extracellular immunoreactive TGF/31 in the deciduum at the mesometrial pole on days 7 and 8 may also be involved with development of the placenta. Because the placenta undergoes continuous changes in size, shape, and internal structure, a role for TGF/3 in placental development has been considered (17). Even though TGF/3 opposes fibroblast growth factor-induced endothelial cell proliferation in vitro (13), TGF/3 has been shown to be angiogenic in vivo, and therefore, this growth factor may be involved with the extensive neovascularization that occurs during decidualization. The embryo is considered an allograft (2). Despite many suggestions as to mechanisms, it remains a challenge to explain the escape of the embryo from the
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immunological responses of the mother (2). Because TGF/3 is a potent immunosuppressant (reviewed in Ref. 13), this growth factor, of embryonic and/or uterine origin, could be involved in the immunological acceptance of the embryo by the mother (23). Furthermore, our present findings and previous reports of expression of TGF/3 in pre- and postimplantation embryos (24, 25) suggest a role for this growth factor in embryogenesis. The role of TGF/3s and fibroblast growth factor in amphibian embryo morphogenesis is well documented (26, 27). The accumulation of TGF/31 in the extracellular matrix of the uterine stroma and deciduum suggests an active uptake. Whether this uptake is receptor mediated or via binding of the protein with the matrix components is not known. It is interesting to note that TGF/3 copurifies with fibronectin (28), and membraneanchored and soluble forms of /3-glycan (proteoglycan) can bind TGF/8 (29). To understand the mechanisms of action of TGF/31 during the periimplantation period, data on temporal and cell type-specific expression of its receptors are needed. In summary, the present finding of cell type-specific expression of TGF/31 in the uterus and deciduum during early murine pregnancy suggests that this multifunctional growth factor plays important roles in various events of blastocyst implantation, decidualization, placentation, and early embryogenesis. Future experiments will determine the effects of P4 and/or E on regulation of the expression of TGF/31 in the uterus, and whether the implanting embryo influences the decidual expression of this growth factor.
MATERIALS AND METHODS Animals All experiments involving animals were conducted in accordance with NIH standards for the care and use of experimental animals. Female CD-1 mice (48 days old; Charles River Laboratories, Raleigh, NC) were mated with fertile males (90 days old) of the same strain. The morning of finding a vaginal plug was defined as day 1 of pregnancy. Mice were killed between 0800-0900 h on specific days of pregnancy. Production of Antisera Polyclonal antibodies were produced in rabbits to different preparations of a peptide corresponding to amino acids 1 -30 of TGF/31. Two antibodies, anti-CC (1-30) and anti-LC (1-30), were produced as described by Ellingsworth ef a/. (30) and Flanders ef al. (16), respectively. In each case, the injected peptide was not coupled to a carrier protein. IgG was isolated from each antiserum by protein-A-Sepharose chromatography (31) for use in immunohistochemistry. A previous study with these antibodies, which seem to recognize different epitopes of TGF/3, has shown that anti-CC-(1-30) stains principally extracellular TGF/3, while anti-LC-(1-30) stains intracellular TGFjS (16). These antibodies do not react with TGF/32 on Western blots (16, 30); however, reactivities with TGF/33, -4, and -5 have not yet been tested. Immunohistochemistry Immunolocalization was based on the procedure described previously (5, 17). In brief, uteri were excised, cleaned of fat,
cut into 4- to 6-mm pieces, and fixed in Bouin's solution for 24 h. Paraffin-embedded tissue blocks were sectioned at 7 ^M and mounted on poly-L-lysine-coated slides. Sections were deparaffinized, hydrated in PBS for 20 min, and then incubated in blocking solution (10% normal goat serum) for 10 min before incubation in primary antibodies at 20 ng/m\ for 24 h at 4 C. Immunostaining was performed using a Zymed Histostain-SP kit for rabbit primary antibody (Zymed Laboratories, San Francisco, CA). This kit uses a biotinylated secondary antibody, a horseradish peroxidase-streptavidin conjugate, and a substrate chromogen mixture (32). Blocking of endogenous peroxidase activity was achieved by a 35-sec incubation in 0.23% periodic acid in PBS after secondary antibody incubation (33). Sections were counterstained lightly with hematoxylin, mounted, and examined under brightfield. Red deposits indicated the sites of immunostaining. Control experiments included incubation of sections with normal rabbit IgG, antisera preincubated with TGF-/?1-Sepharose resin, or primary antibodies neutralized with an excess of appropriate peptides. Hybridization Probes A cDNA clone was obtained from Rik Derynck (Genentech, Inc., San Francisco, CA) containing a 1050-basepair fragment encoding human TGF/31 inserted into the Sp64 plasmid (34) and used as a template for the Sp6. polymerase-directed synthesis of 32P- and 35S-labeled cRNA probes, as described by Melton ef al. (35). Probes had specific activities of about 2 x 109dpm/Mg. In Situ Hybridization The details of this technique have been described by us previously (5, 36). Briefly, hybridization was performed on paraformaldehyde perfusion-fixed tissue sections (7 /IM) using an 35S-labeled cRNA probe, as described above. Hybridization was carried out as described previously (5, 36). After hybridization, the sections were treated with RNase-A (20 A*g/ml). Autoradiography was performed with Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY) for 4-8 days, and sections were poststained lightly with hematoxylin. Isolation of RNA Uterine RNA was extracted using the guanidine isothiocyanate procedure described by Han et al. (37). Total RNA from decidua was extracted using the sodium dodecyl sulfatephenol-chloroform procedure described in detail by Andrews ef al. (38). Northern Blot Hybridization Total RNA (6 ng) was denatured and separated by formaldehyde agarose gel electrophoresis (36, 38, 39). After transfer to MSI nylon membranes (40), the membranes were baked in a vacuum oven at 75 C for 5 h. Northern blots were prehybridized, hybridized, and washed as described by Andrews ef al. (36, 38) with the following modifications. Hybridization was carried out under highly stringent conditions (67 C in the presence of 40% formamide, 3 x SET (1 x SET = 150 mM NaCI, 5 mM EDTA, and 10 mM Tris-HCI, pH 8.0), and 10% dextran sulfate to reduce background signals and ensure specificity. In all experiments, duplicate gels were stained with acridine orange to ensure the integrity of the RNA samples and to confirm that equal amounts of RNA had been loaded onto each lane. Acknowledgments Thanks are due to Susan Strong for technical assistance. Received February 15, 1990. Revision received March 30, 1990. Accepted April 4,1990.
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Address requests for reprints to: Dr. S. K. Dey, Departments of Obstetrics-Gynecology and Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66103. This work was supported in parts by grants from the NICHHD (HD-12304 to S.K.D.), the NIEHS (ES-04725 to G.K.A.), and the Wesley Foundation (to G.K.A. and S.K.D.). * Present address: Department of Animal Reproduction, University of Osaka Prefecture, College of Agriculture, Sakai City, Osaka 591, Japan. t March of Dimes predoctoral fellow.
REFERENCES 1. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EG, Geiger SR (eds) Handbook of Physiology. American Physiological Society, Washington DC, pp 187-215 2. Beer AE, Billingham RE 1978 Immunoregulatory aspects of pregnancy. Fed Proc 37:2374-2378 3. DeFeo VJ 1967 Decidualization. In: Wynn RM (ed) Cellular Biology of the Uterus. North Holland, Amsterdam, pp 191 290 4. Kirby DRS, Cowell TP 1968 Trophoblast-host interactions. In: Fleischmeyer R, Billingham RE (eds) EpithelialMesenchymal Interactions. Williams and Wilkins, Baltimore, pp 64-77 5. Huet-Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK 1990 Estrogen regulates synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 4:510-523 6. Huet-Hudson YM, Andrews GK, Dey SK, Epidermal growth factor and pregnancy in the mouse. In: Heyner S, Wiley LM (eds) Early Embryo Development and Paracrine Relationships. Wiley-Liss, New York, pp 125-136 7. Murphy LJ, Murphy LC, Friesen HG 1987 Estrogen induces insulin-like growth factor-l expression in the rat uterus. Mol Endocrinol 1:445-450 8. Norstedt G, Levinoritz A, Eriksson H 1989 Regulation of uterine insulin-like growth factor I mRNA and insulin-like growth factor II mRNA by estrogen in the rat. Acta Endocrinol (Copenh) 8120:466-472 9. Chakraborty C, Tawfik OW, Dey SK 1988 Epidermal growth factor binding in rat uterus during the periimplantation period. Biochem Biophys Res Commun 154:564569 10. Lingham RB, Stancell GM, Loose-Mitchell DS 1988 Estrogen induction of the epidermal growth factor receptor mRNA. Mol Endocrinol 2:230-235 11. Gardner RM, Verner G, Kirkland JL, Stancell GM 1989 Regulation of uterine growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle. J Steroid Biochem 32:339-343 12. Ghahary A, Murphy LJ 1989 Uterine insulin-like growth factor-l receptors: regulation by estrogen and variation throughout the estrous cycle. Endocrinology 125:597604 13. Roberts AB, Sporn MB 1990 The transforming growth factor-betas. In: Sporn MB, Roberts AB (eds) Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. Springer-Verlag, Heidelberg, vol95, p t l , pp 419-472 14. Heino J, Ignotz RA, Hemler ME, Crouse C, Massague J 1989 Regulation of cell adhesion receptors by transforming growth factor-/?: concomittant regulation of integrins that share a 01 subunit. J Biol Chem 264:380-388 15. Ignotz RA, Heino J, Massague J 1989 Regulation of cell adhesion receptors by transforming growth factor-/3: regulation of vitronectin receptor and LFA-1. J Biol Chem 264:389-392
16. Flanders KC, Thompson NL, Cissel DS, Van ObberghenSchilling EV, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB 1989 Transforming growth factor-/?1: histochemical localization with antibodies to different epitopes. J Cell Biol 108:653-660 17. Thompson NL, Flanders KC, Smith JM, Ellingsworth LR, Roberts AB, Sporn MB 1989 Expression of transforming growth factor-/31 in specific cells and tissues of adult and neonatal mice. J Cell Biol 108:661-669 18. Huet-Hudson YM, Andrews GK, Dey SK 1989 Cell typespecific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period. Endocrinology 125:1683-1690 19. Krehbiel RH 1937 Cytological studies of decidual rection in the rat during pregnancy and in the production of deciduomata. Physiol Zool 10:212-238 20. Moses HL, Tucker RF, Leof EB, Coffey RJ, Halper J, Shipley GD 1985 Type-beta transforming growth factor is growth stimulator and growth inhibitor. In: Feramisco J, Ozanne B, Stiles C (eds) Cancer Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, vol 3:65-71 21. Aplin JD, Charlton AK, Ayad S 1988 An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell Tissue Res 253:231-240 22. Wewer UM, Damjanov A, Weiss J, Liotta LA, Damjanov I 1986 Mouse endometrial stromal cells produce basementmembrane components. Differentiation 32:49-58 23. Clark DA, Falbo M, Rowley RB, Banwatt D, StedronskaClark J 1988 Active suppression of host-vs-graft reaction in pregnant mice. IX. Soluble suppressor activity obtained from allopregnant mouse decidua that blocks the cytolytic effector response to IL-2 is related to transforming growth factor-^. J Immunol 141:3833-3840 24. Rappolee DA, Brenner CA, Schultz R, Mark D, Werb Z 1988 Developmental expression of PDGF, TGFa, and TGF/? genes in preimplantation mouse embryos. Science 242:1823-1825 25. Heine Ul, Flanders KC, Roberts AB, Minoz EF, Sporn MB 1987 Role of transforming growth factor-/? in the development of the mouse embryo. J Cell Biol 105:2861-2876 26. Kimelman D, Kirschner M 1987 Synergistic induction of mesoderm by FGF and TGF/? and the identification of an mRNA coding for FGF in early Xenopus embryo. Cell 51:869-877 27. Rosa F, Roberts AB, Danielpour D, Dart LL, Sporn MB, Dawid IB 1988 Mesoderm induction in amphibians: the role of TGF/?2-like factors. Science 239:783-786 28. Fava RA, McClure DB 1987 Fibronectin-associated transforming growth factor. J Cell Physiol 131:184-189 29. Andres JL, Stanley K, Cheifetz S, Massague J 1989 Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-/?. J Cell Biol 109:3137-3146 30. Ellingsworth LR, Brennan JE, Fork K, Rosen DM, Bentz H, Piez KA, Seyedin SM 1986 Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor /3. J Biol Chem 261:12362-12367 31. Flanders KC, Roberts AB, Ling N, Fleurdelys BE, Sporn MB 1988 Antibodies to peptide determinants in transforming growth factor /3 and their applications. Biochemistry 27:739-746 32. Hsu S-M, Raine L 1984 The use of avidin-biotin-peroxidase complex (ABC) in diagnostic and research pathology. In: DeLellis RA (ed) Advances in Immunocytochemistry. Mason, New York, pp 31-42 33. Kelly J, Whelan CA, Weir DG, Reighery C 1987 Removal of endogenous peroxidase activity from cryostat section for immunoperoxidase visualization of monoclonal antibodies. J Immunol Methods 96:127-132 34. Derynk R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV 1985 Human transforming growth factor-/? complementary DNA
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sequence and expression in normal and transformed cells. Nature 316:701-705 35. Melton DA, Kreig PA, Rebegliati MR, Maniatis T, Kinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing bacteriophage SP6 promoter. Nucleic Acids Res 12:7035-7057 36. De SK, McMaster MT, Dey SK, Andrews GK 1989 Cellspecific metallothionein gene expression in mouse decidua and placentae. Development 107:611-621 37. Han JH, Stratona C, Rutter WJ 1987 Isolation of fulllength putative rat lysophospholipase cDNA using im-
proved methods for mRNA isolation and cDNA cloning. Biochemistry 26:1617-1625 38. Andrews GK, Lehman LD, Huet YM, Dey SK 1987 Metallothionein gene regulation in the preimplantation rabbit blastocyst. Development 100:463-465 39. Lehrach H, Diamond D, Wozney JM, Boedtker H 1977 RNA molecular weight determination by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751 40. Thomas PS 1980 Hybridization of RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205
Symposium on Modes of Action of GnRH and GnRH Analogs February 27-March 2, 1991 The Registry Resort Scottsdale, Arizona Co-Chairmen: William F. Crowley, MD and P. Michael Conn, Ph.D. Sponsored by Serono Symposia, USA This symposium will address the most current data on GnRH physiology, focusing on the basic information regarding its physiology, molecular biology, and normal secretion in man and animals. The program will also feature the area of GnRH antagonists as they apply to the human. Major sessions are I) Overview of GnRH Secretion and Mechanism of Action II) Molecular and Developmental Control of GnRH Expression III) GnRH Physiology: Animal Models IV) GnRH Antagonists. The program will include invited expert speakers, poster presentations of original abstracts, and ample time for informal interaction among participants. Deadline for submission of abstracts is January 11, 1991. Category I CME credits will be available. For further information, please contact L. Lisa Kern, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, MA 02061. Telephone: 800-283-8088 or 617-982-9000. Telefax: 617-982-9481.
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Hiromichi Tamada*, Michael T. McMasterj-, Kathleen C. Flanders, Glen K. Andrews, and Sudhansu K. Dey Departments of Obstetrics-Gynecology and Physiology (H.T., S.K.D.) Department of Biochemistry and Molecular Biology (M.T.M., G.K.A.) University of Kansas Medical Center Ralph L. Smith Research Center Kansas City, Kansas 66103 Laboratory of Chemoprevention National Cancer Institute National Institutes of Health (K.C.F.) Bethesda, Maryland 20892
Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-/? 1 (TGF/31) in the mouse uterus during the periimplantation period. The polyclonal antisera antiLC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF/J1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF/91 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF/J1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-01 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-/31 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4. On day 5 (after initiation of implantation on the evening of day 4), while the intracellular staining was restricted to the luminal epithelium and primary decidual zone (PDZ), intense extracellular staining was noted in the decidualizing stroma
around the PDZ. On days 6 and 7, the PDZ was still positive for intracellular staining, but the extracellular staining was limited to the secondary decidual zone (SDZ) on day 6 and to the decidua capsularis on day 7. The extracellular staining persisted in the decidua capsularis on day 8, while the intracellular staining was diffuse in the regressing decidua. On days 7 and 8, decidua at the mesometrial pole also showed intense extracellular staining. TGF/91 mRNA was diffusely distributed throughout the decidua on days 5-8; however, the presence of intracellular staining in the PDZ provides evidence that this zone is the primary site of TGF/31 synthesis, while extracellular immunostaining in the SDZ and decidua capsularis is indicative of the sites of accumulation. The colocalization of mRNA with intracellular staining of the protein in the implanting embryo indicates that the embryo also synthesizes this growth factor. Northern blot hybridization of total RNA confirmed the presence of 2.4-kilobase TGF/31 mRNA in uteri and decidua during the periimplantation period. The results suggest that TGF/? has a role in regulating interactions between the epithelium and surrounding stroma in the uterus during the preimplantation period, and between the PDZ and SDZ in the deciduum during the postimplantation period. Furthermore, because of TGF/3's role in proliferation and differentiation as well as in the formation of extracellular matrix and cell surface molecules, this TGF is likely to participate in various events of blastocyst implantation, decidualization, placentation, and embryogenesis. (Molecular Endocrinology 4: 965-972, 1990)
0888-8809/90/0965-0972$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
The implantation process involves complex interactions between embryonic and uterine cells. The major events
INTRODUCTION
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of the implantation process are 1) synchronized development of the preimplantation embryo into a blastocyst and establishment of the receptive uterus (1); 2) escape of the embryo from immunological responses of the mother (2); 3) increased endometrial capillary permeability at the site of the blastocyst apposition (1); 4) localized decidualization of the endometrial stroma immediately after blastocyst attachment (1, 3); and 5) controlled uterine invasion by trophoblasts (4). These events are a conglomerate of temporally and spatially regulated proliferation, differentiation, migration, and remodelling of heterogeneous cell types of both embryonic and uterine tissues. Although these critical events are primarily dependent upon temporal and cell typespecific interactions mediated by progesterone (P4) and estrogen (E), the molecular and cellular mechanisms involved in these P4/E-regulated processes are not clearly understood. Regulation of expression of epidermal growth factor, insulin-like growth factor-l, and their receptors in the uterus by ovarian steroids and during the periimplantation period provides circumstantial evidence that P4/E actions in the uterus are at least partially mediated in an autocrine/paracrine fashion by growth factors (5-12). However, an important multifunctional growth factor, transforming growth factor-/? (TGF/3), has not yet been studied in the uterus. TGF/3 belongs to a large gene family, and multiple forms of this growth factor have been identified (reviewed in Ref. 13). Because of its role in cell growth, differentiation, and migration as well as in the formation of extracellular matrix and cell surface molecules (13-15), this family of growth factors is likely to play important roles in various events of the implantation process. Therefore, in the present investigation cell type-specific expression of TGF/31 was studied in the mouse uterus during the periimplantation period.
RESULTS In Situ Hybridization Analysis of TGF01 mRNA The cell type-specific expression of TGF/31 mRNA was examined by in situ hybridization. Autoradiographic signals of TGF/31 hybrids on days 1-8 are shown in Figs. 1-3 as brightfield and darkfield photomicrographs (A and B, respectively). On days 1-4 (preimplantation period), TGF/31 mRNA was localized primarily in the luminal and glandular epithelia. However, on days 3 and 4, stromal cells close to the luminal epithelium also contained TGF/31 mRNA. These observations suggest that during the preimplantation period, epithelial cells are the major sites of TGF/31 synthesis. During the postimplantation period (days 5-8), however, TGFj31 hybrids showed a diffuse distribution throughout the deciduum (Figs. 2 and 3). Positive hybrids were also detected in implanting embryos on these days of pregnancy.
Immunohistochemistry Uterine sections in which endogenous peroxidase was blocked showed no staining after incubation with normal rabbit immunoglobulin G (IgG), antibody preabsorbed with TGF/31 resin, or antibody neutralized with excess antigenic peptide (data not shown). The staining pattern of immunoreactive TGF/31 seen with anti-LC was intracellular, and that with anti-CC was extracellular, as reported previously (16, 17). The staining with anti-LC (intracellular) and anti-CC (extracellular) for days 1-8 are represented by C and D, respectively, in Figs. 1-3. On day 1 of pregnancy, while intense intracellular staining was seen in the luminal and glandular epithelia, extracellular staining was evident in the stroma (Fig. 1). Little extracellular staining of the stroma was noted on day 2, in spite of the presence of intracellular staining in the luminal epithelium (Fig. 1). On day 3, intracellular staining persisted in the luminal and some glandular epithelia, and moderately intense extracellular staining reappeared in the stroma (Fig. 1). The intensity of both the intracellular staining in the epithelia and the extracellular staining in the stroma showed a further increase on day 4 (Fig. 1). After blastocyst attachment on day 5, intracellular staining was limited to the luminal epithelium and the primary decidual zone (PDZ), while extracellular staining was intense in the decidualizing stroma surrounding the PDZ (Fig. 2). On day 6, the staining patterns with these two antibodies became clearly distinct; the intracellular staining persisted in the PDZ, while the extracellular staining was restricted to the secondary decidual zone (SDZ; Fig. 2). On day 7, although the PDZ still showed intracellular staining, extracellular staining was only evident at the periphery of the SDZ (decidua capsularis; Fig. 3). The decidua capsularis on day 8 still showed extracellular staining, but the regressing decidua proper showed diffuse intracellular staining. On days 7 and 8, decidua at the mesometrial pole surrounding the blood islands showed intense extracellular staining (Fig. 3). Furthermore, while intracellular staining was present in the embryo on all days examined, extracellular staining was clearly evident on day 8 only (Figs. 2 and 3). During the periimplantation period, TGFj81 mRNA colocalized with immunostaining for intracellular TGF/31 (anti-LC), but not with extracellular immunostaining for this protein (antiCC). This suggests that anti-LC recognizes the sites of synthesis of TGF/31. Northern Blot Hybridization of TGF/31 mRNA To confirm our findings of TGF/31 mRNA in the uterus and deciduum, as determined by in situ hybridization, uterine and decidual RNAs were analyzed by Northern blotting. Adult female mouse adrenal gland RNA served as a positive control for the 2.4-kilobase (kb) TGF/31 transcript (17). As shown in Fig. 4, a 2.4-kb TGF-/31 transcript was detected in total RNA from the uterus (days 1 -4) and the deciduum (days 7-11). There were no remarkable changes in the steady state levels of
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Fig, 1. In Situ Hybridization of TGF/31 mRNA and Immunohistochemical Localization of TGF/31 in the Mouse Uterus on Days 1-4 of Pregnancy In situ hybridization: Mice on the indicated days of pregnancy were anesthetized, and uteri were perfusion fixed with 4% paraformaldehyde in PBS. Paraffin-embedded samples were sectioned at 7 ^M. TGF/31 mRNA was hybridized in situ for 5 h at 42 C with a 35S-labeled human TGF/31 cRNA probe, and RNase-A-resistant hybrids were detected after 4 days of autoradiography using Kodak NTB-2 liquid emulsion. Slides were poststained lightly in hematoxylin. Brightfield (A) and darkfield (B) photomicrographs (x40) of uterine sections on specific days of pregnancy are shown. Uterine sections from each day of pregnancy were hybridized onto the same microscope slides. LE, Luminal epithelium; GE, glandular epithelium; S, stroma; LM, longitudinal muscle; CM, circular muscle. Immunohistochemistry: Bouin's-fixed paraffin-embedded uterine sections (7 HM) from specific days of pregnancy were mounted onto the same slide. After deparaffinization and hydration, sections were incubated in primary antibodies at a concentration of 20 ^g/ml for 24 h at 4 C. Immunostaining was performed by employing the avidin-biotin-peroxidase complex technique. Photomicrographs (x40) of immunostaining by anti-LC (intracellular staining) and anti-CC (extracellular staining) are represented by C and D, respectively, for each day of pregnancy.
uterine or decidual TGF/?1 transcripts on various days of pregnancy. This is probably a reflection of expression of this gene in a subpopulation of cells in these tissues, as evident from in situ hybridization.
DISCUSSION
The present study shows, for the first time, that expression of TGF/31 occurs in the uterus and deciduum during the periimplantation period. Expression of this gene is
temporally and spatially regulated. Sequential steroid hormone interplays before ovulation and after conception initiate a series of changes that involve temporal and cell type-specific proliferation and differentiation of the uterus (18). In the adult ovariectomized mouse, proliferation of luminal and glandular epithelial cells occurs in response to E, whereas maximal stromal cell proliferation requires the presence of both P4 and E (18). A similar pattern of P4/E-regulated cell type-specific proliferation and differentiation occurs in the mouse uterus during the periimplantation period (18). Preovulatory ovarian E secretion results in intense proliferation
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Fig. 2. In Situ Hybridization of TGFj81 mRNA and Histochemical Localization of TGF/31 in Sections of Implantation Sites on Days 5 and 6 of Pregnancy In situ hybridization: Brightfield (A) and darkfield (B) photomicrographs (x40) of sections of implantation sites on the indicated days of pregnancy are shown. Autoradiographic exposure was for 8 days. Immunohistochemistry: C and D represent photomicrographs (x40) of immunostaining of TGFj81 in sections of implantation sites on the indicated days of pregnancy by anti-LC (intracellular staining) and anti-CC (extracellular staining), respectively. Procedures for in situ hybridization and immunohistochemistry were described in Fig. 1. E, Embryo. The left and right sides of each photomicrograph represent mesometrial and antimesometrial poles, respectively.
of the luminal epithelium early on day 1, and that of the glandular epithelium on day 2 of pregnancy. Rising P4 levels from day 3 onward result in proliferation of the stromal cells and affects differentiation of the luminal epithelium. Stromal cell proliferation is enhanced by preimplantation E secretion early on day 4. After the process of implantation is initiated on the evening of day 4, stromal cells begin to differentiate into decidual cells, a process requiring both preimplantation ovarian steroid hormone secretion and the presence of a blastocyst (1, 18). Decidualization is first initiated at antimesometrial sites where blastocysts are implanting. In
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Fig. 3. In Situ Hybridization of TGFj81 mRNA and Histochemical Localization of TGF/31 in Sections of Implantation Sites on Days 7 and 8 of Pregnancy In situ hybridization: Brightfield (A) and darkfield (B) photomicrographs (x40) of sections of implantation sites on the indicated days of pregnancy are shown. Autoradiographic exposure was for 8 days. Immunohistochemistry: C and D represent photomicrographs (x40) of immunostaining of TGF/31 in sections of implantation sites on the indicated days of pregnancy by anti-LC (intracellular staining) and anti-CC (extracellular staining), respectively. Procedures for in situ and immunohistochemistry were described in Fig. 1. E, Embryo; DC, decidua capsularis. The orientations of mesometrial and antimesometrial poles are the same as in Fig. 2. Sections of implantation sites on days 5-8 of pregnancy were mounted onto the same slides in each experiment for comparison.
the mouse, proliferating and differentiating stromal cells surrounding the blastocyst initially form the PDZ on day 5. By day 6, the SDZ is formed around the PDZ, and the PDZ progressively degenerates. After day 8, the growing placenta and embryo slowly displace the SDZ, which is reduced to a thin layer of cells termed the decidua capsularis. The mesometrial decidual cells ultimately form the decidua basalis, a part of the definitive placenta (19). The colocalization of mRNA with intracellular protein in uterine epithelial cells on days 1-4 provides strong evidence for the synthesis of TGF/31 in these cells during the preimplantation period. On the other hand, the presence of extracellular TGF/31 in the stroma in the absence of mRNA in these cells suggests that this
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TGF/31 in the Uterus
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Fig. 4. Northern Blot Detection of Steady State Levels of TGF/31 mRNA in the Mouse Uterus and Deciduum on the Indicated Days of Pregnancy Total RNA (6 ng) samples were separated by formaldehyde agarose gel electrophoresis and blotted to nylon filters. The filters were hybridized with 32P-labeled cRNA probe for human TGF/31 under highly stringent conditions (67 C in 40% fomamide), as described in Materials and Methods. Adrenal gland (AD) RNA was used as a positive control for its TGF/31 mRNA trancript of 2.4 kb. Decidual tissues were isolated by microdissection. A 2.4-kb transcript of TGF/31 mRNA was present in the uterus and deciduum during the periimplantation period.
growth factor, after being synthesized in the epithelium, is released and binds to the extracellular matrix of the stroma, as has been shown to occur in various other tissues (16,17). This finding suggests that TGF/31 plays an important role in controlling epithelial-mesenchymal interactions in the uterus and could be involved in epithelial and stromal cell proliferation and differentiation during the preimplantation period. TGF/3 has been found to have both positive and negative effects on cell growth, depending on the cell type. Inhibition of proliferation by TGF/3 is often associated with terminal differentiation of cells (reviewed in Refs. 12 and 20). The diffuse distribution of mRNA throughout the deciduum from day 5 onward indicates that expression of TGFjSI gene is not restricted to a particular zone of the deciduum. However, the presence of intracellular immunostaining primarily in the PDZ suggests that these cells are the major site of synthesis of TGF/31. The presence of immunostaining in the extracellular matrix of the SDZ again shows that the sites of synthesis and accumulation of TGF/31 in the deciduum are different. The discordant distribution of the mRNA and protein is not uncommon for TGF/31 (17). This finding suggests that TGF/31 may play a role in mediating interactions between the PDZ and SDZ. However, a caveat to interpreting these data is the recent identification of multiple members of the TGF-/3 gene family which share amino acid sequence homology (13). This raises the possibility that antibodies to TGF/31 may also cross-react with other isoforms of TGF/3. Despite this possibility, the colocalization of TGF-/31 mRNA and intracellular staining for TGF/31 coupled with the fact that anti-CC has been shown to react with TGF/31, but not with TGF/32
(16), suggest that TGF/31 was the primary immunoreactive protein in these studies. Although P4 and E are critical for uterine proliferation and differentiation, the mechanism by which they regulate these processes is not known. Epidermal growth factor, insulin-like growth factor-l, and their receptors are expressed in specific uterine cell types during early pregnancy and under E/P4 stimulation, which has led to the suggestion that these proteins are involved in uterine growth and differentiation (5-12). Because of its roles in the regulation of formation of extracellular matrix and cell surface molecules, its involvement in regulation of cell proliferation and differentiation (1315), and its potent immunoregulatory effects, the selective synthesis and accumulation of TGF/31 in the uterus and deciduum suggest that this growth factor plays critical roles during the periimplantation period. The changing endocrine state of the female during the reproductive cycle as well as during pregnancy causes the uterus to undergo extensive remodelling (tissue formation and repair) that is associated with changes in basement-membrane components (21, 22). For example, the various basement membrane components (collagens, laminin, fibronectin, and proteoglycans) in the human uterus undergo changes throughout the menstrual cycle and during pregnancy (21). Likewise, mouse stromal cells undergoing decidualization also synthesize these extracellular components (22). TGF/3 is known to cause accumulation of matrix proteins not only by enhancing their synthesis, but also by decreasing matrix degradation by down-regulating the secretion of proteases and up-regulating the synthesis of protease inhibitors (13-15). Furthermore, this TGF has recently been shown to control a superfamily of cell surface molecules called integrins (14,15). The integrins mediate cell-cell and cell-matrix adhesions (14,15). The apposition and adhesion of the blastocyst trophectoderm with the uterine luminal epithelium as well as the later invasion of the endometrium by the trophoblast cells are dependent upon cell-cell interactions and cell migration, which could be mediated in an autocrine/ paracrine manner by TGF/3. Because TGF/3 down-regulates a specific laminin-binding integrin and because cell migration depends upon laminin binding (14, 15), we postulate that this growth factor participates in controlled invasion of the uterus by the trophoblast cells. Extracellular immunoreactive TGF/31 in the deciduum at the mesometrial pole on days 7 and 8 may also be involved with development of the placenta. Because the placenta undergoes continuous changes in size, shape, and internal structure, a role for TGF/3 in placental development has been considered (17). Even though TGF/3 opposes fibroblast growth factor-induced endothelial cell proliferation in vitro (13), TGF/3 has been shown to be angiogenic in vivo, and therefore, this growth factor may be involved with the extensive neovascularization that occurs during decidualization. The embryo is considered an allograft (2). Despite many suggestions as to mechanisms, it remains a challenge to explain the escape of the embryo from the
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immunological responses of the mother (2). Because TGF/3 is a potent immunosuppressant (reviewed in Ref. 13), this growth factor, of embryonic and/or uterine origin, could be involved in the immunological acceptance of the embryo by the mother (23). Furthermore, our present findings and previous reports of expression of TGF/3 in pre- and postimplantation embryos (24, 25) suggest a role for this growth factor in embryogenesis. The role of TGF/3s and fibroblast growth factor in amphibian embryo morphogenesis is well documented (26, 27). The accumulation of TGF/31 in the extracellular matrix of the uterine stroma and deciduum suggests an active uptake. Whether this uptake is receptor mediated or via binding of the protein with the matrix components is not known. It is interesting to note that TGF/3 copurifies with fibronectin (28), and membraneanchored and soluble forms of /3-glycan (proteoglycan) can bind TGF/8 (29). To understand the mechanisms of action of TGF/31 during the periimplantation period, data on temporal and cell type-specific expression of its receptors are needed. In summary, the present finding of cell type-specific expression of TGF/31 in the uterus and deciduum during early murine pregnancy suggests that this multifunctional growth factor plays important roles in various events of blastocyst implantation, decidualization, placentation, and early embryogenesis. Future experiments will determine the effects of P4 and/or E on regulation of the expression of TGF/31 in the uterus, and whether the implanting embryo influences the decidual expression of this growth factor.
MATERIALS AND METHODS Animals All experiments involving animals were conducted in accordance with NIH standards for the care and use of experimental animals. Female CD-1 mice (48 days old; Charles River Laboratories, Raleigh, NC) were mated with fertile males (90 days old) of the same strain. The morning of finding a vaginal plug was defined as day 1 of pregnancy. Mice were killed between 0800-0900 h on specific days of pregnancy. Production of Antisera Polyclonal antibodies were produced in rabbits to different preparations of a peptide corresponding to amino acids 1 -30 of TGF/31. Two antibodies, anti-CC (1-30) and anti-LC (1-30), were produced as described by Ellingsworth ef a/. (30) and Flanders ef al. (16), respectively. In each case, the injected peptide was not coupled to a carrier protein. IgG was isolated from each antiserum by protein-A-Sepharose chromatography (31) for use in immunohistochemistry. A previous study with these antibodies, which seem to recognize different epitopes of TGF/3, has shown that anti-CC-(1-30) stains principally extracellular TGF/3, while anti-LC-(1-30) stains intracellular TGFjS (16). These antibodies do not react with TGF/32 on Western blots (16, 30); however, reactivities with TGF/33, -4, and -5 have not yet been tested. Immunohistochemistry Immunolocalization was based on the procedure described previously (5, 17). In brief, uteri were excised, cleaned of fat,
cut into 4- to 6-mm pieces, and fixed in Bouin's solution for 24 h. Paraffin-embedded tissue blocks were sectioned at 7 ^M and mounted on poly-L-lysine-coated slides. Sections were deparaffinized, hydrated in PBS for 20 min, and then incubated in blocking solution (10% normal goat serum) for 10 min before incubation in primary antibodies at 20 ng/m\ for 24 h at 4 C. Immunostaining was performed using a Zymed Histostain-SP kit for rabbit primary antibody (Zymed Laboratories, San Francisco, CA). This kit uses a biotinylated secondary antibody, a horseradish peroxidase-streptavidin conjugate, and a substrate chromogen mixture (32). Blocking of endogenous peroxidase activity was achieved by a 35-sec incubation in 0.23% periodic acid in PBS after secondary antibody incubation (33). Sections were counterstained lightly with hematoxylin, mounted, and examined under brightfield. Red deposits indicated the sites of immunostaining. Control experiments included incubation of sections with normal rabbit IgG, antisera preincubated with TGF-/?1-Sepharose resin, or primary antibodies neutralized with an excess of appropriate peptides. Hybridization Probes A cDNA clone was obtained from Rik Derynck (Genentech, Inc., San Francisco, CA) containing a 1050-basepair fragment encoding human TGF/31 inserted into the Sp64 plasmid (34) and used as a template for the Sp6. polymerase-directed synthesis of 32P- and 35S-labeled cRNA probes, as described by Melton ef al. (35). Probes had specific activities of about 2 x 109dpm/Mg. In Situ Hybridization The details of this technique have been described by us previously (5, 36). Briefly, hybridization was performed on paraformaldehyde perfusion-fixed tissue sections (7 /IM) using an 35S-labeled cRNA probe, as described above. Hybridization was carried out as described previously (5, 36). After hybridization, the sections were treated with RNase-A (20 A*g/ml). Autoradiography was performed with Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY) for 4-8 days, and sections were poststained lightly with hematoxylin. Isolation of RNA Uterine RNA was extracted using the guanidine isothiocyanate procedure described by Han et al. (37). Total RNA from decidua was extracted using the sodium dodecyl sulfatephenol-chloroform procedure described in detail by Andrews ef al. (38). Northern Blot Hybridization Total RNA (6 ng) was denatured and separated by formaldehyde agarose gel electrophoresis (36, 38, 39). After transfer to MSI nylon membranes (40), the membranes were baked in a vacuum oven at 75 C for 5 h. Northern blots were prehybridized, hybridized, and washed as described by Andrews ef al. (36, 38) with the following modifications. Hybridization was carried out under highly stringent conditions (67 C in the presence of 40% formamide, 3 x SET (1 x SET = 150 mM NaCI, 5 mM EDTA, and 10 mM Tris-HCI, pH 8.0), and 10% dextran sulfate to reduce background signals and ensure specificity. In all experiments, duplicate gels were stained with acridine orange to ensure the integrity of the RNA samples and to confirm that equal amounts of RNA had been loaded onto each lane. Acknowledgments Thanks are due to Susan Strong for technical assistance. Received February 15, 1990. Revision received March 30, 1990. Accepted April 4,1990.
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TGF/31 in the Uterus
Address requests for reprints to: Dr. S. K. Dey, Departments of Obstetrics-Gynecology and Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66103. This work was supported in parts by grants from the NICHHD (HD-12304 to S.K.D.), the NIEHS (ES-04725 to G.K.A.), and the Wesley Foundation (to G.K.A. and S.K.D.). * Present address: Department of Animal Reproduction, University of Osaka Prefecture, College of Agriculture, Sakai City, Osaka 591, Japan. t March of Dimes predoctoral fellow.
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16. Flanders KC, Thompson NL, Cissel DS, Van ObberghenSchilling EV, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB 1989 Transforming growth factor-/?1: histochemical localization with antibodies to different epitopes. J Cell Biol 108:653-660 17. Thompson NL, Flanders KC, Smith JM, Ellingsworth LR, Roberts AB, Sporn MB 1989 Expression of transforming growth factor-/31 in specific cells and tissues of adult and neonatal mice. J Cell Biol 108:661-669 18. Huet-Hudson YM, Andrews GK, Dey SK 1989 Cell typespecific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period. Endocrinology 125:1683-1690 19. Krehbiel RH 1937 Cytological studies of decidual rection in the rat during pregnancy and in the production of deciduomata. Physiol Zool 10:212-238 20. Moses HL, Tucker RF, Leof EB, Coffey RJ, Halper J, Shipley GD 1985 Type-beta transforming growth factor is growth stimulator and growth inhibitor. In: Feramisco J, Ozanne B, Stiles C (eds) Cancer Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, vol 3:65-71 21. Aplin JD, Charlton AK, Ayad S 1988 An immunohistochemical study of human endometrial extracellular matrix during the menstrual cycle and first trimester of pregnancy. Cell Tissue Res 253:231-240 22. Wewer UM, Damjanov A, Weiss J, Liotta LA, Damjanov I 1986 Mouse endometrial stromal cells produce basementmembrane components. Differentiation 32:49-58 23. Clark DA, Falbo M, Rowley RB, Banwatt D, StedronskaClark J 1988 Active suppression of host-vs-graft reaction in pregnant mice. IX. Soluble suppressor activity obtained from allopregnant mouse decidua that blocks the cytolytic effector response to IL-2 is related to transforming growth factor-^. J Immunol 141:3833-3840 24. Rappolee DA, Brenner CA, Schultz R, Mark D, Werb Z 1988 Developmental expression of PDGF, TGFa, and TGF/? genes in preimplantation mouse embryos. Science 242:1823-1825 25. Heine Ul, Flanders KC, Roberts AB, Minoz EF, Sporn MB 1987 Role of transforming growth factor-/? in the development of the mouse embryo. J Cell Biol 105:2861-2876 26. Kimelman D, Kirschner M 1987 Synergistic induction of mesoderm by FGF and TGF/? and the identification of an mRNA coding for FGF in early Xenopus embryo. Cell 51:869-877 27. Rosa F, Roberts AB, Danielpour D, Dart LL, Sporn MB, Dawid IB 1988 Mesoderm induction in amphibians: the role of TGF/?2-like factors. Science 239:783-786 28. Fava RA, McClure DB 1987 Fibronectin-associated transforming growth factor. J Cell Physiol 131:184-189 29. Andres JL, Stanley K, Cheifetz S, Massague J 1989 Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-/?. J Cell Biol 109:3137-3146 30. Ellingsworth LR, Brennan JE, Fork K, Rosen DM, Bentz H, Piez KA, Seyedin SM 1986 Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor /3. J Biol Chem 261:12362-12367 31. Flanders KC, Roberts AB, Ling N, Fleurdelys BE, Sporn MB 1988 Antibodies to peptide determinants in transforming growth factor /3 and their applications. Biochemistry 27:739-746 32. Hsu S-M, Raine L 1984 The use of avidin-biotin-peroxidase complex (ABC) in diagnostic and research pathology. In: DeLellis RA (ed) Advances in Immunocytochemistry. Mason, New York, pp 31-42 33. Kelly J, Whelan CA, Weir DG, Reighery C 1987 Removal of endogenous peroxidase activity from cryostat section for immunoperoxidase visualization of monoclonal antibodies. J Immunol Methods 96:127-132 34. Derynk R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV 1985 Human transforming growth factor-/? complementary DNA
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sequence and expression in normal and transformed cells. Nature 316:701-705 35. Melton DA, Kreig PA, Rebegliati MR, Maniatis T, Kinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing bacteriophage SP6 promoter. Nucleic Acids Res 12:7035-7057 36. De SK, McMaster MT, Dey SK, Andrews GK 1989 Cellspecific metallothionein gene expression in mouse decidua and placentae. Development 107:611-621 37. Han JH, Stratona C, Rutter WJ 1987 Isolation of fulllength putative rat lysophospholipase cDNA using im-
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Symposium on Modes of Action of GnRH and GnRH Analogs February 27-March 2, 1991 The Registry Resort Scottsdale, Arizona Co-Chairmen: William F. Crowley, MD and P. Michael Conn, Ph.D. Sponsored by Serono Symposia, USA This symposium will address the most current data on GnRH physiology, focusing on the basic information regarding its physiology, molecular biology, and normal secretion in man and animals. The program will also feature the area of GnRH antagonists as they apply to the human. Major sessions are I) Overview of GnRH Secretion and Mechanism of Action II) Molecular and Developmental Control of GnRH Expression III) GnRH Physiology: Animal Models IV) GnRH Antagonists. The program will include invited expert speakers, poster presentations of original abstracts, and ample time for informal interaction among participants. Deadline for submission of abstracts is January 11, 1991. Category I CME credits will be available. For further information, please contact L. Lisa Kern, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, MA 02061. Telephone: 800-283-8088 or 617-982-9000. Telefax: 617-982-9481.
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