Progesterone-regulated determinants of stromal cell survival and death in uterine decidua are linked to protein kinase C activity.

This study examined the role of protein kinase C enzymatic activity as a physiologic determinant of stromal cell death in decidua basalis (DB) during pregnancy. The expression of epidermal growth factor receptor (EGF-R) and Bcl2 was used as an indicator of stromal cell proliferation/survival, whereas Bax and the occurrence of apoptosis provided an index of cell death. Stromal cell cycle progression during pregnancy and after in vivo administration of phorbol esters was analyzed by flow cytometry. DB were isolated from pregnant rats between Days 8 and 21 of pregnancy and prepared for immunohistochemistry, Western blotting procedures, or flow cytometry. The results showed that stromal cells were actively proliferating on Days 8 and 10, whereas the frequency of cell death by apoptosis increased progressively between Days 14 and 21 (Day 22 is term). The proliferative stage was characterized by low PKC activity and high levels of EGF-R and Bcl2 expression. On the other hand, DB regression (Days 14-21) was marked by an elevation in endogenous PKC activity and Bax expression; EGF-R and Bcl2 were suppressed. Administration of phorbol 12-myristate, 13-acetate (0.4 micromole/kg) induced apoptosis on Day 10. Additionally, antiprogestin (RU-486) given on Day 9 induced PKC activity and Bax expression within 6 h and suppressed Bcl2 and EGF-R. By 12 h, RU-486 enhanced percent apoptotic cells. Thus, enhanced levels of PKC activity were closely linked to stromal cell apoptosis.     

Regression of the decidualized mesometrium and decidual cell apoptosis are associated with a shift in expression of Bcl2 family members

The purpose of this study was to determine whether regression of the decidua basalis (DB), which begins on Day 14 of pregnancy in the rat, results from an intrinsic program of apoptosis regulated by Bax and Bcl2. Expression of Bax and Bcl2 and the incidence of apoptosis were evaluated throughout gestation by Western blot analysis and detection of DNA fragments. Antiprogestin (RU486) was also administered during proliferation of DB to study progesterone regulation of Bax/Bcl2 balance. Bax, the pro-apoptotic protein, was expressed at a low level throughout pregnancy, whereas Bcl2, the pro-survival partner, was most abundantly expressed on Days 8 and 10, which are a time of proliferation and decidualization, and declined to barely detectable levels thereafter. These changes resulted in a 12-fold increase in the Bax:Bcl2 ratio on Day 17 as compared with Day 8 of pregnancy (P < 0.05). DNA laddering and in situ staining of DNA fragments first became visible on Day 14 and involved 2% of cells by Days 17 and 21 (P < 0.05). Treatment with RU486 on Day 9 enhanced Bax and suppressed Bcl2 within 6 h, increasing the Bax:Bcl2 ratio sixfold (P < 0.05). Apoptosis was minimal at 6 h and increased to 9% of cells by 24 h (P < 0.05). Thus, progesterone appears to regulate the apoptotic threshold of stromal cells by modulating Bax and Bcl2 expression.     

Changes in the temporal and spatial expression of H beta 58 during formation and maturation of the chorioallantoic placenta in the Rat

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with H beta 58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. H beta 58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of H beta 58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6-8 of pregnancy) showed H beta 58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed H beta 58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of H beta 58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive H beta 58 localized to erythroid cells within the developing fetal vasculature of the chorioallantoic primordia at Day 10 of pregnancy. By Day 12, the fetal vasculature extended into the placental labyrinth, and the erythroid stem cells continued to strongly express H beta 58. At Day 14 of pregnancy, immunoreactivity became evident in the trophoblast giant cells and syncytiotrophoblast of the fetal placenta. As the chorioallantoic placenta matured (Day 18), H beta 58 mRNA was 3.6-fold higher in the labyrinth compared with the junctional region. Stable cell lines (HRP/LRP) isolated from the rat labyrinthine placenta expressed H beta 58 mRNA and protein. The expression pattern of H beta maternal and fetal placental tissues and its early expression in fetal erythroid stem cells during formation and maturation of the chorioallantoic placenta suggest that H beta 58 plays key roles in the regulatory networks that control hematopoietic development and placentation.     

Expression and regulation of calcitonin gene-related Peptide receptor in rat placentas

Calcitonin gene-related peptide (CGRP), one of the most potent vasodilators known, exerts its biological action by interacting with its receptors. Recent reports suggest the existence of two types of CGRP receptors, CGRP-A and CGRP-B. The current study was designed to examine whether CGRP-B receptors are present in the rat placenta, and if they are, whether they are modulated by gestational age and by sex-steroid hormones. Placentas were obtained from timed pregnant Sprague-Dawley rats that were killed on Days 17-21 and 22 before and during labor (n = 6 for each gestational age). In addition, placentas were also obtained from pregnant rats injected with progesterone (P(4); 4 mg per rat per day s.c. on Days 20-22), antiprogesterone RU-486 (10 mg/rat s.c. on Day 17), 17beta-estradiol (5 micro g/rat s.c. on Day 17), and antiestrogen ICI 182780 (0.3 micro g/rat s.c. on Day 17). Results showed that first, immunoflourescent staining of rat placentas using monoclonal anti-CGRP-B receptor antibody revealed the presence of CGRP-B receptors in the labyrinthine layer of the placenta, specifically to the trophoblast and blood vessel endothelium and underlying smooth muscle cells. The intensity of staining was lower in placentas obtained during labor. Second, a single band of 66 kDa, reactive to CGRP-B receptor antibody, was obtained in Western blotting of the rat placenta; third, densitometric analysis of protein bands showed that CGRP-B receptors were increased from Day 17 to Day 22, with maximal levels obtained on Day 22 before labor, which was 10 times higher than that of Day 17 (P < 0.01); fourth, expression of CGRP-B receptors in rat placenta decreased during labor (8% vs. 100% on Day 22 before labor, P < 0.01); fifth, P(4) given during Days 20-22 attenuated the fall in placental CGRP-B receptors at term labor; sixth, RU-486 given on Day 17 of gestation significantly decreased expression of placental CGRP-B receptors (18% vs. 100% in controls at 6 h, P < 0.01); seventh, a significant decrease in CGRP-B receptor expression was noted 48 h after estrogen administration; and eighth, ICI 182780 treatment on Day 17 increased placental CGRP-B receptors (152% vs. 100% in control at 48 h, P < 0.01). These results indicate that CGRP-B receptors are present in rat placenta and that receptor levels are higher with gestational age and lower at term labor. Progesterone stimulated and estrogen inhibited placental CGRP-B receptor expression. Thus, elevations in placental CGRP-B receptors in late pregnancy could play a role in increasing blood flow through the fetoplacental unit associated with rapid fetal growth during late gestation.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Inhibition of egg development and implantation in rats after post-coital administration of the progesterone antagonist RU 486

RU 486 was administered to rats on Day 1 or Days 1 + 2 of pregnancy. Endometrial sensitivity (i.e. decidualization in response to oil instillation) was delayed by 2.5 mg/kg injected s.c. on Day 1, and almost half of the animals also exhibited a delay in implantation of 1-2 days. Higher doses (5 or 10 mg/kg) administered on Days 1 + 2 reduced the number of implantations to zero in all animals. Apparently normal morulae were found up to the evening of Day 4 in the oviduct and/or the uterus of most animals. However, on the morning of Day 5, ova were detected in only 25% of the animals and all were in the uterus: none was at the blastocyst stage and they appeared to be degenerated or compacted morulae. Egg survival and rate of egg recovery from the uterus was not improved by early ovariectomy, showing that this antiprogestagen acts on these events independently of the presence of circulating oestrogens.     

Gamma‐amino butyric acid and the A‐type receptor suppress decidualization of mouse uterine stromal cells by down‐regulating cyclin D3

Uterine decidualization, characterized by stromal cell proliferation and differentiation into polyploid decidual cells, is critical to the establishment of pregnancy in mice, although the mechanism underlying this process remains poorly understood. This study is the first to investigate the expression of gamma‐amino butyric acid (GABA) and the GABA A‐type receptor π subunit (GABPR) in the early‐pregnancy mouse uterus and their roles in decidualization. The expression of GABRP was detected from Day 4 to 8 of pregnancy. The effects of GABA and GABA A‐type receptor on cell proliferation and apoptosis were investigated using the Cell Titer 96® AQueous One Solution Cell Proliferation Assay and flow cytometry. The levels of cyclin D3 protein were measured in cultured stromal cells artificially induced to undergo decidualization, and treated with GABA and a GABA A‐type receptor agonist or antagonist, respectively, at the same time. mRNA expression of gabrp in implantation sites was lower than that in inter‐implanted sites. GABA and GABRP protein were localized in the luminal and glandular epithelium, stromal cells, and decidual cells. In vitro, GABPR protein level was decreased in cultured stromal cells during the decidualization process. The addition of GABA and the GABA A‐type receptor agonist Muscimol inhibited stromal cell proliferation, promoted apoptosis, and arrested cells in S‐phase, followed by decreased expression of cyclin D3. These results show that in mice, GABA was actively involved in inhibiting stromal cell proliferation and suppresses decidualization progress through GABA A‐type receptors by down‐regulating cyclin D3 level. Mol. Reprod. Dev. 80: 59–69, 2013. © 2012 Wiley Periodicals, Inc.     

Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats

These studies examine the trophic effects of progesterone (P) on the progesterone receptor (Rp) and growth of the decidua basalis (DB) and junctional zone (JZ) in the rat placenta. Pregnant rats were ovariectomized (Ovx) in mid-pregnancy and received steroid replacement therapy consisting of implantation of P pellets (25 mg) and injections of estradiol (E), 2 micrograms s.c., daily. Placental protein synthesis, measured by 3H-leucine incorporation in vitro, decreased more than 99% within 24 h of Ovx. However, treatment with P immediately after castration maintained control levels of synthesis. Delay of P treatment for 4 h caused a 60% decline in protein production measured 20 h later (p less than 0.01). Intraperitoneal implantation of a 50-mg pellet of the antiprogestin, RU-38486, in intact pregnant rats decreased protein synthesis by 50% within 6 h and by more than 90% 12 h and 24 h post-implantation (p less than 0.01). Growth of DB and JZ in Ovx rats treated for 48 h with P and/or E was studied both histologically and by changes in protein and DNA content. Rp binding activity was also measured by exchange assay under equilibrium conditions. Only P was able to reverse the effects of Ovx on growth of the DB and JZ. P also maintained Rp levels in the DB above those observed in Ovx and Ovx + E-treated groups (p less than 0.01). The Rp may be a constitutive product in the JZ since binding activity was not altered by Ovx or by steroid treatments. This study shows that P is clearly a trophic hormone of the maternal and chorioallantoic placenta and is essential for placental growth, cellular differentiation, and histological integrity.     

Insulin-like growth factors and their binding proteins define specific phases of myometrial differentiation during pregnancy in the rat

While the insulin-like growth factor (IGF) system is known to regulate uterine function during the estrous cycle, there are limited data on its role in myometrial growth and development during pregnancy. To address this issue, we defined the expression of the Igf hormones (1 and 2), their binding proteins (Igfbp 1-6), and Igf1r receptor genes in pregnant, laboring, and postpartum rat myometrium by real-time PCR. IGF family genes were differentially expressed throughout gestation. Igf1 and Igfbp1 mRNA levels were upregulated during proliferative phase (Days 6-12) of rat gestation. Igfbp3 gene expression also was elevated in proliferating smooth muscle cells (SMCs) and was highest at the time of transition between proliferative and synthetic phases (Days 12-15). Igfbp6 gene expression profile paralleled plasma progesterone (P4) concentrations, peaking during the synthetic phase (Days 17-19) and decreasing thereafter. Administration of P4 at late pregnancy (starting from Day 20) to maintain elevated plasma P4 concentrations blocked the onset of labor and prevented the fall in Igfbp6 mRNA levels. In contrast, the treatment of pregnant rats with the P4 receptor antagonist RU486 on Day 19 induced preterm labor and the premature decrease of Igfbp6 gene expression. Igfbp2 gene expression was transiently upregulated during the contractile phase of gestation (Days 21-23) solely in the gravid horn of unilaterally pregnant rats, but it was not affected in P4- or RU486-treated animals, supporting a role for mechanical stretch imposed by the growing fetuses. Igfbp5 gene was induced during postpartum involution. Our results suggest the importance of the IGF system in phenotypic and functional changes of myometrial SMCs throughout gestation in preparation for labor.     

Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus

Progesterone (P4) is unequivocally required to maintain a uterine environment conducive to pregnancy. This study investigated the effects of P4 treatment on expression of selected growth factors (fibroblast growth factor 7 [FGF7], FGF10, hepatocyte growth factor [HGF], and insulin-like growth factors [IGF1 and IGF2]), their receptors (MET, FGFR2(IIIB), and IGF1R), and IGF binding proteins (IGFBPs) in the ovine uterus. Ewes received daily injections of corn oil vehicle (CO) or 25 mg of P4 in vehicle from 36 h after mating (Day 0) to hysterectomy on Day 9 or Day 12. Another group received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Day 8 through Day 12. Endometrial FGF10 mRNA levels increased between Day 9 and Day 12 and in response to P4 on Day 9 in CO-treated ewes, which had larger blastocysts, and were substantially reduced in P4+RU486-treated ewes, which had no blastocysts on Day 12. Endometrial FGF7 or HGF mRNA levels were not affected by day or reduced by RU486 treatment, but MET mRNA levels were higher in P4-treated ewes on Day 9 and Day 12. Levels of IGF1, IGF2, and IGF1R mRNA in the endometria were not affected by early P4 treatment. Although stromal IGFBPs were unaffected by P4, levels of IGFBP1 and IGFBP3 mRNA in uterine luminal epithelia were increased substantially between Day 9 and Day 12 of pregnancy in CO-treated ewes and on Day 9 in early P4-treated ewes. Therefore, FGF10, MET, IGFBP1, and IGFBP3 are P4-regulated factors within the endometrium of the ovine uterus that have potential effects on endometrial function and peri-implantation blastocyst growth and development.     

Hmgn5 functions downstream of Hoxa10 to regulate uterine decidualization in mice

Although Hmgn5 is involved in the regulation of cellular proliferation and differentiation, its physiological function during decidualization is still unknown. Here we showed that Hmgn5 was highly expressed in the decidual cells. Silencing of Hmgn5 expression by specific siRNA reduced the proliferation of uterine stromal cells and expression of Ccnd3 and Cdk4 in the absence or presence of estrogen and progesterone, whereas overexpression of Hmgn5 exhibited the opposite effects. Simultaneously, Hmgn5 might induce the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP and progesterone could up-regulate the expression of Hmgn5, but the up-regulation was impeded by H89 and RU486, respectively. Attenuation of Hmgn5 expression could block the differentiation of uterine stromal cells in response to cAMP and progesterone. Further studies found that regulation of cAMP and progesterone on Hmgn5 expression was mediated by Hoxa10. During in vitro decidualization, knockdown of Hmgn5 could abrogate Hoxa10-induced upregulation of Prl8a2 and Prl3c1, while overexpression of Hmgn5 reversed the inhibitory effects of Hoxa10 siRNA on the expression of Prl8a2 and Prl3c1. In the stromal cells undergoing decidualization, Hmgn5 might act downstream of Hoxa10 to regulate the expression of Cox-2, Vegf and Mmp2. Collectively, Hmgn5 may play an important role during mouse decidualization.     

Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.     

Epidermal growth factor inhibits 8-Br-cAMP-induced decidualization of human endometrial stromal cells

The effects of epidermal growth factor (EGF) on human endometrial stromal cells have not been characterized well, although production of EGF in endometrial epithelial and stromal cells and expression of EGF receptors in endometrial stromal cells have been reported. We investigated the effects of EGF on endometrial cell viability, 8-Br-cAMP-induced stromal decidualization, and prolactin secretion from decidualized endometrial stromal cells using an in vitro decidualization activity assay of human endometrial stromal cells. EGF did not show any significant effects on viable cell numbers of nondecidualized and 8-Br-cAMP-induced decidualized cells. Prolactin release from the 8-Br-cAMP-induced decidualized cells was not affected by EGF. However, EGF dose-dependently inhibited prolactin release from the stromal cells that were in the process of decidualization by co-stimulation with 8-Br-cAMP and EGF, though there was no significant change in viable cell numbers of the 8-Br-cAMP-stimulated decidualizing cells. Flow cytometric analysis revealed that 8-Br-cAMP enhanced EGF receptor expression on the endometrial stromal cells. These results indicate that endometrial EGF inhibits decidualization through autocrine/paracrine mechanisms.     

Implantation delay and anti-deciduogenic activity in the rat by the anti-androgen, hydroxyflutamide

Studies were undertaken to determine whether the anti-androgen, hydroxyflutamide, has anti-progestagenic activity by using implantation, maintenance of pregnancy, and decidualization as end points. Prepubertal rats were induced to ovulate with the injection of 4 IU pregnant mare's serum gonadotropin and allowed to mate. Mated females were assigned randomly to various treatment groups. Beginning at 0800 h on Day 4 of pregnancy and at 12-h intervals thereafter, rats received a series of 6 s.c. injections of 5 mg hydroxyflutamide in oil, or oil only. Localized changes in endometrial vascular permeability, indicative of implantation, were assessed on Days 6 and 8 of pregnancy, after an injection of Evans blue dye. By Day 6, implantation has been initiated in the vehicle-treated rats, but not in hydroxyflutamide-treated rats. Hydroxyflutamide treatment was terminated on Day 6, and implantation was initiated by Day 8. The weights of uterine dye sites in hydroxyflutamide-treated rats on Day 8 were similar to those in vehicle-treated rats on Day 6. The number of fetuses and placentae were similar in all groups on Day 19. The weights of fetuses in both hydroxyflutamide-treated and hydroxyflutamide + progesterone-treated rats were similar and significantly lower than those in control rats. Although there were no significant differences between vehicle-treated or hydroxyflutamide-treated rats in the proportion of rats delivering and litter size, the hydroxyflutamide-treated rats delivered pups a mean of one day later than did the controls. Endometrial decidualization in ovariectomized, steroid-treated rats, following artificial stimuli, was significantly suppressed in hydroxyflutamide-treated rats compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of luteinizing hormone-releasing factor (LH-RF) and LH on maintenance of early gestation in rats.

The role of luteinizing hormone (LH) and LH-releasing hormone (LH-RH) in the maintenance of early pregnancy in rats was studied. Serum levels of progesterone (P) and LH were measured daily in untreated pregnant rats from Day 4 through parturition. Serum levels of P and LH were determined on Days 11 and 15 of pregnancy in animals treated with antisera to LH (LH-A/S) and to LH-RH (LH-RH-A/S) on Days 8-10. Serum levels of P peaked on Days 7 and 16 in untreated animals, after which they declined sharply just before delivery. Serum LH fluctuated between 30-160 ng/ml during pregnancy but did not exhibit any distinctive peaks. Treatment with .2 ml LH-A/S on Days 8-10 reduced serum P to virtually undetectable levels on Day 11, and only a slight recovery was evident on Day 15. Lower doses of LH-A/S had no effect. Administration of 1.3 ml LH-RH-A/S had no effect on serum levels of P or LH, and did not impede fetal development. The results indicate that LH is essential to the luteotropic complex of early pregnancy in the rat, and also that LH-RH-A/S can maintain to some extent basal levels of P and LH during early pregnancy.     

The effect of transforming growth factor-alpha on the progression of decidualization in rats

Although transforming growth factor-alpha (TGF-alpha), one of the epidermal growth factor (EGF) family of growth factors, is expressed in the rat decidual cells, its roles in decidualization remain to be elucidated. This study examined the effect of TGF-alpha on the progression of decidualization and a possibility for involvement of prostaglandins (PGs) in its action. Pseudopregnant rats were ovariectomized and given endometrial trauma on Day 5 (vaginal plug = Day 1) and were daily treated with 2 mg progesterone thereafter. Immunocytochemical localization of EGF receptor was distinctly evident in the decidual, stromal and epithelial cells on Day 7. Continuous infusion of TGF-alpha (500 pg/h) into the uterine lumen from Day 7 significantly increased weights of the uterine horns with deciduomata on Day 9. Although injection on Day 7 of indomethacin, an inhibitor of PGs synthesis, decreased the uterine weight, this effect was overridden by the continuous infusion of this growth factor. These results demonstrated the stimulatory action of TGF-alpha on the progression of decidualization. Further, TGF-alpha increased the secretion of prostaglandin E in cultured decidual and/or stromal cells dose-dependently, suggesting the possibility that PGs mediate the action of this growth factor.     

Expression of tenascin-C in stromal cells of the murine uterus during early pregnancy: induction by interleukin-1 alpha, prostaglandin E(2), and prostaglandin F(2 alpha)

Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1 alpha (IL-1 alpha) and prostaglandin E(2) (PGE(2)) and F(2 alpha) (PGF(2 alpha)) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and ss-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1 alpha antibody showed epithelial cells to be positive on Days 2-4 of pregnancy, and addition of progesterone but not ss-estradiol enhanced IL-1 alpha expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1 alpha antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1 alpha secreted by epithelial cells and PGE(2) and PGF(2 alpha) secreted by stromal cells.