Decidualization regulates the expression of the endometrial chorionic gonadotropin receptor in the primate

Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.     

Expression and regulation of cytosolic prostaglandin E synthase in mouse uterus during the peri-implantation period

Prostaglandin E(2) (PGE(2)) is considered important for blastocyst spacing, implantation, and decidualization in rodent uteri. PGE synthase (PGES) catalyzes the isomerization of PGH(2) to PGE(2). Two isoforms of PGES exist: microsomal PGES (mPGES) and cytosolic PGES (cPGES); however, the expression and regulation of cPGES in the mammalian uterus during early pregnancy are still unknown. The aim of this study was to investigate the differential expression of cPGES in mouse uterus during early pregnancy and its regulation under different conditions using in situ hybridization and immunohistochemistry. A strong level of cPGES mRNA signal was exhibited in the stromal cells at the implantation site on Day 5 of pregnancy, whereas cPGES immunostaining was strongly detected in the luminal epithelium. The signals for both cPGES mRNA and immunostaining were strongly detected in the decidualized cells from Days 6-8 of pregnancy. A basal level of cPGES mRNA signal and immunostaining was exhibited in the uterus in delayed implantation. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, cPGES mRNA signal was strongly detected in the stroma underlying the luminal epithelium at the implantation site, and cPGES immunostaining was strongly observed in the luminal epithelium surrounding the implanting blastocyst. A strong cPGES mRNA signal and immunostaining were detected in decidualized cells under artificial decidualization, whereas only a basal level of cPGES mRNA signal and immunostaining were observed in the control horn. Our data suggest that cPGES may play an important role during implantation and decidualization.     

Progesterone regulation of epidermal growth factor receptor in rat decidua basalis during pregnancy.

Ovarian steroid hormones and epidermal growth factor (EGF) play important interactive roles in proliferation and decidualization of mesometrial stromal cells during pregnancy. This study determined the ontogeny of EGF receptor (EGF-R) expression in the decidua basalis (DB) throughout pregnancy and its regulation by estrogen and progesterone (P4). DB were isolated from rats between Days 8-21 of pregnancy and prepared for immunohistochemistry or Western analysis. In one study, rats were ovariectomized (Ovx) on Day 8 or 9 and given estradiol-17beta, P4, or both. In another study, the antiprogestin, mifepristone (RU-486), was administered on Day 9. During normal pregnancy, total EGF-R (phosphorylated and unphosphorylated forms) increased from Day 8 to a maximum level on Days 10 and 12. Tyrosine-phosphorylated EGF-R (pEGF-R), the bioactive form, was also highest on Days 10 and 12. Both forms of receptor decreased to almost undetectable levels during DB regression on Days 17-21. Immunohistochemistry of DB from Ovx rats revealed that only P4 was able to maintain normal expression of EGF-R; RU-486 decreased EGF-R expression within 6 h, and by 24 h EGF-R and pEGF-R were 15% of the Day 10 control group levels. These findings show that EGF-R is a P4-dependent protein associated with stromal cell proliferation and decidualization.     

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.     

Characterization of deciduoma marker proteins in hamster uterus: detection in decidual cell cultures

The objective of this study was first, to identify the proteins associated with decidualization of the hamster uterus by comparing the protein maps of decidualized and nondecidualized endometrium in vivo, and second, to determine whether decidual cell cultures produced these characteristic proteins. Decidualization was induced in one uterine horn, and the contralateral horn was not stimulated (control tissue). Animals were ovariectomized and a subcutaneous progesterone implant was used to maintain decidualization. Uterine proteins from nuclear and cytosol fractions were analyzed by two-dimensional electrophoresis using a highly sensitive protein staining technique. Analysis of nuclear extract and cytosol from decidualized and nondecidualized endometrium from Days 6, 7, and 8 of pseudopregnancy demonstrated the presence of 11 nuclear and five cytosolic deciduoma-associated proteins. Serum and erythrocyte proteins were identified by two-dimensional electrophoresis, and none of the 16 deciduoma-associated proteins was a serum or erythrocyte contaminant. Forty-eight-hour cultures of decidual cells harvested from Day 5 of pseudopregnancy produced all 16 of the deciduoma-associated proteins found in whole tissue in situ. Culture conditions minimized serum and erthrocyte contamination, enhancing the detection of deciduomal cell proteins. Four nuclear and two cytosolic proteins were considered deciduoma specific, i.e., they were not associated with cellular proliferation, as evidenced by their absence from cultures of rapidly dividing fetal hamster fibroblasts. Thus, these studies show that the detection of deciduomal proteins may be a useful criterion for the assessment of decidualization in vitro and in vivo.     

Uterine estradiol and progesterone receptor concentration in relation to circulating hormone levels and histoarchitecture during high endometrial sensitivity and induced decidualization in guinea pigs

Alterations in nuclear and cytosolic estradiol (ER) and progesterone (PR) receptor concentration in the antimesometrial (AM) and mesometrial (M) segments of the uterus in relation to circulating hormone levels, histology and surface topography during the period of high endometrial sensitivity and development of trauma-induced decidualization in cyclic guinea pigs were investigated. The period of high endometrial sensitivity (i.e. day 5 of the estrous cycle) was characterized by elevated plasma estradiol and progesterone and their receptors in the nuclear and cytosolic fractions of the uterus. There was, however, no difference in the concentration of these receptors or the surface ultrastructure in the AM and M segments. Unilateral traumatization by scissor cut along the AM length of the uterus on day 5 of the estrous cycle induced decidual cell reaction resulting in a marked increase in weight of the decidualized (traumatized) uterine horn with advancing decidualization to reach maximum levels (926% of the contralateral nontraumatized uterine horn) 7 days after traumatization. This was associated with decidual transformation and a marked increase in nuclear and cytosolic ER and PR concentration in the AM segment of the traumatized uterine horn. An increase in receptor concentration in the M segment of the traumatized uterine horn or the AM segment of the nontraumatized uterine horn was transitory and of a low order. Receptor concentration in the M segment of the nontraumatized uterine horn remained low throughout days 8–12 of the cycle. Findings indicate a possible role of both estradiol and progesterone in induction of endometrial sensitivity and development and maintenance of decidua in the guinea pig.     

Protein kinase C isoforms during the development of deciduomata in pregnant rats.

In this study, we determined the expression of protein kinase C (PKC) isoforms during pregnancy. At pregnant duration, PKC alpha was down-modulated in the deciduomata but not in the myometrium. Down-modulation was compatible with the increase in cell mitosis, which reached a maximum at 8-9 days. On the other hand, PKC zeta was not down-modulated. It was increased both in the cytosolic and particulate fractions of the deciduomata, and paralleled the frequency of decidual cell mitosis. The other PKC isoform of delta was also increased, but it was associated with the cell regression. Therefore, these findings confirmed that the variable expression of PKC isoforms in decidualizing tissue may be involved in the modulation of decidual cell growth.     

Induction of cytotoxic T-lymphocyte antigen-2beta, a cysteine protease inhibitor in decidua: a potential regulator of embryo implantation

During early pregnancy, the steroid hormone progesterone induces differentiation of uterine stroma to decidual cells, which regulate embryo-uterine interactions. The progesterone-induced signaling molecules that participate in the formation and function of decidua remain poorly understood. We recently utilized high-density oligonucleotide microarrays to identify several genes whose expression is markedly altered in pregnant uterus in response to RU486, a well characterized antagonist of the progesterone receptor (PR). Our study revealed that the gene encoding cytotoxic T-lymphocyte antigen-2beta (CTLA-2beta), a cysteine protease inhibitor, is expressed during PR-induced decidualization. The spatio-temporal expression of CTLA-2beta mRNA precisely overlapped with the decidual phase of pregnancy. Interestingly, administration of progesterone to estrogen-primed ovariectomized mice failed to induce CTLA-2beta expression. A concomitant artificial decidual stimulation was necessary to trigger this expression. Uteri of PR knockout mice failed to express this mRNA, even after a combined administration of steroid hormones and artificial stimulation. The uterine expression of CTLA-2beta was, therefore, dependent on PR as well as other unknown factor(s) associated with decidual response. To identify the molecular target(s) of CTLA-2beta,we analyzed its interaction with proteins present in soluble extracts prepared from day 7 pregnant uteri containing implanted embryos. A protein affinity strategy employing recombinant CTLA-2beta helped us to determine that cathepsin L, a cysteine protease, is one of its targets in the pregnant uterus. Consistent with this finding, expression of cathepsin L was detected in the giant trophoblast cells of the ectoplacental cone on day 7 of pregnancy. Collectively, our results support the hypothesis that expression of CTLA-2beta in the decidua may regulate implantation of the embryo by neutralizing the activities of one or more proteases generated by the proliferating trophoblast.