Previous studies established that in the rat, the uterus can accept a developing blastocyst for implantation only during a limited period of time on day 5 of gestation, termed the receptive phase. Our previous studies showed that the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, is induced in rat uterus between days 3–5 of gestation and is switched off once the implantation process has progressed to day 6. In the present study, we analyze in detail how the expression of calcitonin messenger RNA (mRNA) in the uterus is regulated by the steroid hormones progesterone and estrogen and explore the possibility that calcitonin may serve as a potential marker of uterine receptivity. We demonstrate by in situ hybridization that calcitonin mRNA is synthesized specifically in the glandular epithelial cells between days 3–5 of pregnancy. Interestingly, calcitonin synthesis is also induced in these cells during pseudopregnancy, indicating that this peptide hormone is produced in the endometrium in response to maternal, rather than embryonic, signals. We also demonstrate that calcitonin mRNA expression during pseudopregnancy, like that in normal pregnancy, is under progesterone regulation. We further examined the steroid hormone regulation of uterine calcitonin expression in a delayed implantation model. In pregnant rats in which implantation is blocked upon removal of both ovaries on day 4 of gestation, continued administration of progesterone sustains calcitonin expression in the uterus for several days in the absence of estrogen. Administration of estrogen, which allows delayed implantation, also rapidly reduces calcitonin expression, indicating a role for this steroid hormone in turning off calcitonin gene expression. In gene transfection studies, expression of the progesterone receptor B isoform in cultured endometrial cells induces RNA synthesis from a reporter gene containing a 1.3-kb calcitonin promoter fragment in a hormone-dependent manner. As expected, mifepristone-complexed progesterone receptor B isoform fails to activate the calcitonin promoter. Progesterone acting through its nuclear receptor therefore regulates the expression of the calcitonin gene at the level of transcription. Finally, using RIA we investigated whether calcitonin is secreted from its glandular site of synthesis at the time of implantation by analyzing uterine flushings obtained from pregnant rats. We report the detection of a significant amount of calcitonin in the luminal secretions collected on day 4 and a lower amount on day 5 of gestation, whereas similar samples collected from animals on either day 3 or 6 of gestation did not contain detectable amounts of this peptide hormone. A transient burst of calcitonin secretion into the uterine lumen therefore occurs immediately preceding implantation. Based on these results, we propose that calcitonin is a measurable marker that forecasts the receptive state of rat endometrium during blastocyst implantation.
The abbreviations used are: eIF-2, eukaryotic initiation factor 2, which forms a ternary complex, Met-tRNApeIF-2.GTP; Co-eIF-2, a high molecular weight protein complex that stimulates ternary complex formation by eIF-2 and also promotes GDP displacement from eIF-2.GDP during ternary complex formation; C O -~I F -~A , stimulates ternary complex formation by eIF-2 and also stabilizes the complex formed; Co-eIF-2Am, M, 80,000; C O -~I F -~A ~~, M, 25,000; C O -~I F -~B , promotes dissociation of preformed ternary complexes at high M e ; C O -~I F -~C , promotes GDP displacement from eIF-2.GDP during ternary complex formation and also renders the complex active in Met-tRNAf-40 S complex formation; HRI, heme-regulated translational inhibitor; ATA, aurintricarboxylic acid. 14976This is an Open Access article
List of protein cargoes identified by mass spectroscopic analysis of EVs obtained from MESC isolated from uteri of mice on day 6 of pregnancy. EVs were harvested from conditioned media undergoing in vitro decidualization for 48 h under hypoxic conditions. Only proteins with significant matching (p-value <0.05) are displayed. Accession number, genes name, fasta headers annotated from UniProt (http://www.uniprot.org/).
Abstract Synthetic steroid hormone antagonists are clinically important compounds that regulate physiological responses to steroid hormones. The antagonists bind to the hormone receptors, which are ligand-inducible transcription factors, and modulate their gene-regulatory activities. In most instances, a steroid receptor, such as progesterone receptor (PR) or estrogen receptor (ER), is transcriptionally inactive when complexed with an antagonist and competitively inhibits transactivation of a target steroid-responsive gene by the cognate hormone-occupied receptor. In certain cellular and promoter contexts, however, antagonist-occupied PR or ER acquires paradoxical agonist-like activity. The cellular mechanisms that determine the switch from the negative to the positive mode of transcriptional regulation by an antagonist-bound steroid receptor are unknown. We now provide strong evidence supporting the existence of a cellular inhibitory cofactor that interacts with the B form of human PR (PR-B) complexed with the antiprogestin RU486 to maintain it in a transcriptionally inactive state. In the presence of unliganded thyroid hormone receptor (TR) or ER complexed with the antiestrogen 4-hydroxytamoxifen, which presumably sequesters a limiting pool of the inhibitory cofactor, RU486-PR-B functions as a transcriptional activator of a progesterone-responsive gene even in the absence of hormone agonist. In contrast, hormone-occupied TR or ER fails to induce transactivation by RU486-PR-B. Recent studies revealed that a transcriptional corepressor, NCoR (nuclear receptor corepressor), interacts with unliganded TR but not with liganded TR. Interestingly, coexpression of NCoR efficiently suppresses the partial agonistic activity of antagonist-occupied PR-B but fails to affect transactivation by agonist-bound PR-B. We further demonstrate that RU486-PR-B interacts physically with NCoR in vitro. These novel observations suggest that the inhibitory cofactor that associates with RU486-PR-B and represses its transcriptional activity is either identical or structurally related to the corepressor NCoR. We propose that cellular mechanisms that determine the switch from the antagonistic to the agonistic activity of RU486-PR-B involve removal of the corepressor from the antagonist-bound receptor so that it can effect partial but significant gene activation.
Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.
Ovulation is a key female reproductive event, which involves the release of a fertilizable oocyte from an ovarian follicle. This process is initiated when follicular tissue is stimulated by the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). A surge of LH induces a cascade of gene expression in the FSH-primed ovary that culminates in the rupture of the follicle. In order to explore the pathways that control ovulation, we subjected mice to a superovulation protocol and performed global gene expression profiling at 5 h and 11 h after administration of human chorionic gonadotropin (hCG) following 48 h of equine chorionic gonadotropin (eCG) treatment. We found that the gene encoding the peroxisome proliferator-activated receptor gamma (PPARr) is one of several genes that are induced in the mouse ovary at 5 h post hCG. The expression of PPARr is induced primarily in the granulosa cells of the preovulatory follicles. To address the functional role of this gene during ovulation, we created a conditional knockout mouse model by crossing mice harboring “floxed” PPARr gene with progesterone receptor Cre knockin (PR-Cre) mice. This resulted in the generation of females, in which the PPARr gene undergoes Cre-mediated excision in the mural granulosa cells of the preovulatory follicles. The conditional PPARr knockout (KO) mice exhibited impaired ovulation in response to gonadotropins. Histological analysis of the ovaries of conditional PPARr- KO mice revealed defects in follicular rupture. Using these mutant mice, we identified endothelin 2 (ET-2), a potent vasoactive molecule, and interleukin-6, a proinflammatory molecule, as primary targets of regulation by PPARr in the preovulatory follicles. Recent studies indicated that antagonist-mediated blockade of ET-2 action in the preovulatory phase prevented follicular rupture, supporting the concept that it is a critical mediator of PPARr function during ovulation. Since PPARr is activated by certain metabolites of arachidonic acid in other tissues, we examined the effects of indomethacin, an inhibitor of arachidonic acid metabolism and a suppressor of ovulation, on the expression of PPARr-target genes. Treatment with this drug markedly reduced the expression of ET-2 and IL-6, confirming that PPARr signaling is indeed activated by arachidonic acid metabolites during the ovulatory period. Collectively, our studies indicated that PPARr controls ovulation by generating important biological mediators of vaso-regulatory and inflammatory pathways, which are likely to play critical roles during follicular rupture. (Supported by NIH grant.) (platform)
Abstract Environmental and occupational exposure to bisphenol A (BPA), a chemical widely used in polycarbonate plastics and epoxy resins, has received much attention in female reproductive health due to its widespread toxic effects. Although BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In this study, we addressed the effect of prolonged exposure to an environmental relevant dose of BPA on embryo implantation and establishment of pregnancy. Our studies revealed that treatment of mice with BPA led to improper endometrial epithelial and stromal functions thus affecting embryo implantation and establishment of pregnancy. Upon further analyses, we found that the expression of progesterone receptor (PGR) and its downstream target gene, HAND2 (heart and neural crest derivatives expressed 2), was markedly suppressed in BPA-exposed uterine tissues. Previous studies have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor and the MAPK signaling pathways and inhibiting epithelial proliferation. Interestingly, we observed that down-regulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with enhanced activation of fibroblast growth factor and MAPK signaling in the epithelium, thus contributing to aberrant proliferation and lack of uterine receptivity. Further, the differentiation of endometrial stromal cells to decidual cells, an event critical for the establishment and maintenance of pregnancy, was severely compromised in response to BPA. In summary, our studies revealed that chronic exposure to BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.
The steroid hormone progesterone (P), acting via the progesterone receptor ( PR ) isoforms, PR ‐A and PR ‐B, exerts a profound influence on uterine functions during early gestation. In recent years, chromatin immunoprecipitation‐sequencing in combination with microarray‐based gene expression profiling analyses have revealed that the PR isoforms control a substantially large cistrome and transcriptome during endometrial differentiation in the human and the mouse. Genetically engineered mouse models have established that several PR ‐regulated genes, such as Ihh, Bmp2, Hoxa10, and Hand2, are essential for implantation and decidualization. PR ‐A and PR ‐B also collaborate with other transcription factors, such as FOS , JUN , C/ EBP β and STAT 3, to regulate the expression of many target genes that functions in concert to properly control uterine epithelial proliferation, stromal differentiation, angiogenesis, and local immune response to render the uterus ‘receptive’ and allow embryo implantation. This review article highlights recent work describing the key PR ‐regulated pathways that govern critical uterine functions during establishment of pregnancy.
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