Application of functional genomics to primate endometrium: insights into biological processes

Endometrium is a dynamic tissue that responds on a cyclic basis to circulating levels of the ovarian-derived steroid hormones, estradiol and progesterone. Functional genomics has enabled a global approach to understanding gene regulation in whole endometrial tissue in the setting of a changing hormonal milieu. The proliferative phase of the cycle, under the influence of estradiol, has a preponderance of genes involved in DNA synthesis and cell cycle regulation. Interestingly, genes encoding ion channels and cell adhesion, as well as angiogenic factors, are also highly regulated in this phase of the cycle. After the LH surge, different gene expression profiles are uniquely observed in the early secretory, mid-secretory (window of implantation), and late secretory phases. The early secretory phase is notable for up-regulation of multiple genes and gene families involved in cellular metabolism, steroid hormone metabolism, as well as some secreted glycoproteins. The mid-secretory phase is characterized by multiple biological processes, including up-regulation of genes encoding secreted glycoproteins, immune response genes with a focus on innate immunity, and genes involved in detoxification mechanisms. In the late secretory phase, as the tissue prepares for desquamation, there is a marked up-regulation of an inflammatory response, along with matrix degrading enzymes, and genes involved in hemostasis, among others. This monograph reviews hormonal regulation of gene expression in this tissue and the molecular events occurring therein throughout the cycle derived from functional genomics analysis. It also highlights challenges encountered in using human endometrial tissue in translational research in this context

Regulated expression of matrix metalloproteinases, inflammatory mediators, and endometrial matrix remodeling by 17beta-estradiol in the immature rat uterus

<p>Abstract</p> <p>Background</p> <p>Administration of a single physiological dose of 17beta-estradiol (E2:40 microg/kg) to the ovariectomized immature rat rapidly induces uterine growth and remodeling. The response is characterized by changes in endometrial stromal architecture during an inflammatory-like response that likely involves activated matrix-metalloproteinases (MMPs). While estrogen is known as an inducer of endometrial growth, its role in specific expression of MMP family members in vivo is poorly characterized. E2-induced changes in MMP-2, -3, -7, and -9 mRNA and protein expression were analyzed to survey regulation along an extended time course 0-72 hours post-treatment. Because E2 effects inflammatory-like changes that may alter MMP expression, we assessed changes in tissue levels of TNF-alpha and MCP-1, and we utilized dexamethasone (600 microg/kg) to better understand the role of inflammation on matrix remodeling.</p> <p>Methods</p> <p>Ovariectomized 21 day-old female Sprague-Dawley rats were administered E2 and uterine tissues were extracted and prepared for transmission electron microscopy (TEM), mRNA extraction and real-time RT-PCR, protein extraction and Western blot, or gelatin zymography. In inhibitor studies, pretreatment compounds were administered prior to E2 and tissues were harvested at 4 hours post-hormone challenge.</p> <p>Results</p> <p>Using a novel TEM method to quantitatively assess changes in stromal collagen density, we show that E2-induced matrix remodeling is rapid in onset (< 1 hour) and leads to a 70% reduction in collagen density by 4 hours. Matrix remodeling is MMP-dependent, as pretreatment with batimastat ablates the hormone effect. MMP-3, -7, and -9 and inflammatory markers (TNF-alpha and MCP-1) are transiently upregulated with peak expression at 4 hours post-E2 treatment. MMP-2 expression is increased by E2 but highest expression and activity occur later in the response (48 hours). Dexamethasone inhibits E2-modulated changes in collagen density and expression of MMPs although these effects are variable. Dexamethasone upregulates MMP-3 mRNA but not protein levels, inhibiting E2-induced upregulation of MMP-7, and -9, and MCP-1 mRNA and protein but not inhibiting the hormone-induced increase in TNF-alpha mRNA.</p> <p>Conclusion</p> <p>The data demonstrate that E2-regulated endometrial remodeling is rapid in onset (<1 hour) and peak expression of MMPs and inflammatory mediators correlates temporally with the period of lowest stromal collagen density during uterine tissue hypertrophy.</p

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site

Comprehensive Analysis of Leukocytes, Vascularization and Matrix Metalloproteinases in Human Menstrual Xenograft Model

In our previous study, menstrual-like changes in mouse were provoked through the pharmacologic withdrawal of progesterone with mifepristone following induction of decidualization. However, mouse is not a natural menstruation animal, and the menstruation model using external stimuli may not truly reflect the occurrence and development of the human menstrual process. Therefore, we established a model of menstruation based on human endometrial xenotransplantation. In this model, human endometrial tissues were transplanted subcutaneously into SCID mice that were ovarectomized and supplemented with estrogen and progestogen by silastic implants with a scheme imitating the endocrinological milieu of human menstrual cycle. Morphology, hormone levels, and expression of vimentin and cytokeratin markers were evaluated to confirm the menstrual-like changes in this model. With 28 days of hormone treatment, transplanted human endometrium survived and underwent proliferation, differentiation and disintegration, similar to human endometrium in vivo. Human CD45+ cells showed a peak of increase 28 days post-transplantation. Three days after progesterone withdrawal, mouse CD45+ cells increased rapidly in number and were significantly greater than human CD45+ cell counts. Mouse CD31+ blood vascular-like structures were detected in both transplanted and host tissues. After progesterone withdrawal, the expression levels of matrix metalloproteinases (MMP) 1, 2, and 9 were increased. In summary, we successfully established a human endometrial xenotransplantation model in SCID mice, based on the results of menstrual-like changes in which MMP-1, 2 and 9 are involved. We showed that leukocytes are originated from in situ proliferation in human xenografts and involved in the occurrence of menstruation. This model will help to further understand the occurrence, growth, and differentiation of the endometrium and the underlying mechanisms of menstruation

Periodic Accumulation of Regulatory T Cells in the Uterus: Preparation for the Implantation of a Semi-Allogeneic Fetus?

BACKGROUND: Naturally occurring Foxp3(+)regulatory T cells play an important role in the inhibition of an immunological attack of the fetus. As implantation of the fetus poses an immediate antigenic challenge, the immune system has to prepare itself for this event prior to implantation. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we show using quantitative RT-PCR and flow cytometry that regulatory T cells accumulate in the uterus not only during pregnancy, but also every time the female becomes fertile. Their periodic accumulation is accompanied by matching fluctuations in uterine expression of several chemokines, which have been shown to play a role in the recruitment and retention of regulatory T cells. CONCLUSIONS/SIGNIFICANCE: The data lead us to propose that every time a female approaches estrus, regulatory T cells start to accumulate in the uterus in preparation for a possible implantation event. Once pregnancy is established, those regulatory T cells that have seen alloantigen need to be retained at their site of action. Whilst several chemokines appear to be involved in the recruitment and/or retention of regulatory T cells during estrus, in pregnancy this role appears to be taken over by CCL4

Regulation of human endometrial function: mechanisms relevant to uterine bleeding

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function – including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding

Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion

Expression of CD82 in Human Trophoblast and Its Role in Trophoblast Invasion

BACKGROUND: Well-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development. METHODOLOGY/PRINCIPAL FINDINGS: CD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P<0.01), as well as invasion and migration of HTR8/SVneo cells (P<0.05), whereas the trophoblast proliferation was not affected. The enhanced effect of CD82 siRNA on invasion and migration of trophoblast cells was found associated with increased gelatinolytic activities of matrix metalloproteinase MMP9 while over-expression of CD82 markedly decreased trphoblast cell invasion and migration as well as MMP9 activities. CONCLUSIONS/SIGNIFICANCE: These findings suggest that CD82 is an important negative regulator at maternal-fetal interface during early pregnancy, inhibiting human trophoblast invasion and migration