胚胎着床的分子调控网络

胚胎着床是活性胚胎与接受态子宫相互对话,并建立紧密联系的过程。在雌激素和孕酮的协同调控下,一些粘附分子、细胞因子和生长因子等呈时空特异性表达,许多信号通路间相互协作对于胚胎着床至关重要。近年来发现,miRNA等非编码RNA也参与胚胎着床的分子调控网络。本文旨在综述近年来胚胎着床分子调控网络方面的研究进展。     

子宫内膜接受性的建立和分子调控

胚泡着床是一个复杂的生理过程,依赖于胚泡发育和子宫内膜获得接受能力的同步进行.着床只发生在具有接受性的子宫内膜,而子宫内膜只在很短的时间内具有接受性.被称为"着床窗口".子宫内膜接受性的建立涉及子宫腔上皮的形态学改变,以及甾类激素和许多细胞因子复杂的调控作用.本文综述了子宫内膜接受性的建立及其分子调控.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

Lipid signaling in embryo implantation

A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is required for successful implantation. Although various molecular pathways are known to participate in this cross-talk, a comprehensive understanding of the implantation process is still missing. Gene expression studies and genetically engineered mouse models have provided evidence that lipid mediators serve as important signaling molecules in coordinating the series of events during early pregnancy including preimplantation embryo formation and development, implantation and postimplantation growth. This review focuses on the roles of two groups of lipid mediators, prostaglandins (PGs) and endocannabinoids, during early pregnancy. Our laboratory has shown that while PGs generated by the cPLA2-cyclooxygenase (COX) system are essential to ovulation, fertilization, and implantation, endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. A better understanding of these molecular signaling pathways is hoped to generate new strategies to correct implantation failure and improve pregnancy rates in women.     

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.     

Heparanase expression and function during early pregnancy in mice

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.     

Immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha and growth factor-beta s in the caprine peri-implantation period

Control over the action of steroid hormones in the uterus and conceptus during the initial period of gestation appears to be regulated locally by growth factors. This study involved immunohistochemical detection of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta s (TGF-beta s), to determine their role in the caprine peri-implantation period. Epidermal growth factor was expressed in the luminal and glandular endometrial epithelium of goats on all days studied (Days 22 to 30 post coitum), but it was not detected in trophoblastic cells or in other embryonic structures. Between Days 22 and 30 post coitum, TGF-alpha was detected in the epithelial cells and superficial stroma of the uterus and in the trophoendodermic cells of the embryo. Transforming growth factor-beta s expression, observed in the endometrium, embryo and extraembryonic membranes on Day 22 post coitum, decreased by Day 24 post coitum and disappeared in the embryo by Day 30 post coitum, while remaining in the other structures. The presence of these growth factors during the peri-implantation period in the goat suggests their participation in proliferation and differentiation phenomena which occur during implantation and embryonic development.     

Developmental biology of uterine glands.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.     

Inhibiting uterine PC6 blocks embryo implantation: an obligatory role for a proprotein convertase in fertility

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.     

Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.     

Human trophoblast function during the implantation process

The implantation process involves complex and synchronized molecular and cellular events between the uterus and the implanting embryo. These events are regulated by paracrine and autocrine factors. Trophoblast invasion and migration through the uterine wall is mediated by molecular and cellular interactions, controlled by the trophoblast and the maternal microenvironment. This review is focused on the molecular constituents of the human trophoblast, their actions and interactions, including interrelations with the uterine endometrium.     

胚泡着床窗口的分子调控

着床窗口是指当胚胎发育到胚泡阶段时,子宫也增殖和分化到可接受状态,二者相互作用使胚泡着床的短暂时间.雌激素和孕酮是该过程的综合调控分子,它们通过多种局部信号分子的介导,使子宫中的各种细胞类型增殖、分化,为着床窗口的开放做出相互协调的反应.子宫与胚胎在着床窗口通过前列腺素、组织胺、降钙素、多种细胞因子和生长因子的旁分泌作用进行分子对话,使胚泡滋养层与子宫内膜上皮发生附着反应.着床窗口一旦开放,即自动向非接受态转化.     

Uterine leukocytes: key players in pregnancy

In species with hemochorial placentation, which includes humans, mice and rats, antigen-specific T and B lymphocytes which are responsible for acquired immunity are virtually absent from the maternal-fetal interface. In contrast, non-antigen specific natural killer cells and macrophages which provide innate immunity are abundant and highly specialized. Autocrine/paracrine factors such as steroid and polypeptide hormones, prostaglandins and anti-inflammatory cytokines that are present in the uterine environment during pregnancy re-program their secretory profiles. Recent studies using transgenic mice and other approaches indicate that these environmentally modified leukocytes have major pregnancy-associated functions that include facilitation of implantation, modulation of the maternal uterine vasculature, supply of growth factors to the placenta, promotion of trophoblast differentiation and facilitation of parturition.     

New perspectives on growth factor-sex steroid interaction in the prostate

Many organs respond to both sex steroids and growth factors. Regulation of these pathways is integral to cell-cell communications during development and aberrant changes cause disease pathogenesis. Traditionally, paracrine and endocrine actions of growth factors and steroid hormones are considered independently. Recently, new data indicated that activin/TGFbeta and sex steroid signalling are linked; explicitly, that the pathways cross-talk intracellularly. Here we present new perspectives on these interactions, using examples predominantly from the prostate, as it is a well-characterised organ in this context. While this information provides insight to the potential mechanisms behind these interactions, it also presents a new challenge; the action of any of these factors cannot be considered exclusively without considering the impact on the other biological pathways.     

Seminal plasma and male factor signalling in the female reproductive tract

In mammals, insemination results in the transmission of seminal factors that act, in the female reproductive tract, to promote sperm survival, to “condition” the female immune response to tolerate the conceptus and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. These events are initiated when signalling agents, including transforming growth factor-β and other cytokines and prostaglandins secreted by seminal vesicle and prostate glands, interact with epithelial cells in the cervix and uterus to activate cytokine synthesis and to induce cellular and molecular changes resembling a classical inflammatory cascade. The consequences are the recruitment and activation of macrophages, granulocytes and dendritic cells, which have immune-regulatory and tissue-remodelling roles that culminate in improved endometrial receptivity to the implanting embryo. Cytokines elicited by seminal activation have embryotrophic properties and also contribute directly to the optimal development of the early embryo. This review summarises our current understanding of the physiology of responses to seminal plasma in the female reproductive tract and considers the evolutionary significance of seminal plasma in influencing female tissues to promote the success of pregnancy.The author acknowledges the support of the NHMRC of Australia Fellowship and Program Grant schemes.     

Effect of IL-1alpha on prostaglandin synthesis of oestrogenized rat uterus is mediated by nitric oxide

We examined the possible relationship between cytokines, nitric oxide (NO) and prostaglandins in the oestrogenized rat uterus. Results indicate that: IL-1alpha but not IL-2 enhances the synthesis of prostaglandins in oestrogenized rat uteri; IL-1alpha but not IL-2 induced an augmention of NO production in this tissue; the effect of IL-1alpha on prostaglandin synthesis is abolished by NMMA, an NO antagonist; NS-398, a COX-2 inhibitor, prevents the augmention of prostaglandins produced by IL-1alpha. These results suggest that there is an interaction between IL-1alpha, NO and prostaglandins and that this interrelationship involves COX-2. This mechanism might be important during implantation and labor.     

Induction of tryptophan 2,3-dioxygenase in the mouse endometrium during implantation

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site.