Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice

The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.     

The effect of fludrocortisone on the uterine receptivity partially mediated by ERK1/2-mTOR pathway

Implantation of embryos needs endometrial receptivity. Mineralocorticoids is one of the causes influencing the implantation window. This study targeted to evaluation fludrocortisone different properties on endometrial receptivity. The objective of this study was to assess whether treatment with fludrocortisone could impact the expression of diverse genes and proteins that are involved in uterine receptivity in mice. In this study, 40 female adult BALB/c mice were used. The samples were allocated to four groups of ten. Control group (C) received: vehicle; fludrocortisone group (FCA): received 1.5 mg/kg fludrocortisone; PP242 group (PP242): received 30 mg/kg PP242; fludrocortisone+PP242 group (FCA+PP242): received fludrocortisone and PP242. Mice were killed on window implantation day after mating and confirmed pregnancy. The endometrial epithelium of mouse was collected to assess mRNA expression of leukemia inhibitory factor (LIF), mucin-1 (MUC1), heparin-binding epidermal growth factor (HB-EGF), (Msx.1), miRNA Let-7a, and miRNA 223-3p as well as protein expression of extracellular signal-regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the uterine using real-time PCR and western blot, respectively. In comparison with the control group, fludrocortisone administration upregulated the expression of LIF, HB-EGF, Msx.1, miRNA Let-7a, ERK1/2, and mTOR in the epithelial endometrium. The PP242-treated group demonstrated a significant rise in the expression of MUC1, miRNA 223-3p and a remarkable decline in ERK1/2 and p-4E-BP1 levels in comparison with the control group. Combination therapy of (FCA+PP242) resulted in a remarkable rise in LIF, Msx-1, HB-EGF, ERK1/2, and mTOR levels, in comparison with the PP242 group. Furthermore, combination therapy of (FCA+PP242) downregulated the expression of MUC1 in comparison with the PP242-treated group. According to the results, fludrocortisone affected uterine receptivity possibly by means of modulating the expression of genes involved in the uterine receptivity and activation of the ERK1/2-mTOR pathway.     

Uterine sensitization-associated gene-1: a novel gene induced within the rat endometrium at the time of uterine receptivity/sensitization for the decidual cell reaction

For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.     

Dysregulated cannabinoid signaling disrupts uterine receptivity for embryo implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so in CB1(-/-)/CB2(-/-) double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.     

Expression and estradiol regulation of Wnt genes in the mouse blastocyst identify a candidate pathway for embryo-maternal signaling at implantation

Implantation of mammalian embryos depends on differentiation of the blastocyst to a competent state and of the uterine endometrium to a receptive state. Communication between the blastocyst and uterus ensures that these changes are temporally coordinated. Although considerable evidence indicates that the blastocyst induces expression of numerous genes in uterine tissue, potential signaling mechanisms have yet to be identified. Moreover, whereas a surge of maternal estradiol occurring on Day 4 of pregnancy in the mouse is critically required for many of the peri-implantation uterine changes, whether this surge also affects blastocyst gene expression has not been established. We show here that mouse morulae express genes encoding several members of the Wnt family of signaling molecules. Additional Wnt genes are newly expressed following development to blastocyst. Unexpectedly, Wnt5a and Wnt11 are expressed in embryos that undergo the morula-to-blastocyst transition in vivo, but only weakly or not at all in embryos that do so in vitro. Upregulation of Wnt11 is temporally coordinated with the surge of maternal estradiol on Day 4. Wnt11 fails to be upregulated in blastocysts obtained from mice ovariectomized early on Day 4 or from mice treated with the estradiol antagonist, ICI 182,780. Administration of estradiol-17beta or its metabolite, 4-OH-estradiol, to ovariectomized mice restores Wnt11 expression. Moreover, Wnt11 expression is not upregulated when blastocysts are trapped in the oviduct following ligation of the utero-tubal junction, nor when estradiol-17beta or 4-OH-estradiol are administered to blastocysts in vitro. These results establish a comprehensive profile of Wnt gene expression during late preimplantation development, demonstrate that estradiol regulates gene expression in the blastocyst via uterine factors, and identify Wnts as potential mediators of embryo-uterine communication during implantation.     

Msx1 is required for dorsal diencephalon patterning

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1(nlacZ) mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.     

Deciphering the molecular basis of uterine receptivity

Uterine receptivity is defined as a limited time period during which the uterus enters into an appropriately differentiated state that is ready for the initiation of implantation by competent blastocysts. Although various cellular aspects and molecular pathways involved in uterine receptivity have been identified by gene expression studies and genetically engineered mouse models, a comprehensive understanding of the window of uterine receptivity is still missing. This review focuses on the recent progress in this area, with particular focus on the molecular basis of stromal‐epithelial dialogue and crosstalk between the blastocyst and the uterus during implantation. A better understanding of the underlying mechanisms governing the window of uterine receptivity is hoped to generate new strategies to correct implantation failure and to improve pregnancy rates in women. Mol. Reprod. Dev. 80: 8–21, 2013. © 2012 Wiley Periodicals, Inc.     

Inactivation of nuclear Wnt-beta-catenin signaling limits blastocyst competency for implantation

The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-beta-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-beta-catenin signaling with attenuation of the non-canonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.     

Effect of cyclooxygenase on "window of implantation" in mouse

The major determinants of uterine receptivity are the ovarian progesterone and estrogen hormones, respectively. Different prostaglandins (PGs) have been elucidated in reproduction and also in this process of implantation in various ways. The blastocyst undergoes implantation on the uterine epithelium in defined hormone prepared period known as "implantation window". However, any definitive role of PGs in the window of receptivity remains elusive. It is demonstrated herein that selective COX1 inhibitor (SC560) and selective COX2 inhibitor (nimesulide) separately had no significant effect on blastocyst implantation while combination of both inhibitors in lower dose showed partial delay in implantation by more than 24h and became implanted beyond the window of implantation, i.e. on D6 but these implantation sites were significantly reduced on D10 and the pregnancy is lost in significant number. However, the higher doses of inhibitors in combination completely prevented implantation. Embryos retrieved from these treated mice showed significantly lower number of embryonic cells (77+/-3.3 and 65.2+/-3.9) than the optimum number of embryonic cells (93.4+/-2.6). The lower doses of both the inhibitors reduced uterine PGE2 and PGI2 content on D5 but did not inhibit as efficiently as higher doses. In addition, our immunohistochemistry result shows that there was no COX1 and COX2 localization on D5 of treated mice but COX2 begins expressing on D6 like normal D5 of pregnancy. Therefore, we can conclude that embryos implanted after the delay showed defective post-implantation development because of lower number of embryonic cells of implanting blastocyst and implantation beyond the proper time in window of receptivity.     

Induction of dual-specificity phosphatase 1 (DUSP1) by pulsatile gonadotropin-releasing hormone stimulation: role for gonadotropin subunit expression in mouse pituitary LbetaT2 cells

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.     

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.