In mice WNT signaling regulates cell fate, differentiation, and growth in the conceptus (embryo and associated extra-embryonic membranes), as well as implantation. We studied various components of the WNT signaling pathway in the ovine uterus during the estrous cycle (C) and pregnancy (P) and in the peri-implantation conceptus. Expression of WNT2, WNT2B, and WNT4 mRNAs was very low in endometria of C and P ewes from Days 10 to 16 and in conceptus trophectoderm (Tr). WNT5A/5B mRNAs were abundant in the stratum compactum stroma, whereas WNT11 mRNA was detected in endometrial epithelia of C and P ewes, but not in conceptus Tr. WNT7A mRNA was localized specifically to luminal (LE) and superficial glandular (sGE) epithelia of Day 10 C and P ewes, was undetectable by Day 12, and then increased up to Day 16 and was maximum on Day 20 only in P ewes. Frizzled receptor (FZD6/8) mRNAs were detected primarily in conceptus Tr and uterine LE and GE, whereas the co-receptor LRP5/6 (low density lipoprotein receptor-related protein) mRNAs were expressed in all uterine cell types and conceptus Tr. Dickkopf (DKK1), a negative regulator of WNT signaling, was detected in stratum compactum stroma after Day 14 in P ewes. CTNNB1 (beta-catenin), a key mediator of canonical WNT signaling, and GSK3B (glycogen synthase kinase-3 beta) and CHD1 (E-cadherin) mRNAs were abundant in endometrial epithelia and in conceptus Tr. Immunoreactive CTNNB1 protein was abundant in LE and GE, and present at lower levels in stroma and myometrium in uteri from C and P ewes. In the conceptus Tr, immunoreactive CTNNB1 protein was abundant in nuclei of the mononuclear and binuclear cells (BNC), as well as in cell adherens junctions. Nuclear CTNNB1 interacts with transcription factors, most notably LEF1/TCF7 (lymphoid enhancer-binding factor 1/transcription factor 7), to regulate gene transcription. LEF1 mRNA was detected in LE and sGE, whereas nuclear TCF7L2 protein was particularly abundant in trophoblast giant BNC. WNT/beta-catenin/TCF7 target genes were also studied. MSX2 mRNA was abundant in conceptus Tr, and MYC mRNA was abundant in BNC of conceptus Tr and endometrial epithelia. Next, ovine Tr (oTr) cells and endometrial stromal (oST) cells were used for mechanistic studies that revealed that transfection of mouse WNT7A stimulated a LEF/TCF-responsive reporter (TOPFLASH), and co-transfection of either dnTCF or SFRP2 (a secreted FZD inhibitor) inhibited WNT7A effects. WNT7A stimulated expression of MSX2 and MYC in oTr cells, and this effect was inhibited by SFRP2. These results implicate the canonical WNT system as a regulator of peri-implantation conceptus growth and differentiation in sheep. This work was supported by NIH HD38274 and 5 P30 ES09106 funding.
This study was performed to examine whether prenatal exposure to bisphenol (BP) A analogues, BPE and BPS, negatively impacts female reproductive functions and follicular development using mice as a model. CD-1 mice were orally exposed to control treatment (corn oil), BPA, BPE, or BPS (0.5, 20, or 50 µg/kg/day) from gestational day 11 (the presence of vaginal plug = 1) to birth. Exposure to BPA, BPE, and BPS accelerated the onset of puberty and exhibited abnormal estrous cyclicity, especially with lower doses. Females exposed to BPA, BPE, and BPS exhibited mating difficulties starting at 6 months of age. By 9 months, mice exhibited various fertility problems including reduced pregnancy rate, parturition issues, and increased dead pups at birth. Furthermore, the levels of serum testosterone were elevated by BPE or BPS exposure at the age of 9 months, whereas estrogen levels were not affected. On the other hand, the dysregulated expression of steroidogenic enzymes was observed in the ovary at 3, 6, or 9 months of age by BPE or BPS exposure. When we examined neonatal ovary on postnatal day 4, BPA, BPE, and BPS exposure inhibited germ cell nest breakdown and reduced number of primary and secondary follicles. These results suggest that prenatal exposure to BPA analogues, BPE, and BPS, have effects on fertility in later reproductive life probably due to the disruption of early folliculogenesis.
Development of uterine glands (adenogenesis) in mammals typically begins during the early post-natal period and involves budding of nascent glands from the luminal epithelium and extensive cell proliferation in these structures as they grow into the surrounding stroma, elongate and mature. Uterine glands are essential for pregnancy, as demonstrated by the infertility that results from inhibiting the development of these glands through gene mutation or epigenetic strategies. Several genes, including forkhead box A2, beta-catenin and members of the Wnt and Hox gene families, are implicated in uterine gland development. Progestins inhibit uterine epithelial proliferation, and this has been employed as a strategy to develop a model in which progestin treatment of ewes for 8 weeks from birth produces infertile adults lacking uterine glands. More recently, mouse models have been developed in which neonatal progestin treatment was used to permanently inhibit adenogenesis and adult fertility. These studies revealed a narrow and well-defined window in which progestin treatments induced permanent infertility by impairing neonatal gland development and establishing endometrial changes that result in implantation defects. These model systems are being utilized to better understand the molecular mechanisms underlying uterine adenogenesis and endometrial function. The ability of neonatal progestin treatment in sheep and mice to produce infertility suggests that an approach of this kind may provide a contraceptive strategy with application in other species. Recent studies have defined the temporal patterns of adenogenesis in uteri of neonatal and juvenile dogs and work is underway to determine whether neonatal progestin or other steroid hormone treatments might be a viable contraceptive approach in this species.
Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.
Prolactin (PRL) acts through its receptor (PRLR) via both endocrine and local paracrine/autocrine pathways to regulate biological processes including reproduction and lactation. We analyzed the tissue- and stage of gestation-specific regulation of PRL and PRLR expression in various tissues of pigs. Abundance of p PRLR -long form (LF) mRNA increased in the mammary gland and endometrium during gestation while in other tissues it remained constant. There was a parallel increase in the abundance of the pPRLR-LF protein in the mammary gland and endometrium during gestation. We determined the hormonal regulation of p PRLR -LF mRNA expression in various tissues from ovariectomized, hypoprolactinemic gilts given combinations of the replacement hormones estrogen (E 2 ), progestin (P), and/or haloperidol-induced PRL. Abundance of p PRLR -LF mRNA in kidney and liver was unaffected by hormone treatments. Expression of uterine p PRLR -LF mRNA was induced by E 2 whereas the effect of E 2 was abolished by co-administering P. The expression of p PRLR -LF mRNA in the mammary gland stroma was induced by PRL, whereas E 2 induced its expression in the epithelium. In contrast to these changes in p PRLR expression, p PRL expression was relatively constant and low during gestation in all tissues except the pituitary. Taken together, these data reveal that specific combinations of E 2 , P, and PRL differentially regulate pPRLR-LF expression in the endometrium and mammary glands, and that the action of PRL on its target tissues is dependent upon pPRLR-LF abundance more so than the local PRL expression.
WNT signaling is well known to play an important role in the regulation of development, cell proliferation and cell differentiation in a wide variety of normal and cancerous tissues. Despite the wealth of knowledge concerning when and where various WNT genes are expressed and downstream events under their control, there is surprisingly little published evidence of how they are regulated. We have recently reported that aberrant WNT7A is observed in serous ovarian carcinomas, and WNT7A is the sole ligand accelerating ovarian tumor progression through CTNNB1 (β-catenin)/TCF signaling in the absence of CTNNB1 mutations. In the present study, we report that WNT7A is a direct target of miR-15b in ovarian cancer. We showed that a luciferase reporter containing the putative binding site of miR-15b in the WNT7A 3'-UTR was significantly repressed by miR-15b. Mutation of the putative binding site of miR-15b in the WNT7A 3'-UTR restored luciferase activity. Furthermore, miR-15b was able to repress increased levels of TOPFLASH activity by WNT7A, but not those induced by S33Y. Additionally, miR-15b dose-dependently decreased WNT7A expression. When we evaluated the prognostic impact of WNT7A and miR-15b expression using TCGA datasets, a significant inverse correlation in which high-expression of WNT7A and low-expression of miR-15b was associated with reduced survival rates of ovarian cancer patients. Treatment with decitabine dose-dependently increased miR-15b expression, and silencing of DNMT1 significantly increased miR-15b expression. These results suggest that WNT7A is post-transcriptionally regulated by miR-15b, which could be down-regulated by promoter hypermethylation, potentially via DNMT1, in ovarian cancer.
Secreted phosphoprotein 1 (SPP1, osteopontin) is an extracellular matrix (ECM) protein that binds integrins to affect cell-cell and cell-matrix interactions including adhesion, migration, proliferation and survival. Secreted-protein-acidic-and-rich-in-cysteine (SPARC, osteonectin) is a calcium-binding matricellular glycoprotein known to regulate ECM interaction during tissue remodeling and repair. We developed a Spp1-/-/Sparc-/- double null mouse in which 50% fewer pups are present at birth than are found in utero on days 18-20 of gestation due to postpartum maternal cannibalization of the pups, suggesting that these pups are stillborn or exhibit severe birth defects. Indeed, hemorrhages were observed prominently in lower right limbs and distal region of the tail in many embryos; in a few embryos, hemorrhages were present the intestine and right foreheads. In order to gain insight into the developmental abnormalities and decreased litter sizes seen in double null litters, we evaluated the expression of both Spp1 and Sparc in the mouse uterus throughout pregnancy. Ten-week old outbred CD-1 female mice were mated to an intact male of the same strain (copulatory plug = Day 1 of gestation). Pregnant females were sacrificed and uteri collected on Day 4, 4.5, 5, 9, 10, 11, 12, and 16 of pregnancy. Cell-specific expression of Spp1 and Sparc mRNA in serial sections of mouse uteri was determined by radioactive in situ hybridization analysis. On Day 4 of pregnancy, Spp1 was expressed in central cells of the inner cell mass (ICM) of the embryo, in scattered immune cells in uterine stroma, and in luminal epithelium (LE) directly contacting the embryo within the implantation chamber. LE expression within the implantation chamber was absent by Days 4.5-5 of pregnancy, but remained in LE adjacent to the implantation chamber and in interimplantation sites, suggesting that Spp1 may be involved in adhesion during the initial phase of embryo attachment to the LE, but subsequently must be down-regulated at sites of attachment to allow embryo invasion. Sparc was expressed diffusely in the uterine stroma, in peripheral cells of the ICM, and was not expressed in LE through Days 4.5-5. When we utilized a model of delayed implantation to determine the effect of nidatory estrogen, localization of Spp1 and Sparc was identical to that of pregnancy. On Day 9 of gestation, Spp1 was expressed by uterine natural killer (NK) cells and LE, whereas Sparc was expressed by decidual cells and LE. On Day 10, NK cells and LE continued to express Spp1, while Sparc was localized to the stroma directly beneath the LE. On Day 12, Spp1 was also expressed by trophoblast giant cells, and Sparc was expressed in the Reichert's Membrane adjacent to the trophoblast giant cells. By Day 16 of pregnancy, both Spp1 and Sparc were expressed in adjacent but non-overlapping regions of developing fetal bone and cartilage. Because Spp1 and Sparc often have antagonistic roles during tissue remodeling and are localized to adjacent but different cell types during mouse pregnancy, we hypothesize that simultaneous deletion of both Spp1 and Sparc leads to disruption of cell-cell, cell-matrix and inter-tissue communication essential to normal conceptus development that is not observed when the genes are deleted individually. This study was supported by NIH grants R01 CA90920 and RO1 CA137091. (platform)
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