To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2(tm3(cre)Bhr) strain to target deletion of Wnt4 to granulosa cells. Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis.

Whereas the roles of the canonical winglesstype MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation of Wnt5a (but not Wnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genes in vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signaling via the WNT/Ca 2+ or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both b-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decrease via a glycogen synthase kinase-3b-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. FASEB J. 30, 1534-1547 (2016 The pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) represent the major endocrine regulators of ovarian function and are essential for follicle development beyond the secondary stage (1, 2). In addition to the endocrine level of control, follicle development is regulated by several paracrine and autocrine factors produced within the ovary itself. These factors notably include IGF-1, steroid hormones, prostaglandins, epidermal growth factor-like molecules and several TGF-b superfamily members, all of which are indispensable for normal ovarian function and female fertility. The gonadotropins can affect the expression and function of these intraovarian factors; conversely, these factors can modulate follicular responses to the gonadotropins (1-3).Recent studies have established the wingless-type MMTV (mouse mammary tumor virus) integration site (WNT) family of secreted glycoproteins as yet another class of signaling molecules that act to modulate and coordinate follicular responses to the gonadotropins and whose activities are indispensable for ovarian...

Recent evidence has suggested that vascular endothelial growth factor A (VEGFA) is an important regulator of ovarian follicle development and survival. Both LH and FSH regulate Vegfa expression in granulosa cells and signal via the transcription factor hypoxia inducible factor 1 (HIF1). To further study the mechanism of action of HIF1 in the regulation of Vegfa, we studied Vegfa(delta/delta) mice, which lack a hypoxia response element in the Vegfa promoter. Granulosa cells from Vegfa(delta/delta) mice failed to respond to FSH or LH with an increase in Vegfa mRNA expression in vitro, and granulosa cells isolated from eCG-treated immature Vegfa(delta/delta) mice had significantly lower Vegfa mRNA levels compared to controls. However, normal Vegfa mRNA levels were detected in the granulosa cells from immature Vegfa(delta/delta) mice following hCG treatment. Vegfa(delta/delta) females produced infrequent litters, and their pups died shortly after birth. Ovaries from Vegfa(delta/delta) mice were much smaller than controls and contained few antral follicles and corpora lutea. Antral follicles numbers were decreased by nearly 50% in ovaries from Vegfa(delta/delta) mice relative to controls, and 74% of antral follicles in Vegfa(delta/delta) ovaries were atretic. Serum progesterone levels in adult Vegfa(delta/delta) females were significantly lower, apparently reflecting reduced numbers of corpora lutea. This study demonstrates for the first time the requirement of HIF1 for FSH-regulated Vegfa expression in vivo and that HIF1 acts via a single hypoxia response element in the Vegfa promoter to exert its regulatory functions. Our findings also further define the physiological role of VEGFA in follicle development.

The efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in the treatment of chronic aortic regurgitation (AR) is not well established and remains controversial. The mechanisms by which ACEIs may protect against left-ventricular (LV) volume overload are not well understood, and clinical trials performed until now have yielded conflicting results. This study was therefore performed to assess the effectiveness of two different doses of the ACEI captopril in a rat model of chronic AR. We compared the effects of a 6-month low-dose (LD) (25 mg/kg) or higher dose (HD) (75 mg/kg) treatment with captopril on LV function and hypertrophy in Wistar rats with severe AR. Untreated animals developed LV eccentric hypertrophy and systolic dysfunction. LD treatment did not prevent hypertrophy and provided modest protection against systolic dysfunction. HD treatment preserved LV systolic function and dimensions and tended to slow hypertrophy. The cardiac index remained high and similar among all AR groups, treated or not. Tissue renin-angiotensin system (RAS) analysis revealed that ACE activity was increased in the LVs of AR animals and that only HD treatment significantly decreased angiotensin II receptor mRNA levels. Fibronectin expression was increased in the LV or AR animals, but HD treatment almost completely reversed this increase. The ACE inhibitor captopril was effective at high doses in this model of severe AR. These effects might be related to the modulation of tissue RAS and the control of fibrosis.

WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine whether it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1(-/-) females were sterile because of uterine fibrosis and ovarian tubulostromal hyperplasia, most were subfertile, producing ≈1 fewer pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1(-/-) mice had normal weights, numbers of follicles, steroid hormone production, and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1(-/-) mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, and Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, and Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4(tm1.1Boer/tm1.1Boer);Tg (CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1(-/-) COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but it is unlikely to function as the sole ovarian WNT4 receptor.

Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.

No abstract

One of the major functions of the ovary is the biosynthesis of steroid hormones, which are essential for the development of secondary sexual characteristics at puberty, for subsequent ovarian function, and for the establishment and maintenance of pregnancy. Increases in our understanding of the molecular mechanisms governing the control of ovarian steroidogenesis have greatly improved our understanding of the female reproductive cycle, as well as the pathogenesis of reproductive disorders such as polycystic ovarian syndrome and premature ovarian failure. The pituitary gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are the main endocrine regulators of ovarian steroidogenesis, and act by directly or indirectly modulating the activity of a multitude of intracellular signaling pathways. The WNT/CTNNB1 pathway, which is now believed to be a significant contributor to the regulation of ovarian steroidogenesis, could be one of the pathways modulated by gonadotropin signaling. This review will focus on the emerging role of WNT/CTNNB1 signaling in the regulation of steroidogenesis, with emphasis on potential mechanisms of interaction with FSH/LH signaling in ovarian granulosa and luteal cells.