Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

Redundant roles of AKT1 and AKT2 in fertility, estrous cyclicity and endometrial gland development.

In brief: The regulation of AKT in the endometrium during many cellular processes such as apoptosis and cell survival is crucial during the estrous cycle to ensure fertility. This research shows the specific function of AKT isoforms in the mouse endometrium for litter size, estrous cyclicity and endometrial gland development. Abstract: Apoptosis and cell survival regulation are crucial processes during the estrous cycle to prepare a receptive uterus during implantation for successful recognition of pregnancy. PI3K/AKT signaling has a crucial role during gestation, and AKT isoforms (1, 2 or 3) are regulated differently in the endometrium during the estrous cycle and embryo implantation. However, the specific roles of these isoforms are still unclear. We have previously shown that AKT isoforms expression during the rat estrous cycle and gestation is differently regulated. The present study aimed to establish the specific role of AKT isoforms in the mouse uterus. The hypothesis is that dysregulation of AKT isoforms expression could cause fertility-related issues in an isoform-specific manner. With four different mouse models and in-house crossbreeding, all isoforms KO combinations (single, double and triple) were obtained in progesterone receptor-expressing tissues. The results demonstrated that in absence of one or more AKT isoforms, female fertility was decreased. Mainly, we have observed smaller litter size, specifically in Akt1-2 KO mice. Additionally, we have found Akt1-2-3 KO mice to be fully infertile. Estrous cyclicity was also disrupted in Akt1-2 KO mice with longer diestrus stage. Moreover, the number of endometrial glands was decreased throughout the estrous cycle suggesting an important role in gland development for AKT1 and AKT2. Our results suggest not only specific roles between each isoform but also a partially redundant function of AKT1 and AKT2 in litter size, estrous cyclicity and endometrial gland development. This highlights the importance of AKT in the physiological regulation of mouse fertility.

Effect of Inactivation of Mst1 and Mst2 in the Mouse Adrenal Cortex.

Recent conditional knockout of core components of the Hippo signaling pathway in the adrenal gland of mice has demonstrated that this pathway must be tightly regulated to ensure proper development and maintenance of the adrenal cortex. We report herein that the most upstream kinases of the pathway, the mammalian STE20-like protein kinases 1 and 2 (MST1and MST2, respectively), are expressed in the mouse adrenal cortex with MST2 expression being restricted to the zona glomerulosa (zG). To further explore the role of Hippo signaling in adrenocortical cells, we conditionally deleted Mst1/2 in steroidogenic cells using an Nr5a1-cre strain (Mst1 flox/flox ; Mst2 flox/flox ; Nr5a1-cre). Our results show that the loss of MST1/2 leads to the premature and progressive accumulation of subcapsular GATA4+, WT1+ adrenal gonadal primordium (AGP)-like progenitor cells starting at 2 months of age without affecting aldosterone and corticosterone secretion. To help us understand this phenotype, microarray analyses were performed on adrenal glands from 2-month-old mutant and control mice. Gene expression analyses revealed that loss of Mst1/2 leads to the overexpression of known downstream target genes (Ajuba, Aqp1, Fn1, Ibsp, Igf1, Igfbp2, Mmp2, Thbs1) of the main effector of Hippo signaling, YAP; and underexpression of genes (Agtr1b, Ecgr4, Hsd3b6, Nr0b1, Tesc, Vsnl1) that are normally specifically expressed in the zG or overexpressed in the zG compared to the zona fasciculata (zF). Together, these results suggest that MST1/2 regulates Hippo signaling activity in the adrenal cortex and that these two kinases are also involved in the fine tuning of zG cell function or differentiation.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Testis Development and Alters Somatic Cell Fate.

Hippo signaling plays an essential role in the development of numerous tissues. Although it was previously shown that the transcriptional effectors of Hippo signaling Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) can fine-tune the regulation of sex differentiation genes in the testes, the role of Hippo signaling in testis development remains largely unknown. To further explore the role of Hippo signaling in the testes, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in the somatic cells of the testes using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that early stages of testis somatic cell differentiation were not affected in this model but progressive testis cord dysgenesis was observed starting at gestational day e14.5. Testis cord dysgenesis was further associated with the loss of polarity of the Sertoli cells and the loss of SOX9 expression but not WT1. In parallel with testis cord dysgenesis, a loss of steroidogenic gene expression associated with the appearance of myofibroblast-like cells in the interstitial space was also observed in mutant animals. Furthermore, the loss of YAP phosphorylation, the accumulation of nuclear TAZ (and YAP) in both the Sertoli and interstitial cell populations, and an increase in their transcriptional co-regulatory activity in the testes suggest that the observed phenotype could be attributed at least in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper differentiation of testis somatic cells.

The granulosa cell response to luteinizing hormone is partly mediated by YAP1-dependent induction of amphiregulin.

BACKGROUND: The LH surge is a pivotal event that triggers multiple key ovarian processes including oocyte maturation, cumulus expansion, follicular wall rupture and luteinization of mural granulosa and theca cells. Recently, LH-dependent activation of the Hippo signaling pathway has been shown to be required for the differentiation of granulosa cells into luteal cells. Still, the precise interactions between Hippo and LH signaling in murine granulosa cells remain to be elucidated. METHODS: To detect the expression of effectors of the Hippo pathway, western blot, immunohistochemical and RT-qPCR analyses were performed on granulosa cells treated with LH in vitro or isolated from immature mice treated with eCG and hCG. Cultured granulosa cells were pretreated with pharmacologic inhibitors to identify the signaling pathways involved in Hippo regulation by LH. To study the roles of Yap1 and Taz in the regulation of the LH signaling cascade, RT-qPCR and microarray analyses were done on granulosa cells from Yap1f/f;Tazf/f mice treated with an adenovirus to drive cre expression. RT-qPCR was performed to evaluate YAP1 binding to the Areg promoter following chromatin immunoprecipitation of granulosa cells collected from mice prior to or 60 min following hCG treatment. RESULTS: Granulosa cells showed a transient increase in LATS1, YAP1 and TAZ phosphorylation levels in response to the ovulatory signal. This Hippo activation by LH was mediated by protein kinase A. Furthermore, Yap1 and Taz are required for the induction of several LH target genes such as Areg, Pgr and Ptgs2, and for the activation of the ERK1/2 pathway. Consistent with these results, there was a substantial overlap between genes that are upregulated by LH and those that are downregulated following loss of Yap1/Taz, highlighting a major role for Hippo in mediating LH actions in the ovulation process. Finally, we showed that there is a marked recruitment of YAP1 to the Areg promoter of granulosa cells in response to hCG stimulation. CONCLUSIONS: Overall, these results indicate that Hippo collaborates with the cAMP/PKA and ERK1/2 pathways to participate in the precise regulation of the LH cascade, and that Areg, as a direct transcriptional target of YAP1, is involved in mediating its actions in the ovary. Video Abstract.

The Hippo Pathway Effectors YAP and TAZ Regulate LH Release by Pituitary Gonadotrope Cells in Mice.

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LßT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

Statins downregulate YAP and TAZ and exert anti-cancer effects in canine mammary tumour cells.

Canine mammary tumours (CMTs) are the most common neoplasms in intact bitches, and few chemotherapeutic options are available for highly invasive and metastatic tumours. Recent studies have shown the potential involvement of dysregulated Hippo signalling in CMT development and progression. Statins can activate the Hippo pathway by blocking protein geranylgeranylation (GGylation), resulting in decreased expression and activity of the transcriptional co-activators YAP and TAZ. In this study, we therefore sought to determine if statins could exert anti-cancer effects in CMT cells. Our results demonstrate that Atorvastatin and Fluvastatin are cytotoxic to two CMT cell lines (CMT9 and CMT47), with ED50 values ranging from 0.95 to 23.5 µM. Both statins acted to increase apoptosis and promote cell cycle arrest. Both statins also decreased YAP and TAZ expression and reduced the mRNA levels of key Hippo transcriptional target genes known to be involved in breast cancer progression and chemoresistance (CYR61, CTGF and RHAMM). Moreover, both statins effectively inhibited cell migration and anchorage independent growth, but did not influence matrix invasion. Taken together, our results demonstrate for the first time that statins act upon the Hippo pathway in CMT cells to counteract several molecular and cellular hallmarks of cancer. These findings suggest that targeting the Hippo pathway with statins represents a novel and promising approach for the treatment canine mammary gland cancers.

Addition of a carboxy-terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice.

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G-protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

Constitutive activation of CTNNB1 results in a loss of spermatogonial stem cell activity in mice.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Slit/Robo signaling regulates Leydig cell steroidogenesis.

BACKGROUND: First identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored. METHODS: Immunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice. RESULTS: Slit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism. CONCLUSION: Taken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis. Video Abstract.

Non-canonical WNT5a regulates Epithelial-to-Mesenchymal Transition in the mouse ovarian surface epithelium.

The ovarian surface epithelium (OSE) is a monolayer that covers the ovarian surface and is involved in ovulation by rupturing and enabling release of a mature oocyte and by repairing the wound after ovulation. Epithelial-to-mesenchymal transition (EMT) is a mechanism that may promote wound healing after ovulation. While this process is poorly understood in the OSE, in other tissues wound repair is known to be under the control of the local microenvironment and different growth factors such as the WNT signaling pathway. Among WNT family members, WNT4 and WNT5a are expressed in the OSE and are critical for the ovulatory process. The objective of this study was to determine the potential roles of WNT4 and WNT5a in regulating the OSE layer. Using primary cultures of mouse OSE cells, we found WNT5a, but not WNT4, promotes EMT through a non-canonical Ca2+-dependent pathway, up-regulating the expression of Vimentin and CD44, enhancing cell migration, and inhibiting the CTNNB1 pathway and proliferation. We conclude that WNT5a is a stimulator of the EMT in OSE cells, and acts by suppressing canonical WNT signaling activity and inducing the non-canonical Ca2+ pathway.

YAP and TAZ are required for the postnatal development and the maintenance of the structural integrity of the oviduct.

The development of the Müllerian ducts into the female reproductive tract requires the coordination of multiple signaling pathways that regulate proliferation, apoptosis and differentiation. The Hippo pathway has been reported to interact with several pathways with established roles in Müllerian duct development; yet, its potential roles in reproductive tract development and function remain mostly uncharacterized. The objective of this study was therefore to characterize the roles of the Hippo transcriptional coactivators YAP and TAZ in the female reproductive tract using transgenic mouse models. This report shows that the concomitant conditional inactivation of Yap and Taz in the mouse Müllerian duct mesenchyme results in postnatal developmental defects of the oviduct. Most notably, discontinuities in the myosalpinx layer lead to the progressive formation of cystic dilations of the isthmus. These defects prevented embryo transport and subsequent implantation in older animals, causing infertility. The loss of YAP/TAZ did not appear to affect other biological processes known to be required for the maintenance of oviductal wall integrity, such as TGF-ß/SMAD and Notch signaling and the biogenesis of miRNA, suggesting that the Hippo pathway acts independently of these processes to direct oviduct development. Taken together, these results suggest redundant and essential roles for YAP and TAZ in the postnatal development of the oviduct and the maintenance of its structural integrity.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate.

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.

Lats1 and Lats2 are required for the maintenance of multipotency in the Müllerian duct mesenchyme.

WNT signaling plays essential roles in the development and function of the female reproductive tract. Although crosstalk with the Hippo pathway is a key regulator of WNT signaling, whether Hippo itself plays a role in female reproductive biology remains largely unknown. Here, we show that conditional deletion of the key Hippo kinases Lats1 and Lats2 in mouse Müllerian duct mesenchyme cells caused them to adopt the myofibroblast cell fate, resulting in profound reproductive tract developmental defects and sterility. Myofibroblast differentiation was attributed to increased YAP and TAZ expression (but not to altered WNT signaling), leading to the direct transcriptional upregulation of Ctgf and the activation of the myofibroblast genetic program. Müllerian duct mesenchyme cells also became myofibroblasts in male mutant embryos, which impeded the development of the male reproductive tract and resulted in cryptorchidism. The inactivation of Lats1/2 in differentiated uterine stromal cells in vitro did not compromise their ability to decidualize, suggesting that Hippo is dispensable during implantation. We conclude that Hippo signaling is required to suppress the myofibroblast genetic program and maintain multipotency in Müllerian mesenchyme cells.

Yes-associated protein expression in germ cells is dispensable for spermatogenesis in mice.

Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is expressed in the nucleus of spermatogonia in mice, suggesting a potential role in spermatogenesis. Here, we report the generation of a conditional knockout mouse model (Yapflox/flox ; Ddx4cre/+ ) that specifically inactivates Yap in the germ cells. The inactivation of Yap in spermatogonia was found to be highly efficient in this model. The loss of Yap in the germ cells had no observable effect on spermatogenesis in vivo. Histological examination of the testes showed no structural differences between mutant animals and age-matched Yapflox/flox controls, nor was any differences detected in gonadosomatic index, expression of germ cell markers or sperm counts. Cluster-forming assay using undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), also showed that YAP is dispensable for SSC cluster formation in vitro. However, an increase in the expression of spermatogenesis and oogenesis basic helix-loop-helix 1 (Sohlh1) and neurogenin 3 (Ngn3) was observed in clusters derived from Yapflox/flox ; Ddx4cre/+ animals. Taken together, these results suggest that YAP fine-tunes the expression of genes associated with spermatogonial fate commitment, but that its loss is not sufficient to alter spermatogenesis in vivo.

Lats1 and Lats2 are required for ovarian granulosa cell fate maintenance.

Recent reports suggest that the Hippo signaling pathway influences ovarian follicle development; however, its exact roles remain unknown. Here, we examined the ovarian functions of the Hippo kinases large tumor suppressors (LATS)1 and 2, which serve to inactivate the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Inactivation of Lats1/2 in murine granulosa cells either in vitro or in vivo resulted in a loss of granulosa cell morphology, function, and gene expression. Mutant cells further underwent changes in structure and gene expression suggestive of epithelial-to-mesenchymal transition and transdifferentiation into multiple lineages. In vivo, granulosa cell-specific loss of Lats1/2 caused the ovarian parenchyma to be mostly replaced by bone tissue and seminiferous tubule-like structures. Transdifferentiation into Sertoli-like cells and osteoblasts was attributed in part to the increased recruitment of YAP and TAZ to the promoters of sex-determining region Y box 9 and bone γ-carboxyglutamate protein, key mediators of male sex determination and osteogenesis, respectively. Together, these results demonstrate for the first time a critical role for Lats1/2 in the maintenance of the granulosa cell genetic program and further highlight the remarkable plasticity of granulosa cells.-Tsoi, M., Morin, M., Rico, C., Johnson, R. L., Paquet, M., Gévry, N., Boerboom, D. Lats1 and Lats2 are required for ovarian granulosa cell fate maintenance.

SFRP4 Is a Negative Regulator of Ovarian Follicle Development and Female Fertility.

WNT signaling regulates a variety of ovarian processes, including follicle development, granulosa cell (GC) proliferation and differentiation, steroidogenesis, and ovulation. The secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists. Sfrp4 expression was previously reported to be induced in ovarian GCs and cumulus cells in vivo following human chorionic gonadotropin treatment, suggesting that it may play key roles in cumulus expansion, ovulation/luteinization, and corpus luteum (CL) function. In this study, we aimed to define the physiological roles of Sfrp4 in the ovary by gene targeting. Sfrp4-null female mice were found to produce larger litters than did their wild-type littermates. Although previous studies had suggested roles of Sfrp4 in luteal cell survival, no differences in CL formation, morphology, steroidogenesis, involution, or luteal cell apoptosis were found in Sfrp4-null mice. Likewise, cumulus expansion occurred normally in Sfrp4-null mice, with minimal changes in cumulus cell gene expression. Hyperfertility in the Sfrp4-null model was ultimately attributed to decreased antral follicle atresia, leading to an enhanced ovulatory rate. Increased expression of FSH- and LH-responsive genes was found in GCs from Sfrp4-null mice, and GCs isolated from Sfrp4-null mice were found to be hyperresponsive to FSH and LH in vitro. Although Sfrp2 was found to be overexpressed in the GCs of Sfrp4-null mice (suggesting a compensatory mechanism), Sfrp2-null mice had normal fertility and ovulatory rates, and Sfrp2/4 double knockout mice did not differ from Sfrp4-null mice. Taken together, our results suggest that SFRP4 acts to attenuate GC responsiveness to gonadotropins, thereby decreasing follicle survival, ovulatory rate, and fertility.

The endogenous hydrogen sulfide generating system regulates ovulation.

The generation of free-radicals such as nitric oxide has been implicated in the regulation of ovarian function, including ovulation. Tissues that generate nitric oxide typically generate another free-radical gas, hydrogen sulfide (H2S), although little is known about the role of H2S in ovarian function. The hypothesis of this study was that H2S regulates ovulation. Treatment with luteinizing hormone (LH) increased the levels of mRNA and protein of the H2S generating enzyme cystathionine γ-lyase (CTH) in granulosa cells of mice and humans in vivo and in vitro. Pharmacological inhibition of H2S generating enzymes reduced the number of follicles ovulating in mice in vivo and in vitro, and this inhibitory action was reversed by cotreatment with a H2S donor. Addition of a H2S donor to cultured mouse granulosa cells increased basal and LH-dependent abundance of mRNA encoding amphiregulin, betacellulin and tumor necrosis alpha induced protein 6, proteins important for cumulus expansion and follicle rupture. Inhibition of CTH activity reduced abundance of mRNA encoding matrix metalloproteinase-2 and -9 and tissue-type plasminogen activator, and cotreatment with the H2S donor increased the levels of these mRNA above those stimulated by LH alone. We conclude that the H2S generating system plays an important role in the propagation of the preovulatory cascade and rupture of the follicle at ovulation.

Targeted ablation of Wnt4 and Wnt5a in Müllerian duct mesenchyme impedes endometrial gland development and causes partial Müllerian agenesis.

Wnt4 and Wnt5a have well-established roles in the embryonic development of the female reproductive tract, as well as in implantation, decidualization, and ovarian function in adult mice. Although these roles appear to overlap, whether Wnt5a and Wnt4 are functionally redundant in these tissues has not been determined. We addressed this by concomitantly inactivating Wnt4 and Wnt5a in the Müllerian mesenchyme and in ovarian granulosa cells by crossing mice bearing floxed alleles to the Amhr2cre strain. Whereas fertility was reduced by ∼50% in Wnt4flox/flox; Amhr2cre/+ and Wnt5aflox/flox; Amhr2cre/+ females, Wnt4flox/flox; Wnt5aflox/flox; Amhr2cre/+ mice were either nearly or completely sterile. Loss of fertility was not due to an ovarian defect, as serum ovarian hormone levels, follicle counts, and ovulation rates were comparable to controls. Conversely, the uterus was abnormal in Wnt4flox/flox; Wnt5aflox/flox; Amhr2cre/+ mice, with thin myometrial and stromal layers, frequent fibrosis and a >90% reduction in numbers of uterine glands, suggesting redundant or additive roles of Wnt4 and Wnt5a in uterine adenogenesis. Loss of fertility in Wnt4flox/flox; Wnt5aflox/flox; Amhr2cre/+ mice was attributed to defects in decidualization, implantation, and placental development, the severity of which were proportional to the extent of gland loss. Furthermore, a third of Wnt4flox/flox; Wnt5aflox/flox; Amhr2cre/+ females had a partial agenesis of Müllerian duct-derived structures, but with normal oviducts and ovaries. Together, our results suggest that Wnt4 and Wnt5a play redundant roles in the development of the female reproductive tract, and may provide insight into the etiology of certain cases of Müllerian agenesis in women.