Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

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.

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.

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.

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.

In Vitro Validation of the Hippo Pathway as a Pharmacological Target for Canine Mammary Gland Tumors.

Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Some clinical and molecular similarities between certain CMT subtypes and breast cancer make them a potential model for the study of the human disease. As misregulated Hippo signaling is thought to play an important role in breast cancer development and also occurs in CMTs, we sought to determine if Hippo represents a valid pharmacological target for the treatment of CMTs. Six CMT cell lines were assessed for their expression of the Hippo pathway effectors YAP and TAZ and for their sensitivity to verteporfin, an inhibitor of YAP-mediated transcriptional coactivation. Four cell lines that expressed YAP (CMT-9, -12, -28, -47) were found to be very sensitive to verteporfin treatment, which killed the cells through induction of apoptosis with ED50 values of 14-79 nM. Conversely, two YAP-negative cell lines (CF-35, CMT-25) were an order of magnitude more resistant to verteporfin. Verteporfin suppressed the expression of YAP/TAZ target genes, particularly CYR61 and CTGF, which play important roles in breast cancer development. Verteporfin was also able to inhibit cell migration and anchorage-independent growth. Likewise, verteporfin efficiently suppressed tumor cell invasiveness in the CMT-28 and -47 lines, but not in CF-35 cells. Together, our findings provide proof of principle that pharmacological targeting of the Hippo pathway compromises the viability and attenuates the malignant behavior of CMT cells. These results will serve as the basis for the development of novel chemotherapeutic approaches for CMTs that could translate to human medicine.