The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.M üllerian inhibiting substance (MIS), also known as antiMüllerian hormone (AMH), has long been appreciated for its role in sex differentiation and reproduction, and sensitive ELISAs measuring blood levels are used in fertility clinics around the world as a measure of ovarian reserve (1-6). MIS plays important roles in the development of the gonad and the differentiation of the urogenital ridge. In the male fetus, MIS produced by the developing testes causes regression of the Müllerian duct (7). In the female fetus, MIS may play a role in early follicle assembly in the gonad by primordial germ cells (not to be confused with primordial follicles), since mice overexpressing MIS are devoid of germ cells shortly after birth (8), and, similarly, ex vivo incubation of fetal ovaries with MIS results in the inhibition of follicle assembly (9). These data highlight a role of MIS during fetal development that is distinct from its regulatory role of folliculogenesis postnatally.In the adult, MIS is produced predominantly by the cumulus (less so by the mural) granulosa cells of secondary and early antral...

Sertoli and germ cell interactions are essential for spermatogenesis and, thus, male fertility. Sertoli cells provide a specialized microenvironment for spermatogonial stem cells to divide, allowing both self-renewal and spermatogenesis. In the present study, we used mice with a conditional activated allele of the beta-catenin gene (Ctnnb1(tm1Mmt)(/+)) in Sertoli cells expressing Cre recombinase driven by the anti-Müllerian hormone (AMH; also known as Müllerian-inhibiting substance) type II receptor promoter (Amhr2(tm3(cre)Bhr)(/+)) to show that constitutively activated beta-catenin leads to their continuous proliferation and compromised differentiation. Compared to controls, Sertoli cells in mature mutant mice continue to express high levels of both AMH and glial cell-derived neurotrophic factor (GDNF), which normally are expressed only in immature Sertoli cells. We also show evidence that LiCl treatment, which activates endogenous nuclear beta-catenin activity, regulates both AMH and GDNF expression at the transcriptional level. The epididymides were devoid of sperm in the Amhr2(tm3(cre)Bhr)(/+);Ctnnb1(tm1Mmt)(/+) mice at all ages examined. We show that the mutant mice are infertile because of defective differentiation of germ cells and increased apoptosis, both of which are characteristic of GDNF overexpression in Sertoli cells. Constitutive activation of beta-catenin in Amhr2-null mice showed the same histology, suggesting that the phenotype was the result of persistent overexpression of GDNF. These results show that dysregulated wingless-related MMTV integration site/beta-catenin signaling in Sertoli cells inhibits their postnatal differentiation, resulting in increased germ cell apoptosis and infertility.

Leiomyomas and other mesenchymally derived tumors are the most common neoplasms of the female reproductive tract. Presently, very little is known about the etiology and progression of these tumors, which are the primary indication for hysterectomies. Dysregulated WNT signaling through beta-catenin is a well-established mechanism for tumorigenesis. We have developed a mouse model that expresses constitutively activated beta-catenin in uterine mesenchyme driven by the expression of Cre recombinase knocked into the Müllerian-inhibiting substance type II receptor promoter locus to investigate its effects on uterine endometrial stroma and myometrium. These mice show myometrial hyperplasia and develop mesenchymal tumors with 100% penetrance that exhibit histological and molecular characteristics of human leiomyomas and endometrial stromal sarcomas. By immunohistochemistry, we also show that both transforming growth factor beta and the mammalian target of rapamycin are induced by constitutive activation of beta-catenin. The prevalence of the tumors was greater in multiparous mice, suggesting that their development may be a hormonally driven process or that changes in uterine morphology during pregnancy and after parturition induce injury and repair mechanisms that stimulate tumorigenesis from stem/progenitor cells, which normally do not express constitutively activated beta-catenin. Additionally, adenomyosis and endometrial gland hyperplasia were occasionally observed in some mice. These results show evidence suggesting that dysregulated, stromal, and myometrial WNT/beta-catenin signaling has pleiotropic effects on uterine function and tumorigenesis.