The Yin and Yang of WNT4 Signaling in the Adult Gonad.

Abstract

The WNTs are a family of secreted glycoprotein signaling molecules best known for the multiple roles that they play during embryonic development. WNT signaling first attracted the attention of the reproductive biology research community a little over a decade ago, when a groundbreaking study by Vainio et al. demonstrated that the ablation of Wnt4 in female mice causes partial sex-reversal and oocyte loss. More recently, the regulated expression of several WNTs and WNT signaling effectors has been shown to occur in adult male and female gonads. For instance, in mice, WNT4 expression persists in ovarian granulosa cells after birth, and is markedly induced during ovulation/luteinization, strongly suggesting a role for WNT4 in adult ovarian physiology. To define the function(s) of WNT4 in postnatal granulosa cells, we used a conditional gene targeting approach. We showed that Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had reduced ovary weights, produced smaller litters, and had only a quarter of the normal number of healthy antral follicles at puberty. RTPCR analyses of Wnt4flox/-;Amhr2tm3(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 (β-catenin). Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice. Together, these data indicated that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. In male mice, ourselves and others have shown that constitutive activation of CTNNB1 in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model results in progressive germ cell loss and sterility. More recently, we sought to determine if the latter phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes lose SSC activity over time, and cannot support colonization by donor SSCs. Microarray analyses showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway in Sertoli cells, including Wnt4. We therefore tested the effects of exogenous WNT4 on SSC-enriched germ cell cultures, and found that WNT4 reduced germ cell numbers due to increased apoptosis and reduced SSC capacity to colonize recipient testes. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Taken together, our data indicate diametrically opposed functions for WNT4/CTNNB1 signaling in the adult female and male gonad: a powerful positive regulator of follicle development on the one hand, an equally powerful negative regulator of spermatogenesis on the other. (Supported by the Canadian Institutes of Health Research and the National Sciences and Engineering Research Council of Canada). (platform)