WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PR(cre) mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PR(cre/+)Wnt4(f/f) (Wnt4(d/d)) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4(d/d) mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.

Misregulation of the Wnt/B-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/ B-catenin pathway are expressed in ovarian granulosa cells; nevertheless, its potential involvement in granulosa cell tumorigenesis has not been examined. To this end, human (n = 6) and equine (n = 18) granulosa cell tumors (GCT) were analyzed for B-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of B-catenin, suggesting that activation of the Wnt/B-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnb flox(ex3)/+ ; Amhr2 cre/+ mice that express a dominant stable B-catenin mutant in their granulosa cells were generated. These mice developed follicle-like structures containing disorganized, pleiomorphic granulosa by 6 weeks of age. Even in older mice, these follicle-like lesions grew no larger than the size of antral follicles and contained very few proliferating cells. Similar to corpora lutea, the lesions were highly vascularized, although they did not express the luteinization marker Cyp11a1. Catnb flox(ex3)/+ ; Amhr2 cre/+ females were also found to be severely subfertile, and fewer corpora lutea were found to form in response to exogenous gonadotropin compared with control mice. In older mice, the ovarian lesions often evolved into GCT, indicating that they represent a pretumoral intermediate stage. The GCT in Catnb flox(ex3)/+ ; Amhr2 cre/+ mice featured many histopathologic similarities to the human disease, and prevalence of tumor development attained 57% at 7.5 months of age. Together, these studies show a causal link between misregulated Wnt/B-catenin signaling and GCT development and provide a novel model system for the study of GCT biology. (Cancer Res 2005; 65(20): 9206-15)

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Remodeling of ovarian follicle extracellular matrix is essential for ovulation and vascularization of the corpus luteum (CL). Formation of the cumulus matrix around oocytes also plays an important role in ovulation and subsequent fertilization of oocytes. ADAMTS1 is an extracellular metalloprotease induced in ovarian follicles by ovulatory hormones and is required for fertility. In this study, we identified ADAMTS1-mediated structural and morphological changes in remodeling of the follicle and cumulus oocyte complex (COC). In Adamts1(-/-) mice, the ovulation rate was 77% reduced and fertilization of ovulated oocytes was reduced a further 63%, resulting in a reduced number of litters and pups per litter. Morphological assessment of peri-ovulatory ovaries revealed abnormal morphogenesis with a lack of thecal/vascular invagination in the basal region of follicles. Cleavage of the ADAMTS1 substrate, versican, at these invaginating regions was abundant in Adamts1(+/-) but undetectable in Adamts1(-/-) ovaries, indicating that processing of versican by ADAMTS1 is involved in ovulating follicle remodeling. Versican and hyaluronan localization was abnormal during COC matrix expansion, and versican persisted beyond the expected time of fertilization in Adamts1(-/-) but was catabolized and cleared from control COC. The results demonstrate that ADAMTS1 is critical in both ovulation and fertilization processes in vivo. The protease activity of ADAMTS1 mediates neomorphogenesis of the ovulating follicle wall and COC matrix necessary for successful ovulation and fertilization, as well as subsequent catabolism of versican required for degradation of COC matrix after fertilization.