The Gonadal Supporting Cell Lineage and Mammalian Sex Determination: The Differentiation of Sertoli and Granulosa Cells

The Wnt/β-catenin signaling system plays essential roles in embryonic development and in the self-renewal and maintenance of adult stem cells. R-spondins (RSPOs) are a group of secreted proteins that enhance Wnt/β-catenin signaling and have pleiotropic functions in development and stem cell growth. LGR5, an orphan receptor of the G protein-coupled receptor (GPCR) superfamily, is specifically expressed in stem cells of the intestinal crypt and hair follicle. Knockout of LGR5 in the mouse results in neonatal lethality. LGR4, a receptor closely related to LGR5, also has essential roles in development, as its knockout leads to reduced viability and retarded growth. Overexpression of both receptors has been reported in several types of cancer. Here we demonstrate that LGR4 and LGR5 bind the R-spondins with high affinity and mediate the potentiation of Wnt/β-catenin signaling by enhancing Wnt-induced LRP6 phosphorylation. Interestingly, neither receptor is coupled to heterotrimeric G proteins or to β-arrestin when stimulated by the R-spondins, indicating a unique mechanism of action. The findings provide a basis for stem cell-specific effects of Wnt/β-catenin signaling and for the broad range of functions LGR4, LGR5, and the R-spondins have in normal and malignant growth.

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.

In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Here we demonstrate in the mouse that a single factor, the forkhead transcriptional regulator FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. Inducible deletion of Foxl2 in adult ovarian follicles leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Our results show that maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.