Oocyte-derived R-spondin2 and GDF9:BMP15 heterodimer coordinate to promote the development of granulosa cells and oocytes in mice

Oocyte-derived paracrine factors, of particular the TGF-beta superfamily members growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play prominent roles in this communication. However, whether there are other essential ODPFs functioning in the follicles, and how different components of the ODPF coordinate to regulate the development of ovarian follicles remains largely elusive. Here, we report that RSPO2, a member of the R-spondin family of proteins, is an essential ODPF that cooperates with the GDF9BMP15 heterodimer to regulate the development of granulosa cells, which in turn promotes the oocyte\u2019s own development in mice. We observed that Rspo2 was predominantly expressed in oocytes within mouse follicles, and in vitro, it coordinated with GDF9 BMP15 heterodimers to precisely maintain the integrity of the transcriptome of preantral granulosa cells. Most intriguingly, we observed that the coordinative effect between RSPO2 and GDF9BMP15 heterodimers on granulosa cell gene expression was either in concert or opposite depending on the specific genes that they regulate, and the crosstalk between the CTNNB1 and SMAD2 dependent signaling pathways was involved in this process. In vivo, conditional knockout of Rspo2 in oocytes starting either from the non-growing primordial-stage or growing primary-stage follicles caused severe defects in granulosa cell and oocytes, which resulted in subfertility in females before the 8-month age, and premature ovarian insufficiency and the ultimate sterility thereafter. Mechanistically, Rspo2 cKO in oocytes caused severe alteration of the transcriptomes of both the granulosa cells and oocytes, of which the downregulation of a number of transcripts essential for follicular development and function in granulosa cell could account for the defects of granulosa cells and follicles, while the upregulation of transcripts involved in mitochondrial function and the compromised distribution and function of mitochondria could be one of the key reasons for the decreased oocyte developmental competence observed in the cKO oocytes. We further demonstrated that RSPO2 interacted directly with LGR4, 5, 6 to enhance the activation of the CTNNB1 signaling and the downstream induction of the expression of Amh in granulosa cells. Taken together, this study revealed that RSPO2 is another key component of the ODPF cocktail within the follicle, which plays a crucial role in the control of granulosa cell development by coordination with GDF9:BMP15 heterodimers. Given that receptors for RSPO2 are barely detectable in mouse oocytes, the effects of oocyte-specific depletion of RSPO2 on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides a new genetic evidence buttressing the existence of an oocyte-granulosa cell regulatory loop functioning within the mammalian ovarian follicle.