Identification of WNT4 alternative splicing patterns and effects on proliferation of granulosa cells in goat.

Increasing ovulation numbers is one of the most important ways to promote reproduction in mammals, and follicular granulosa cells (GCs) provide the necessary nutrients and microenvironment for oocytes to ovulate. WNT4 has been shown to be a key factor in regulating the proliferation of GCs in mammalian ovarian tissues. Our previous transcriptome sequencing (RNA-seq) results have identified two alternatively spliced products of WNT4;however, little is known about the splicing mechanism and its effect on GC proliferation. In this study, two alternatively spliced products of WNT4, designated WNT4-α and WNT4-β, were identified by cloning and analyzed for their function by bioinformatics. The RT-qPCR and Western blot results showed that the expression of WNT4-α was significantly higher than that of WNT4-β in the ovary tissues and GCs of Yunshang black goats. We therefore hypothesized that WNT4-α was the main isoform affecting the proliferation of goat GCs. Subsequently, goat GC proliferation assays showed that overexpression of WNT4-α significantly promoted GC proliferation, and the opposite was true after WNT4-α inhibition. The expression of marker genes of the Wnt signaling pathway was also examined and WNT4-α was found to affect the proliferation and hormone secretion of goat GCs by regulating the Wnt signaling pathway. In addition, a series of splicing factors were involved in in the alternative splicing; in this study, SRSF6 was found to be involved as a splicing factor in the generation of WNT4 alternative splicing. In summary, WNT4 alternative splicing was mediated by the splicing factor SRSF6, and WNT4-α alternative splicing played an important role in follicle development and had a significant effect on the proliferation of goat GCs. The results of this study provide a theoretical foundation for further understanding the molecular regulatory mechanisms of the WNT4 in follicle development in goats.