Identification And Functional Analysis Of WNT4 Alternative Splicing,a Gene Related To Multiple Kidding Traits In Goats

Wingless MMTV integration site family member 4(WNT4)is an important member of the WNT protein family,which is involved in the growth and differentiation of a variety of cells and plays an important role in embryonic development and cellular homeostasis maintenance.Previous study has identified the presence of WNT4 alternative splicing in goats.However,the expression and mechanism of action of WNT4 alternative splicing in goat ovaries has not been reported.In this study,5 Yunshang black goats in follicular stage and 5 Yunshang black goats in luteal stage were selected from high yield group(average kidding number was 3.00±0.38)and low yield group(average kidding number was 1.32±0.19),with a total of 20 Yunshang black goats.Goat ovaries and other tissues were slaughtered and collected for subsequent experiments.In this study,we identified WNT4 alternative splicing in a population of Yunshang black goats;overexpression/disruption of different alternative splicing of WNT4 by culturing goat granulosa cells(GCs)in vitro.Resolve the molecular mechanism of WNT4 alternative spliceosome on GCs proliferation.The main results of the study are as follows:1.Two alternative splices of WNT4,WNT4-α and WNT4-β,were successfully cloned from ovarian tissue of Yunshang black goats.The results showed that the full length CDS region of WNT4-α and WNT4-β were 1055 bp and 986 bp,respectively,encoding 350 and317 amino acids.The prediction of secondary structure and tertiary structure showed that the protein structure of WNT4-β changed compared with that of WNT4-α,and the tertiary structure of WNT4-α was more stable.The expression levels of both WNT4-α and WNT4-β were significantly higher in high-fertility Yunshang black goats than that in low-fertility group(P < 0.05).The expression level of WNT4-α the ovaries with high-fertility Yunshang black goats was significantly higher than that of WNT4-β at the follicular phase(P < 0.05).2.To investigate the effect of different splices of WNT4 on the performance of goats.Immunofluorescence(IF)results showed that WNT4 protein was highly expressed in goat granulosa cells,and the expression level of WNT4-α in goat GCs was significantly higher than that of WNT4-β(P < 0.05).Overexpression/disruption of WNT4-α and WNT4-β in goat GCs showed that overexpression of both WNT4-α and WNT4-β significantly increased the expression of the GC proliferation marker genes Cyclin D2 and Cyclin dependent kinase 4,while the opposite after inhibition of their expression(P < 0.05).Both WNT4-α and WNT4-β significantly promoted GCs proliferation(P < 0.05)and significantly promoted estradiol and progesterone secretion(P < 0.05).To investigate whether WNT4 affects goat granulosa cell proliferation through the WNT signaling pathway,the m RNA of WNT signaling pathway marker genes RAC family small GTPase1 and Ras homolog family member A and the protein level of the key signaling factor Beta-catenin were examined in goat GCs after overexpression or inhibition.The results showed that the expression of RAC1 and RHOA genes related to WNT signaling pathway and the protein level of key factor β-catenin were significantly increased after overexpression of WNT4(P < 0.05).The WNT4 can regulate the proliferation of goat GCs through the WNT/β-catenin signaling pathway.3.The role of serine and arginine-rich splicing factor 6(SRSF6)in the generation of WNT4 alternative splicing and its effect on GCs proliferation were investigated.The expression level of SRSF6 was significantly higher than that of other splicing factors(P< 0.05).The dual luciferase activity assay reported that the luciferase activity of SRSF6 can bind to the WNT4 wild-type splice site sequence(P < 0.05).The m RNA and protein expression levels of WNT4-α alternative splicing were significantly reduced and those of WNT4-β alternative splicing were significantly increased after overexpression of SRSF6(P < 0.05).The expression levels of the cell proliferation markers CCND2 and CDK4 genes were significantly decreased(P < 0.05).Significantly inhibited the proliferation of goat GCs(P < 0.05).These results suggested that the splicing factor SRSF6 regulated the production of WNT4 alternative splicing and inhibited the proliferation of goat GCs proliferation.In conclusion,two alternative splices(WNT4-α and WNT4-β)of WNT4 in the Yunshang black goats and investigated the effects of the two alternative splices of WNT4 on goat GCs and the molecular mechanism of alternative splices generation.This study provides a theoretical basis for an in-depth analysis of the potential molecular mechanisms by which WNT4 regulates the proliferation of goat GCs.