Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats

Cathepsin D(CTSD),the major lysosomal aspartic protease that is widely expressed in different tissues,potentially regulates the biological behaviors of various cells.Follicular granulosa cells are responsive to the increase of ovulation number,hence indirectly influencing litter size.However,the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated.This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues.Moreover,western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats.Subsequently,the effects of CTSD knockdown on cell proliferation,apoptosis,cell cycle,and the expression of candidate genes of the prolific traits,including bone morphogenetic protein receptor IB(BMPR-IB),follicle-stimulating hormone(FSHR),and inhibinα(INHA),were determined in granulosa cells.Results showed that CTSD was expressed in corpus luteum,follicle,and granulosa cells.Notably,CTSD expression in the monotocous group was significantly higher than that in the polytocous group.In addition,CTSD knockdown could improve granulosa cell proliferation,inhibit cell apoptosis,and significantly elevate the expression of proliferating cell nuclear antigen(PCNA)and B cell lymphoma 2(Bcl-2),but it lowered the expression of Bcl-2-associated X(Bax)and caspase-3.Furthermore,CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase.The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression.However,the expression levels of BMPR-IB,FSHR,and INHA clearly increased as a result of CTSD knockdown.Hence,our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation,apoptosis,cell cycle,and the expression of candidate genes of the prolific trait.