Identification of TSHB gene expression profile in Duolang sheep and its functional role in granulosa cells

Abstract Objective Thyroid-stimulating hormone beta subunit (TSHB) gene, a member of the glycoprotein hormone beta subunit family, has been detected in ovarian tissues and granulosa cells (GCs) of several species, suggesting a potential involvement in ovarian function. However, its molecular regulatory mechanisms and functional roles in Duolang sheep remain unclear. Methods Clone the coding sequence of the TSHB gene in Duolang sheep and identify the development stage when its expression is high. Models with TSHB overexpression and siRNA interference were constructed to investigate their effects on the proliferation, apoptosis, and hormone secretion during the follicular phase in ovarian GCs in Duolang sheep. Results CCK-8 assays, conducted at four distinct time points, showed that TSHB overexpression significantly enhanced the proliferation of GCs, accompanied by the upregulation of Cyclin E/CDK2 and Bcl-2, and downregulation of Bax and Caspase3. Interference showed a trend opposite to that of overexpression (p < 0.05). ELISA results revealed that TSHB overexpression significantly reduced estrogen levels. Interference showed a trend opposite to that of overexpression (p < 0.05), which was consistent with decreased expression of steroidogenic acute regulatory protein and CYP19A1. Additionally, qPCR analysis demonstrated that TSHB significantly upregulated the expression of TSHR and FSHR (p < 0.05). Conclusion Specifically, the above results indicate that the TSHB gene promotes the proliferation of GCs in Duolang sheep, providing a new perspective on their follicular development.