Anti-Müllerian hormone regulates ovarian granulosa cell growth in PCOS rats through SMAD4

Objective

Polycystic ovary syndrome (PCOS) is a diverse condition with an unknown cause. The precise mechanism underlying ovulatory abnormalities in PCOS remains unclear. It is widely believed that malfunction of granulosa cells is the primary factor contributing to aberrant follicular formation in PCOS.

Methods

A DHEA-induced PCOS rat model was established, and ovarian granulosa cells were extracted and identified. Anti-Müllerian hormone (AMH) and SMAD family member 4 (SMAD4) expression was detected in the serum, ovarian tissue and ovarian granulosa cells of each group, and proliferating cell nuclear antigen (PCNA), BCL2-associated 2 (BAX), cleaved caspase-3 and BCL-2 protein expression was detected by Western blot in ovarian granulosa cells. Recombinant anti-Müllerian hormone (rAMH) was administered at different concentrations to act on normal rat ovarian granulosa cells, cell proliferation was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SMAD4, caspase-3, BCL-2 and cyclin A proteins were detected by Western blot. SMAD4-siRNA was transfected into rat ovarian granulosa cells of the PCOS group, and PCNA and BAX were detected by Western blot.

Results

Compared with those in the control group, the expression of AMH and SMAD4 was increased in the ovarian tissues and granulosa cells of rats in the PCOS group, the expression of PCNA and BCL-2 proteins was decreased in the ovarian granulosa cells of the PCOS group, the expression of BAX proteins was increased, and the expression of cleaved caspase-3 was increased. Western blot results indicated that rAMH upregulated SMAD4 and caspase-3 protein expression in granulosa cells and downregulated cyclin A and BCL-2 protein expression. CCK-8 and flow cytometry results indicated that AMH decreased granulosa cells proliferation and increased apoptosis. SiRNA knockdown of SMAD4 gene increased PCNA and BCL-2 protein expression in the granulosa cells of PCOS rats and decreased BAX and cleaved caspase-3 protein expression.

Conclusion

AMH may be involved in regulating impaired ovarian granulosa cells development in PCOS rats via SMAD4.