Dihydrotestosterone regulates oestrogen secretion, oestrogen receptor expression, and apoptosis in granulosa cells during antral follicle development

Dihydrotestosterone (DHT) is involved in the development of preantral follicles. However, the effect of DHT on the development of antral follicles has yet to be fully investigated. Herein, we used enzyme-linked immunosorbent assays, immunofluorescence assays, quantitative real time-polymerase chain reaction, immunohistochemical staining, and western blotting to investigate the effect of DHT on antral follicle development. First, we detected the concentration of DHT and the expression of the androgen receptor (AR) in different antral follicles. Second, multiple DHT concentration (10^-10-10^-7 M) were added to granulosa cells cultured in vitro to examine the influence of DHT on AR expression. Third, to study changes in the expression of oestrogen (E2) synthase and receptors during the development of antral follicles, we divided them according to their diameters into small (≤ 2 mm), medium (2-5 mm), and large (≥ 5 mm) groups. Fourth, we added DHT (10^-8 M) and flutamide (Flu, 10^-7 M) to granulosa cells to determine whether DHT regulates the expression of cytochrome P450 aromatase (CYP19A1) and the associated receptors through the AR pathway. Fifth, we tested the effect of DHT and Flu on the expression of apoptotic genes and proteins in granulosa cells. We found that AR was expressed in sheep antral follicle granulosa cells and was regulated by DHT. During antral follicle development, the concentration of E2 and the expression of CYP19A1 and E2 receptors significantly increased in granulosa cells. DHT influenced this increase, at least partially, through the AR. Moreover, DHT regulated the expression of apoptotic genes and proteins through the AR. Our study expands our knowledge on the regulatory mechanism of DHT in antral follicle development and guides further research on the androgen regulation of ovarian function.