Search

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2 mm), medium (2-5 mm) and large (≥5 mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1 IU/ml) and testosterone (T, 10 -7  M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development., (© 2020 Wiley-VCH GmbH.)

Corpus luteum (CL) regression is a complex physiological process. Previous studies have shown that dihydrotestosterone (DHT) may be involved in regulating CL regression, but the mechanism is still unclear. In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α‐reductase (5α‐red1 and 5α‐red2) and androgen receptor (AR) in sheep CL, and investigated 5α‐red1, 5α‐red2, AR, and DHT levels at different luteal stages of CL (early, middle, and late phase) by immunohistochemistry, quantitative real‐time polymerase chain reaction, and western blot analysis. Moreover, we cultured luteal cells from middle phase CL and treated them with different concentrations of DHT (10−10–10 −6 M) and the AR antagonist flutamide (10 −5 M), to evaluate whether DHT is involved in the regulation of progesterone (P4) secretion and progesterone nuclear receptor (PGR) expression and whether these effects are regulated by the AR pathway. We also investigated the effects of DHT and flutamide on prostaglandin F2α (PGF2α) secretion and apoptotic gene and protein expression. Our results showed that 5α‐red1, 5α‐red2, and AR were expressed in the CL, and their expression and DHT levels were changed during the luteal phase. DHT was involved in mediating P4 and PGF2α secretion and PGR and apoptotic gene and protein expression. The effects of DHT on CL were at least partially regulated by the AR pathway. This study reveals the mechanism of action of DHT on sheep CL regression and lays the foundation for further exploration of androgen regulation of CL function. [ABSTRACT FROM AUTHOR]

• The synthesis and receptor expression of estradiol increased with the development of antral follicles. • Dihydrotestosterone (DHT) participates in follicular development via, at least in part, the androgen receptor. • The upregulation of DHT on GPER in sheep granulosa cells was not mediated by androgen receptor pathway. 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. [ABSTRACT FROM AUTHOR]