Effects of prolactin on the proliferation and hormone secretion of ovine granulosa cells in vitro

Objective: The objective of this study was to investigate the effects of prolactin (PRL) on the proliferation and apoptosis of ovine ovarian granulosa cells (GCs) and the secretion of estrogen (E-2) and progesterone (P-4),as well as to explore the effects of PRL on related genes and proteins. Methods: We isolated ovarian GCs from 1-year-old small-tail Han sheep and identified PRL receptor (PRLR) on ovaries and follicle stimulating hormone receptor (FSHR) on ovarian GCs, respectively, using immunohistochemistry. PRL (0, 0.05, 0.50, 5.00 mu g/mL) were added to GCs in vitro along with FSH, cell proliferation was measured by cell counting Kit-8 (CCK-8) and apoptosis by flow cytometry. The measurement of E-2 and P-4 content by enzyme-linked immunosorbent assays after 48 h and 72 h. The expression of functional genes and proteins was identified by real-time quantitative polymerase chain reaction (RTqPCR) and Western-blot after 48 h. Results: PRLR was expressed in both follicular GCs and corpus luteum, whereas FSHR was expressed specifically. The proliferative activity was lower on day 1 while higher on day 4 and day 5. The apoptosis rate of GCs in the 0.05 mu g/mL group was significantly higher than that in the control group after treatment with PRL for 24 h (p<0.05). Compared with the control group, the secretion of E-2 in GCs was reduced significantly (p<0.05) in PRL treatment for 48 h and 72 h, while the secretion of P-4 was significantly increased (p<0.05). The mRNA expression levels of PRLR, FSHR, LHR, CYP11A1, HSD3B7, and STAR were significantly higher than those in the control group (p<0.01), and the relative abundance of BCL2 in all PRL group were increased after PRL treatment. Conclusion: PRL promoted the proliferation of GCs and supraphysiological concentrations inhibited apoptosis caused by down-regulation of BAX and up-regulation of BCL2. PRL inhibited E(2 )by down-regulating CYP19A1 and promoted P-4 by up-regulating CYP11A1, STAR, and HSD3B7.