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 (E2) and progesterone (P4), 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 μg/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 E2 and P4 content by ELISA after 24h and 48h. The expression of functional genes and proteins was identified by RT-qPCR and Western-blot after 24h.

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 μg/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 E2 in GCs was reduced significantly (p<0.05) in PRL treatment for 24h and 48h, while the secretion of P4 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 E2 by down-regulating CYP19A1 and promoted P4 by up-regulating CYP11A1, STAR and HSD3B7.