He Haiying, Su Xiaohui, Yang Huiguo, Zhang Yingjie, Duan Chunhui, Yang Ruochen, Xie Fengmei, Liu Yueqin, Liu Wujun
Department of Animal Science and Biotechnology, Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China.
Moyu Bibang Sheep Industry Development Co. LTD, Hotan Prefecture, Xinjiang 848100, China.
Anim Biosci. 2024 Oct;37(10):1712-1725. doi: 10.5713/ab.23.0448. Epub 2024 Apr 26.
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.
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 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.
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 48 h and 72 h, 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.
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.
本研究旨在探讨催乳素（PRL）对绵羊卵巢颗粒细胞（GCs）增殖、凋亡及雌激素（E2）和孕酮（P4）分泌的影响，并探究PRL对相关基因和蛋白的作用。
从1岁小尾寒羊中分离卵巢GCs，分别采用免疫组织化学法鉴定卵巢上的PRL受体（PRLR）和卵巢GCs上的促卵泡激素受体（FSHR）。将PRL（0、0.05、0.50、5.00μg/mL）与促卵泡激素一起体外添加到GCs中，用细胞计数试剂盒-8（CCK-8）检测细胞增殖，用流式细胞术检测细胞凋亡。48小时和72小时后通过酶联免疫吸附测定法测量E2和P4含量。48小时后通过实时定量聚合酶链反应（RTqPCR）和蛋白质免疫印迹法鉴定功能基因和蛋白的表达。
PRLR在卵泡GCs和黄体中均有表达，而FSHR仅在卵泡GCs中特异性表达。第1天增殖活性较低，第4天和第5天较高。用PRL处理24小时后，0.05μg/mL组GCs的凋亡率显著高于对照组（p<0.05）。与对照组相比，PRL处理48小时和72小时后，GCs中E2的分泌显著减少（p<0.05），而P4的分泌显著增加（p<0.05）。PRLR、FSHR、LHR、CYP11A1、HSD3B7和STAR的mRNA表达水平显著高于对照组（p<0.01），PRL处理后所有PRL组中BCL2的相对丰度均增加。
PRL促进GCs增殖，超生理浓度通过下调BAX和上调BCL2抑制细胞凋亡。PRL通过下调CYP19A1抑制E2分泌，并通过上调CYP11A1、STAR和HSD3B7促进P4分泌。
