标题：PGRMC1介导孕酮抑制卵泡发育的作用及机制研究

      作者：袁晓华 1，张莉莉 1，盛喜霞 2，杨春荣 1，王亚琴 1    1.陕西省人民医院妇产病院,陕西 西安 710068；2.西安兵器工业251医院，陕西 西安 710068

      卷次： 2019年30卷1期

      【摘要】 目的 探讨孕酮膜受体(PGRMC1)在孕激素抑制卵泡发育及颗粒细胞分化中的作用及作用机制。方法 将10只21 d的SD大鼠按随机数表法分为两组(n=5)，其中一组注射孕马血清促性腺激素(PMSG)(20 IU/mL)48 h后取卵巢，另外一组注射PMSG 24 h后注射孕酮(30 μg/只)，再经过24 h后取卵巢，通过测量卵巢大小、HE染色研究孕酮对卵泡发育的影响。PMSG (20 IU/mL)促排卵 48 h后取卵巢，后通过免疫组化观察PGRMC1受体分布情况。用PMSG促排卵 48 h后分离卵泡颗粒细胞，加入孕酮(100 ng/mL、250 ng/mL)和PGRMC1抑制剂AG205(10 μmol/L)后，采用Western blot检测卵巢颗粒细胞甾体激素生成相关的细胞外信号调节酶(ERK1/2)、甾体激素快速调节蛋白(StAR)、细胞色素P450胆固醇侧链切割酶(P450scc)的表达情况，采用ELISA试剂盒检测培养上清中的孕烯醇酮分泌情况。结果 PMSG处理组卵巢大小为(8.6±0.93) mm，明显大于 PMSG联合孕酮处理组的(5.2±0.58) mm，差异有统计学意义(P<0.05)；PMSG联合孕酮处理组始基卵泡数明显高于PMSG处理组[(62±8.6) vs(39±2.9)]，而有腔卵泡及成熟卵泡显著低于PMSG处理组[(3.8±1.0) vs (8.0±1.2)、(3.7±0.4) vs (8.1±0.6)]，差异均有统计学意义(P<0.05)；孕酮(100 ng/mL、250 ng/mL)抑制甾体激素生成相关的pERK1/2、StAR、p450scc的表达，进而抑制甾体激素的前体孕烯醇酮的合成[(2.9±0.23) ng/mL vs (5.3±0.18) ng/mL、[(3.0±0.67) ng/mL vs (5.3±0.18) ng/mL]，差异均有统计学意义(P<0.05)；然而PGRMC1抑制剂AG205(10 μM)逆转孕酮的这种抑制作用升高pERK1/2、StAR、p450scc的表达，差异均具有统计学意义(P<0.05)；AG205逆转孕酮(100 ng/mL、250 ng/mL)对孕烯醇酮合成的抑制作用[(9.6±0.67) ng/mL vs (2.9±0.18) ng/mL、(9.8±0.91) ng/mL vs (3.0±0.67) ng/mL]差异均具有统计学意义(P<0.05)。结论 孕酮抑制卵泡的发育，孕酮的这种抑制作用主要体现在对窦前卵泡的抑制作用。孕酮对卵巢颗粒细胞甾体激素的合成有抑制作用，PGRMC1的抑制剂AG205可以逆转孕酮的抑制作用，提示孕酮抑制卵泡发育，抑制颗粒细胞分化作用部分依赖于PGRMC1。
      【关键词】 卵巢颗粒细胞；孕酮膜受体；孕酮；孕烯醇酮；卵泡发育
      【中图分类号】 R329.2 【文献标识码】 A 【文章编号】 1003—6350（2019）01—0001—05

Role of PGRMC1 in the process of progesterone inhibiting follicle development and the related mechanism.
YUANXiao-hua 1, ZHANG Li-li 1, SHENG Xi-xia 2, YANG Chun-rong 1, WANG Ya-Qin 1. 1. Obstetrics and Gynecology Hospital,Shaanxi Provincial People's Hospital, Xi'an 710068, Shaanxi, CHINA; 2. Xi 'an Military Engineering 251 Hospital, Xi'an710068, Shaanxi, CHINA
【Abstract】 Objective To explore the role of progesterone receptor membrane component 1 (PGRMC1) in theprocess that progesterone inhibits follicle development and granulosa cells differentiation. Methods Ten SD rats (21days old) were randomly assigned into two groups to receive pregnant mare serum gonadotropin (PMSG, 20 IU/mL) orto receive progesterone 24 h after PMSG injection (30 g/mL). The ovaries were collected after 48 hours. The effect ofprogesterone on follicle development was studied by measuring the ovarian size and HE staining. The distribution ofPGRMC1 was studied through immunohistochemistry. 48 hours after injection of PMSG, granulosa cells were harvestedby puncturing follicles of ovary and then treated with progesterone (100 ng/mL, 250 ng/mL) and PGRMC1 inhibitorAG205 (10 μ mol/L). Then the proteins related with steroidogenesis, including extracellular signal-regulated kinase(ERK1/2), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme(P450scc), were examined by western blot. The secretion of pregnenolone in culture supernatant was examinedthrough ELISA kit. Results The size of ovaries in the PMSG group was (8.6±0.93) mm, significantly larger than(5.2±0.58) mm in the PMSG combined with progesterone group (P<0.05). The number of primordial follicles in PMSGcombined with progesterone group was significantly higher than that in PMSG group, (62±8.6) vs (39±2.9), P<0.05. Thenumber of antral follicles and mature follicles in PMSG combined with progesterone group were lower than those inPMSG group: (3.8±1.0) vs (8.0±1.2), P<0.05; (3.7±0.4) vs (8.1±0.6), P<0.05. Progesterone significantly inhibited the ex-pression of p-ERK1/2, StAR, and p450scc (P<0.05), and further inhibited the synthesis of pregnenolone (the precursor ofsteroid hormones): (2.9±0.23) ng/mL vs (5.3±0.18) ng/mL, (3.0±0.67) ng/mL vs (5.3±0.18) ng/mL, P<0.05. However,