Department of Physiology and Toxicology of Reproduction, Institute of Zoology, Jagiellonian University, Gronostajowa 9, Cracow, Poland.
Theriogenology. 2012 May;77(8):1505-12. doi: 10.1016/j.theriogenology.2011.11.017. Epub 2012 Feb 14.
Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.
最近的研究表明，ghrelin 在控制女性生殖中发挥直接作用。本研究的目的是通过实时 PCR、Western blot 和免疫组织化学分析，分别研究 ghrelin 和其受体（ghrelin receptor）在猪黄体（corpora lutea，CL）中的 mRNA 和蛋白表达（via real time PCR, Western blot and immunohistochemistry analysis, respectively）。在黄体期（luteal phase），CL 分别在排卵后 1-2 天（CL1）、排卵后 7-10 天（CL2）和排卵后 13-15 天（CL3）被收集。ghrelin 的表达和酰化及非酰化形式的 ghrelin 浓度在 CL 发育过程中显著增加。免疫组织化学分析显示，ghrelin 蛋白定位于大黄体细胞的细胞质中。ghrelin 受体的表达没有变化。然后分析了 ghrelin 对孕酮（P4）分泌和 3-β-羟甾脱氢酶（3β-HSD）活性的直接体外影响，通过将 pregnenolone（P5）转化为 P4 并测量 3β-HSD 蛋白表达来进行分析。为了评估 3β-HSD 活性，首先将成熟黄体细胞用 ghrelin 在 100、250、500 和 1000 pg/mL 与 P5 或氨基导眠能（aminoglutethimide，AMG）孵育 24 小时。AMG 是 CYP11A1 介导的羟化的抑制剂；加入 AMG 和 P5 使 P4 产生可作为 3β-HSD 活性的指标。ghrelin 对 P4 分泌、3β-HSD 活性和蛋白表达的抑制作用。总之，ghrelin 和其受体在猪黄体中的存在以及 ghrelin 对黄体 P4 分泌和 3β-HSD 的直接抑制作用表明，ghrelin 在卵巢功能黄体期可能具有自分泌/旁分泌调节作用。
