Rak-Mardyła Agnieszka, Wróbel Anna, Gregoraszczuk Ewa L
Department of Physiology and Toxicology of Reproduction, Institute of Zoology, Jagiellonian University in Krakow, Kraków, Poland
Department of Physiology and Toxicology of Reproduction, Institute of Zoology, Jagiellonian University in Krakow, Kraków, Poland.
Reprod Sci. 2015 Apr;22(4):469-75. doi: 10.1177/1933719114549854. Epub 2014 Sep 11.
We previously showed that expression of ghrelin messenger RNA is significantly increased in the ovaries of cycling pigs but not in prepubertal animals and that ghrelin stimulates estradiol (E2) secretion by ovarian follicles in prepubertal animals. The present study investigated in vitro the role of ghrelin in regulating the ovarian steroidogenesis during estrus cycle in mature pigs. Small (SFs), medium (MFs), and large (LFs) ovarian follicles were collected on days 4 to 6, 10 to 12, and 16 to 18 of the estrous cycle from cycling pigs and exposed to 20, 100, and 500 pg/mL ghrelin for 24 hours. In additional experiments, MFs were exposed to ghrelin plus 100 ng/mL follicle-stimulating hormone (FSH) or luteinizing hormone (LH). Levels of progesterone (P4), testosterone (T), and E2 in culture medium were determined by enzyme-linked immunosorbent assay, and the expression of the steroid pathway enzymes 3β hydroxysteroid dehydrogenase (HSD), 17β-HSD, and cytochrome P450 aromatase (CYP19) was evaluated by Western blotting. Ghrelin had no effect on steroid secretion when present at 20 pg/mL, its concentration in follicular fluid, whereas at 100 pg/mL and 500 pg/mL, its concentration in serum, ghrelin significantly decreased secretion of P4, T, and E2. Moreover, all concentrations of ghrelin decreased steroid secretion in FSH- and LH-stimulated follicles. Western blot analysis showed that ghrelin inhibited expression of 3β-HSD, 17β-HSD, and CYP19 proteins. These results suggest that ghrelin, by direct inhibition of 3β-HSD, 17β-HSD, and CYP19 protein expression, inhibits LH- and FSH-stimulated steroid secretion by ovarian follicles, thus negatively affecting ovarian steroidogenesis in mature pigs.
我们之前的研究表明，生长激素释放肽信使核糖核酸在处于发情周期的母猪卵巢中显著增加，但在青春期前的动物卵巢中未增加，并且生长激素释放肽可刺激青春期前动物卵巢卵泡分泌雌二醇（E2）。本研究在体外探讨了生长激素释放肽在调节成熟母猪发情周期中卵巢类固醇生成的作用。在发情周期的第4至6天、第10至12天和第16至18天，从处于发情周期的母猪收集小卵泡（SFs）、中卵泡（MFs）和大卵泡（LFs），并将其暴露于20、100和500 pg/mL的生长激素释放肽中24小时。在额外的实验中，将中卵泡暴露于生长激素释放肽加100 ng/mL促卵泡激素（FSH）或促黄体生成素（LH）。通过酶联免疫吸附测定法测定培养基中孕酮（P4）、睾酮（T）和E2的水平，并通过蛋白质印迹法评估类固醇途径酶3β羟类固醇脱氢酶（HSD）、17β-HSD和细胞色素P450芳香化酶（CYP19）的表达。当生长激素释放肽浓度为20 pg/mL（卵泡液中的浓度）时，对类固醇分泌无影响，而当浓度为100 pg/mL和500 pg/mL（血清中的浓度）时，生长激素释放肽显著降低P4、T和E2的分泌。此外，所有浓度的生长激素释放肽均降低了FSH和LH刺激的卵泡中的类固醇分泌。蛋白质印迹分析表明，生长激素释放肽抑制3β-HSD、17β-HSD和CYP19蛋白的表达。这些结果表明，生长激素释放肽通过直接抑制3β-HSD、17β-HSD和CYP19蛋白的表达，抑制LH和FSH刺激的卵巢卵泡类固醇分泌，从而对成熟母猪的卵巢类固醇生成产生负面影响。
