College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, Gansu, China; Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou, 730070, Gansu, China.
Animal Science and Technology College, Beijing University of Agriculture, Beijing, 102206, China.
Theriogenology. 2024 Jan 15;214:342-351. doi: 10.1016/j.theriogenology.2023.11.010. Epub 2023 Nov 13.
The corpus luteum is primarily responsible for the production and secretion of progesterone. Melatonin has been established to regulate autophagy and induce progesterone secretion in luteal cell. However, whether melatonin affects progesterone secretion by interfering with autophagy is yet to be reported. In the present study, the expression levels of melatonin receptors (MT1 and MT2), autophagy-related protein Beclin1 (Bec1), microtubule-associated protein light chain 3 B (LC3B), progesterone and steroidogenic acute regulatory protein (StAR), and cytochrome P450scc (CYP11A1) were analyzed in the corpus luteum of sheep at different stages (early, middle, and late); specifically, enzyme-linked immunosorbent assays, immunohistochemical staining, and western blotting were utilized for this expression analysis. In addition, to determine whether melatonin regulated progesterone secretion via the regulation of autophagy, luteal cells were cultured before being exposed to different concentrations of melatonin (0.01-100 nM) and the autophagy inhibitor chloroquine (50 μM). Next, luteal cells were treated with the melatonin receptor inhibitors 4-phenyl-2-propionamidotetralin (1 μM) and luzindole (1 μM) before detecting Bec1, LC3B2, AMPK/mTOR, and progesterone secretion levels to ascertain whether the effect of melatonin on autophagy and progesterone secretion is mediated by its corresponding receptors in luteal cells. Finally, to determine the significance of the AMPK/mTOR pathway in this process, an AMPK inhibitor, Compound C (10 μM), was added to luteal cells. Overall, the highest expression of melatonin receptors, autophagy and progesterone secretion was observed in the middle-phase corpus luteum; additionally, melatonin promoted autophagy, at least partially, through its receptor-mediated AMPK/mTOR pathway, which thereby promoting progesterone secretion in luteal cells in vitro. Ultimately, this study is the first to clarify the important role of autophagy in the melatonin-mediated regulation of progesterone secretion in the corpus luteum of sheep; it also lays a foundation for further exploration into the role of melatonin in regulating sheep's ovarian function.
黄体主要负责孕激素的产生和分泌。褪黑素已被证实可调节自噬并诱导黄体细胞分泌孕激素。然而，褪黑素是否通过干扰自噬来影响孕激素的分泌尚未有报道。在本研究中，我们分析了绵羊黄体在不同阶段（早期、中期和晚期）中褪黑素受体（MT1 和 MT2）、自噬相关蛋白 Beclin1（Bec1）、微管相关蛋白轻链 3B（LC3B）、孕激素和类固醇生成急性调节蛋白（StAR）以及细胞色素 P450scc（CYP11A1）的表达水平；具体来说，我们使用酶联免疫吸附测定、免疫组织化学染色和蛋白质印迹法进行了这些表达分析。此外，为了确定褪黑素是否通过调节自噬来调节孕激素的分泌，我们在培养黄体细胞之前，将其暴露于不同浓度的褪黑素（0.01-100 nM）和自噬抑制剂氯喹（50 μM）中。接下来，我们用褪黑素受体抑制剂 4-苯基-2-丙酰胺四氢萘（1 μM）和鲁米诺（1 μM）处理黄体细胞，然后检测 Bec1、LC3B2、AMPK/mTOR 和孕激素分泌水平，以确定褪黑素对自噬和孕激素分泌的影响是否由其在黄体细胞中的相应受体介导。最后，为了确定 AMPK/mTOR 通路在这个过程中的意义，我们向黄体细胞中添加了 AMPK 抑制剂 Compound C（10 μM）。总的来说，在黄体中期，褪黑素受体、自噬和孕激素分泌的表达最高；此外，褪黑素通过其受体介导的 AMPK/mTOR 通路促进自噬，至少部分促进黄体细胞体外孕激素的分泌。最终，本研究首次阐明了自噬在褪黑素调节绵羊黄体孕激素分泌中的重要作用；它也为进一步探索褪黑素在调节绵羊卵巢功能中的作用奠定了基础。
