Laboratory of Biotechnology and Physiology of Reproduction (LABIREP), Federal University of Ceara, Av. Maurocélio Rocha Ponte 100, postal code 62041-040, Sobral, CE, Brazil.
Department of Veterinary Medicine, Sousa Campus, Federal Institute of Education, Science and Technology of Paraíba, Sousa, Paraíba, Brazil.
Domest Anim Endocrinol. 2024 Jan;86:106824. doi: 10.1016/j.domaniend.2023.106824. Epub 2023 Nov 8.
This study aims to evaluate the effects of melatonin and its mechanisms of action on preantral follicle activation and survival, stromal cell density and collagen distribution in extracellular matrix (ECM). The involvement of melatonin receptors and mTORC1 pathway in these procedures were also investigated. To this end, ovarian fragments were cultured for six days in α-MEM alone or supplemented with 1000 pM melatonin, 1000 pM melatonin with 1000 pM luzindole (inhibitor of melatonin receptors), or 1000 pM melatonin with 0.16 µg/ml rapamycin (mTORC1 inhibitor). At the end of culture period, tissues were processed for classical histology, and the follicles were classified as normal or degenerated, as well as in primordial or growing follicles. The ovarian stromal cell density and ECM collagen distribution were also evaluated. Samples of ovarian tissues were also destined to measure the levels of thiol and mRNA for CAT, SOD, GPX1 and PRDX1, as well as the activity of antioxidant enzymes CAT, SOD, and GPX1. The results demonstrated that ovarian tissues cultured with melatonin, melatonin with luzindole or melatonin with rapamycin had significantly higher percentage of morphologically normal follicles than those cultured in control medium (α-MEM). However, the presence of either luzindole or rapamycin, did not block the positive effects of melatonin on follicle survival (P > 0.05). Although the presence of melatonin in culture medium reduced the percentage of primordial follicles and increased the percentage of development follicles, these positive effects of melatonin were blocked by either luzindole or rapamycin (P < 0.05). Melatonin, melatonin with luzindole or melatonin with rapamycin did not influence the number of ovarian stromal cells. In contrast, melatonin significantly increased the percentages of collagen in ovarian tissues, but the positive effects of melatonin were blocked by either luzindole or rapamycin. Tissues cultured with melatonin and rapamycin had higher levels of mRNA for CAT and lower GPx activity when compared to those cultured in control medium. In conclusion, melatonin promotes primordial follicle activation, increases collagen fiber in ECM of in vitro cultured bovine ovarian tissue through its membrane-coupled receptors and mTORC1. Oppositely, melatonin increase follicles survival by acting through other pathways, since it can pass through cell membranes and directly regulate oxidative stress.
本研究旨在评估褪黑素及其作用机制对原始卵泡激活和存活、基质细胞密度以及细胞外基质 (ECM) 中胶原分布的影响。还研究了褪黑素受体和 mTORC1 通路在此过程中的参与情况。为此，将卵巢组织片段在 α-MEM 中单独培养或添加 1000 pM 褪黑素、1000 pM 褪黑素加 1000 pM 鲁米诺（褪黑素受体抑制剂）或 1000 pM 褪黑素加 0.16 µg/ml 雷帕霉素（mTORC1 抑制剂）培养六天。在培养期末，对组织进行经典组织学处理，并将卵泡分类为正常或退化，以及原始或生长卵泡。还评估了卵巢基质细胞密度和 ECM 胶原分布。卵巢组织样本还用于测量硫醇水平和 CAT、SOD、GPX1 和 PRDX1 的 mRNA 水平，以及 CAT、SOD 和 GPX1 抗氧化酶的活性。结果表明，与在对照培养基（α-MEM）中培养的组织相比，用褪黑素、褪黑素加鲁米诺或褪黑素加雷帕霉素培养的卵巢组织中形态正常的卵泡比例显著更高（P<0.05）。然而，鲁米诺或雷帕霉素的存在并不能阻断褪黑素对卵泡存活的积极影响（P>0.05）。尽管培养基中褪黑素的存在减少了原始卵泡的比例并增加了发育卵泡的比例，但褪黑素的这些积极作用被鲁米诺或雷帕霉素阻断（P<0.05）。褪黑素、褪黑素加鲁米诺或褪黑素加雷帕霉素对卵巢基质细胞的数量没有影响。相反，褪黑素显著增加了卵巢组织中胶原的百分比，但褪黑素的积极作用被鲁米诺或雷帕霉素阻断。与在对照培养基中培养的组织相比，用褪黑素和雷帕霉素培养的组织中 CAT 的 mRNA 水平更高，而 GPx 活性更低。总之，褪黑素通过其膜偶联受体和 mTORC1 促进原始卵泡激活，并增加体外培养牛卵巢组织 ECM 中的胶原纤维。相反，褪黑素通过其他途径增加卵泡存活，因为它可以穿过细胞膜并直接调节氧化应激。
