Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Theriogenology. 2022 Oct 15;192:62-72. doi: 10.1016/j.theriogenology.2022.08.030. Epub 2022 Aug 28.
Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 μM), and the effect of cortisol addition (0.25 μM, 0.5 μM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.
卵母细胞体外成熟（IVM）和玻璃化程序会对卵母细胞的整体质量产生不利影响。在 IVM 期间添加抗氧化剂，如辅酶 Q10（Q10），已被证明由于其在氧化损伤保护和调节基因转录中的作用而对卵丘-卵母细胞复合物产生积极影响。此外，糖皮质激素（GC）在生殖早期调节基因转录、能量代谢和细胞凋亡。在这方面，大多数 GC 作用是通过糖皮质激素受体（NR3C1）介导的，NR3C1 是一种转录因子。然而，GC 在卵巢生理学和卵母细胞成熟中的具体作用仍不清楚。在这方面，更好地了解 IVM 和玻璃化过程中与 GC 相关和凋亡相关基因的表达可能有助于改进牛的辅助生殖技术。本研究旨在探讨新鲜和玻璃化牛卵母细胞和卵丘细胞中 NR3C1 mRNA 的表达情况，以及 Q10（50μM）对其的影响，并研究添加皮质醇（0.25μM，0.5μM）对 NR3C1 表达的影响。我们还研究了属于 GC 调节途径的 NR3C1 相关基因的 mRNA 表达，如羟甾脱氢酶（HSD11B1；HSD11B2）、免疫亲和素（FKBP4；FKBP5）、信号转导和转录激活因子（STAT3；STAT5A）、矿物质皮质激素受体（NR3C2），以及凋亡途径中的抗凋亡（BCL2）和促凋亡（BAX）mRNA 转录本，1）在 IVM 后，2）在玻璃化后，无论 IVM 期间是否补充 Q10。我们的结果表明，卵母细胞玻璃化后 NR3C1 受体表达增加，但 IVM 期间外源性皮质醇补充后 NR3C1 受体表达没有增加。此外，Q10 降低了不成熟卵母细胞中 HSD11B1 和 FKBP5 的 mRNA 表达（卵丘细胞中的 HSD11B1 mRNA 表达也降低），以及 BAX:BCL2 比值的 mRNA 表达。在 Q10 存在下进行玻璃化后，卵丘细胞中 HSD11B2 的 mRNA 表达增加，而卵母细胞和卵丘细胞中 HSD11B1 和 BAX:BCL2 的 mRNA 表达均显著降低。总之，我们的研究结果首次表明 IVM、玻璃化和 Q10 补充对与 GC 相关和凋亡基因的 mRNA 相对表达的影响，以及玻璃化对 NR3C1 蛋白表达的影响。
