Bezerra M É S, Monte A P O, Barberino R S, Lins T L B G, Oliveira Junior J L, Santos J M S, Bezerra D O, Neves C A, Silva G C, Carvalho M A M, Matos M H T
Nucleus of Biotechnology Applied to Ovarian Follicle Development, Federal University of São Francisco Valley, 56300-990, Petrolina, PE, Brazil.
Integrated Nucleus of Morphology and Research with Stem Cells, Federal University of Piauí, 64049-550, Teresina, PI, Brazil.
Theriogenology. 2019 Feb;125:56-63. doi: 10.1016/j.theriogenology.2018.10.012. Epub 2018 Oct 13.
The aim of this study was to evaluate the effect of the conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells (oWJ-MSCs) on the morphology, growth, reactive oxygen species (ROS) and glutathione (GSH) intracellular levels, active mitochondria, and meiotic resumption of isolated ovine secondary follicles in vitro. The oWJ-MSCs were isolated and the medium where they were cultured was recovered (conditioned medium). Isolated ovine secondary follicles were cultured for 6 days in 1) supplemented α-MEM (control); 2) 50% α-MEM + 50% conditioned medium (α-MEM + CM group) or 3) conditioned medium only (CM group). The parameters analyzed were morphology, antrum formation, follicle and oocyte growth, ROS and GSH levels, mitochondrial activity and meiotic resumption. The percentage of normal follicles, antrum formation, and fully grown oocytes did not differ (P > 0.05) among treatments. Follicles cultured in α-MEM + CM group had greater (P < 0.05) diameter than other treatments after culture. Moreover, the diameter of the follicles cultured in CM alone was higher (P < 0.05) than in the α-MEM. In addition, α-MEM + CM and CM treatments increased the growth rate compared to the α-MEM. Treatments containing conditioned medium (α-MEM + CM or CM) significantly reduced ROS levels compared to the control medium. Moreover, mitochondrial activity was higher in α-MEM and α-MEM + CM than in CM alone. All treatments showed oocytes in GV, GVBD and MI. In conclusion, oWJ-MSCs conditioned medium, especially when associated with α-MEM, improves the growth of secondary follicles and reduces ROS generation after short-term culture.
本研究的目的是评估绵羊脐带华通氏胶间充质干细胞（oWJ-MSCs）条件培养基对体外分离的绵羊次级卵泡的形态、生长、活性氧（ROS）和谷胱甘肽（GSH）细胞内水平、活性线粒体以及减数分裂恢复的影响。分离出oWJ-MSCs，并收集其培养的培养基（条件培养基）。将分离出的绵羊次级卵泡在以下条件下培养6天：1）添加α-MEM的培养基（对照）；2）50%α-MEM + 50%条件培养基（α-MEM + CM组）或3）仅使用条件培养基（CM组）。分析的参数包括形态、腔形成、卵泡和卵母细胞生长、ROS和GSH水平、线粒体活性以及减数分裂恢复。各处理组之间正常卵泡的百分比、腔形成和完全成熟的卵母细胞没有差异（P > 0.
05）。培养后，α-MEM + CM组培养的卵泡直径大于其他处理组（P < 0.05）。此外，仅在CM中培养的卵泡直径高于α-MEM中的卵泡（P < 0.05）。另外，与α-MEM相比，α-MEM + CM和CM处理提高了生长速率。与对照培养基相比，含有条件培养基的处理组（α-MEM + CM或CM）显著降低了ROS水平。此外，α-MEM和α-MEM + CM中的线粒体活性高于仅CM中的线粒体活性。所有处理组的卵母细胞均呈现生发泡（GV）、生发泡破裂（GVBD）和第一次减数分裂中期（MI）。总之，oWJ-MSCs条件培养基，特别是与α-MEM联合使用时，在短期培养后可改善次级卵泡的生长并减少ROS的产生。
