Departamento de Medicina Animal, Grupo de Investigación Medicina Interna Veterinaria (MINVET), Instituto de Investigación INBIO G+C, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
Departamento de Reproducción Animal, INIA-CSIC, Madrid, Spain.
Res Vet Sci. 2024 May;171:105222. doi: 10.1016/j.rvsc.2024.105222. Epub 2024 Mar 11.
In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.
卵母细胞体外成熟（IVM）在临床上被用于马以产生囊胚，但目前用于马的条件并不理想。我们分析了马促卵泡成熟激素（FF）分泌组的组成，并测试了其对 IVM 中卵母细胞减数分裂能力和基因表达的影响。采集发情前 FF，使用 10 kDa 超滤浓缩，-80°C 储存。分析其代谢和蛋白质组组成，并通过冷冻传输显微镜评估其超微结构组成。死后或通过卵母细胞抽吸（OPU）获得的卵母细胞在不存在（对照）或存在 20 或 40 μg/ml（S20 或 S40）分泌组的情况下进行 IVM。然后分析卵母细胞的染色质构型或冷冻用于基因表达分析。蛋白质组分析在 Equus caballus 数据库中检测到 255 种蛋白质，主要与补体级联和胆固醇代谢有关。代谢组学分析得到 14 种代谢物，冷冻传输电子显微镜分析显示存在细胞外囊泡（EVs）。在成熟率方面，各处理组之间没有差异。然而，与死后获得的卵母细胞相比，OPU 来源的卵母细胞中 GDF9 和 BMP15 的表达显著增加（倍数增加±SEM：BMP15 为 9.4±0.1 对 1±0.5，GDF9 为 9.9±0.3 对 1±0.5；p<0.05）。添加分泌组可增加 S40 中 TNFAIP6 的表达，而与卵母细胞来源无关。需要进一步研究以充分了解添加分泌组是否会影响马卵母细胞的发育能力。
