University of Agriculture in Krakow, Department of Animal Reproduction, Anatomy and Genomics, Mickiewicza 24/28, 30-059, Krakow, Poland.
University of Agriculture in Krakow, Department of Animal Reproduction, Anatomy and Genomics, Mickiewicza 24/28, 30-059, Krakow, Poland.
Theriogenology. 2022 Aug;188:116-124. doi: 10.1016/j.theriogenology.2022.05.022. Epub 2022 May 27.
The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24-38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.
马卵母细胞的体外成熟（IVM）仍然效率不高，也不能产生一致的结果。在这个过程中，马卵母细胞的具体要求在很大程度上仍然未知，这阻碍了该物种辅助生殖技术（ART）的发展。由于卵巢卵泡微环境支持卵母细胞获得发育能力，卵泡液似乎是支持 IVM 过程的大量生物活性因子的来源。细胞外囊泡（EVs）是体液中细胞分泌的分子，能够在供体细胞和受体细胞之间传递分子信号和遗传信息（mRNA、miRNA）。因此，我们的假设是，来自小（<20mm）卵泡的卵泡液 EVs（ffEVs）可以提高母马卵母细胞的体外成熟率。为了验证我们的假设，抽吸马卵巢卵泡液，并通过超速离心分离 ffEVs，然后使用纳米颗粒跟踪分析和流式细胞术进行表征。此外，使用 ExoGlow-蛋白 EV 标记试剂盒（System Biosciences，Palo Alto，CA）对 ffEVs 进行标记。使用一步法（方法 I，连续培养 24-38 小时）或两步法（方法 II，培养 24 小时后初次去卵丘细胞）对卵丘-卵母细胞复合物（COCs）进行成熟，在存在（200μg 蛋白/ml）或不存在 ffEVs 的情况下进行成熟。结果显示 ffEVs 被马卵丘细胞内化，这也是首次在卵母细胞中观察到。与对照组相比，两步培养中 ffEV 处理对致密 COCs 的成熟率有积极影响（分别为 45.7%和 20.5%；p<0.05）。在一步培养中，补充 ffEV 对成熟率没有影响。我们的结果表明，从小卵泡卵泡液中分离的 EVs 补充培养基可以提高母马卵母细胞的 IVM 率，这表明 ffEVs 在这个过程中发挥着重要作用，并可能促进马的 ART 发展。
