College of Basic Medicine, Jining Medical University, Jining 272000, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
College of Second Clinical Medical, Jining Medical University, Jining 272000, China.
Anim Reprod Sci. 2024 Nov;270:107576. doi: 10.1016/j.anireprosci.2024.107576. Epub 2024 Aug 12.
As a new mechanism of intercellular communication, the uptake of extracellular vesicles (EVs) by receptor cells has become a hot topic in the field. Previously, research on the uptake of EVs has focused on the mechanism of small EVs (sEVs, also known as exosomes). As sEVs represent a mixed heterogeneous population, the issue of whether there are different uptake mechanisms for different subsets of sEVs by recipient cells urgently need to be addressed. There are EVs in follicular fluid, which play an important role in the communication between follicular cells and the development of oocytes. Previously, we isolated two subtypes of sEVs in follicular fluid: low density-sEVs (LD-sEVs) and high density-sEVs (HD-sEVs). The current study aimed to explore the uptake characteristics of these two subtypes of sEVs by granulosa cells. First, PKH67 was used to label the two sEVs subtypes, and we observed their uptake by granulosa cells using confocal microscopy and flow cytometry. We then explored the specific mechanisms underlying uptake of these two sEV subtypes by granulosa cells using specific inhibitors and RNA interference. The results showed that granulosa cells took up both kinds of sEVs through a clathrin-independent pathway. In addition to requiring caveolin, cholesterol, and Na+/H+ exchange, the uptake of HD-sEVs also depended on the activity of tyrosine kinase and phosphoinositide 3-kinase. A better understanding of the mechanism of granulosa cell uptake of different subtypes of sEVs in follicular fluid is of considerable significance leading to more accurate use of EVs for targeted treatment of infertility and other related diseases.
作为细胞间通讯的一种新机制，受体细胞摄取细胞外囊泡 (EVs) 已成为该领域的热门话题。此前，EVs 摄取的研究主要集中在小 EVs（sEVs，也称为外泌体）的机制上。由于 sEVs 代表了一种混合的异质群体，因此迫切需要解决不同子集的 sEVs 是否通过受体细胞具有不同摄取机制的问题。卵泡液中存在 EVs，它们在卵泡细胞之间的通讯和卵母细胞的发育中发挥重要作用。此前，我们在卵泡液中分离出两种 sEVs 亚型：低密度-sEVs（LD-sEVs）和高密度-sEVs（HD-sEVs）。本研究旨在探讨这两种 sEV 亚型被颗粒细胞摄取的特点。首先，使用 PKH67 标记这两种 sEV 亚型，并用共聚焦显微镜和流式细胞术观察它们被颗粒细胞摄取的情况。然后，我们使用特异性抑制剂和 RNA 干扰探索这两种 sEV 亚型被颗粒细胞摄取的具体机制。结果表明，颗粒细胞通过网格蛋白非依赖途径摄取这两种 sEV。除了需要网格蛋白、胆固醇和 Na+/H+交换外，HD-sEV 的摄取还依赖于酪氨酸激酶和磷酯酰肌醇 3-激酶的活性。更深入地了解卵泡液中颗粒细胞摄取不同亚型 sEV 的机制具有重要意义，这将有助于更准确地利用 EVs 进行靶向治疗不孕等相关疾病。
