Department of Biological Sciences, University of Delaware, Newark, Delaware.
Department of Biomedical Engineering, University of Delaware, Newark, Delaware.
J Cell Physiol. 2019 Aug;234(8):12745-12756. doi: 10.1002/jcp.27894. Epub 2018 Dec 7.
Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as "Vaginosomes" (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca -ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10-30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics.
通过对超排卵小鼠阴道腔液（VLF）进行超速离心分离出细胞外囊泡（EVs），并首次使用透射电子显微镜对其进行鉴定。EVs 通过大小和生化标志物（CD9 和 HSC70）进行特征鉴定，被证明是微泡和外泌体，并被命名为“阴道小体”（VGS）。分析阴道切片以在原位可视化 EVs：EVs 存在于腔中，也嵌入在鳞状上皮和角化细胞之间，与其内源性起源一致。Western blot 检测到 VGS 货物中的质膜 Ca2+-ATP 酶 1（PMCA1）和酪氨酸磷酸化蛋白，以及 uterosomes 中的这些蛋白。流式细胞术显示，在尾部精子与 VLF 共孵育 30 分钟后，具有最高精子粘附分子 1（SPAM1）、PMCA1/4 和 PMCA1 水平的细胞频率分别增加了 16.4 倍、8.2 倍和 27 倍；与在磷酸盐缓冲盐水（PBS）中对照共孵育相比。在相同条件下，VLF 共孵育后，精子酪氨酸磷酸化蛋白升高约 3.3 倍。与 PBS 相比，与 VGS 共孵育 10-30 分钟后，孕酮诱导的顶体反应（AR）率显著（p<0.001）升高。将这些时间段内人工沉积在超排卵雌性阴道中的精子也显示出 AR 率显著（p<0.01）升高，与 PBS 相比。因此，在体外和体内，精子从阴道环境中获得诱导获能的因素，这解释了它们在峡部中顶体状态的最近发现。总的来说，VGS 似乎提供了更高水平的参与防止过早获能和 AR 的蛋白质，而不是促进它们的蛋白质。我们的研究结果对人类有影响，为利用外泌体治疗不孕症开辟了新的途径。
