Department of Clinical & Experimental Medicine (IKE), BHK/O&G Linköping University, SE-58185, Linköping, Sweden.
Occupational & Environmental Medicine Centre, Linköping University, SE-58185, Linköping, Sweden.
Theriogenology. 2019 Dec;140:143-152. doi: 10.1016/j.theriogenology.2019.08.023. Epub 2019 Aug 21.
Seminal plasma (SP) is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles (EVs). The SP interacts with spermatozoa and the inner cell lining of the female genital tract, adsorbing proteins and exosomes that modulate sperm functions and female immune responsiveness. In the present study, boar sperm-free SP was studied using flow cytometry (FC) after membrane tetraspanins (CD9, CD63 and CD81) and membrane receptor CD44 marking of non-enriched (whole SP) or gradient fractions enriched through two-step discontinuous KBr-density-gradient ultracentrifugation, in whole ejaculate or in selected ejaculate fractions. The results, evaluated by transmission electron microscopy, confirmed the presence of exosomes in all fractions of the pig SP. Noteworthy, these pig SP-exosomes were CD44-bearing when analysed by FC, with bands detected by western blotting (WB) at the expected 85 kD size. The two-step discontinuous KBr-density-gradient ultracentrifugation enriched the population of exosomes in two specific gradient fractions, indicating exosomes (either prostasomes or epididymosomes) could be separated from low-density lipoprotein (LDL) but they co-sediment with the high-density lipoprotein (HDL)-bearing fraction. The findings pave for the selective isolation of exosomes in functional studies of their function when interacting with spermatozoa, the oocyte and/or the female genitalia, including hyaluronan-CD44 interplay.
精浆（SP）是一种复杂的液体，包含蛋白质、肽、酶、激素以及细胞外囊泡（EVs）。SP 与精子和女性生殖道的内细胞层相互作用，吸附调节精子功能和女性免疫反应的蛋白质和外泌体。在本研究中，使用流式细胞术（FC）研究了无精子的猪 SP，在对非富集（整个 SP）或通过两步不连续 KBr 密度梯度超速离心梯度富集的全精子或所选精子部分进行膜四跨膜蛋白（CD9、CD63 和 CD81）和膜受体 CD44 标记后。通过透射电子显微镜评估的结果证实了猪 SP 所有部分都存在外泌体。值得注意的是，通过 FC 分析，这些猪 SP 外泌体带有 CD44，Western blot（WB）检测到的条带在预期的 85 kD 大小处。两步不连续 KBr 密度梯度超速离心在两个特定的梯度部分富集了外泌体群体，表明外泌体（前列腺小体或附睾小体）可以与低密度脂蛋白（LDL）分离，但它们与富含高密度脂蛋白（HDL）的部分共沉淀。这些发现为在与精子、卵子和/或女性生殖道相互作用时对外泌体功能的选择性分离铺平了道路，包括透明质酸-CD44 相互作用。
