**3.2 EVs derived from the epididymis**

Epididymosomes are produced by epididymal epithelial cells through apocrine secretion. They are a kind of exosomes with a diameter of 50–250 nm and characterized by high cholesterol/phospholipid ratio. Epididymosomes were first found in the intraluminal compartment of the epididymis of Chinese hamsters, and then in mice, rats, cattle, sheep, and humans. Epididymosomes can carry and transport numerous proteins with biological functions (including enzymes, adhesion molecules, transport, and signal transduction proteins) and non-coding RNA (such as miRNA). These proteins and non-coding RNA play an important biological role in the process of sperm maturation and fertilization, participating in the acquisition of motility, the acquisition of fertilization ability, and protection against oxidative stress [50, 53–55].

The proteins of epididymosomes including aldo-keto reductase family 1 member B (AKR1B1), phosphatidylethanolamine binding protein 1 (PEBP1), macrophage migration inhibitory factor (MIF), polyol pathway enzymes, glutathione peroxidase 5 (GPX5), plasma membrane <sup>2</sup> *Ca* <sup>+</sup> -ATPase 4a (PMCA4), ubiquitin, and SPAM1/ PH-20. AKR1B1 and PEBP1 can jointly regulate the sperm state, keeping sperm at a quiescent state during transportation until ejaculation [56]. MIF and polyol pathway enzymes are involved in sperm maturation and fertilization. GPX5, together with ubiquitin, is transferred to the sperm acrosome region during sperm epididymal transport, which protects sperm from oxidative stress, maintains the integrity of DNA, and prevents premature acrosome reaction. As a calcium efflux pump, PMCA4 carried by epididymosomes can regulate calcium concentration, affect the activation of calcium signaling pathway and maintain the homeostasis of <sup>2</sup> *Ca* <sup>+</sup> in spermatozoa. Sperm delete PMCA4 leads to loss of hyperactivation and capacitation. GPI anchoring proteins include P34h and SPAM1/PH-20. As mentioned earlier, they are sperm binding proteins related to epididymosomes, which locate on the sperm surface during sperm epididymal transport, mediating the docking between epididymosomes and sperm and providing a "highway" for epididymosomes to transport their cargos to participate in the process of fertilization. Similar to their function are the P25b/P26 that facilitate sperm binding to the zona pellucida [33, 57–61].

Epididymosomes are also mediator of cell communication. New surface antigens are obtained on the sperm surface during epididymal transport, which are related to the acquisition of fertilization ability. Epididymosomes transmit Notch signals between epididymal epithelial cells and between epididymis and SPZ to influence sperm motility [62].

Epididymosomes are also rich in non-coding RNA. Epididymosomes contain many kinds of miRNA, such as miR-888, miR-182, miR-24, and miR-15b. MiR-888 of epididymosomes maintains sperm flagella peristalsis and mature sperm structure by regulating SPAG6 and SPAG1. Target gene prediction shows that miR-15b can specifically regulate the expression of IDH3A and control energy metabolism in TCA cycle, while miR-182 and miR-24 can specifically regulate the expression of glycogen synthase kinase 3α (GSK3A), and phosphorylation of GSK3A can affect sperm motility. Functional enrichment analysis shows that these miRNAs play important roles in embryonic development. There are also inflammation-related miRNAs in epididymosomes, such as miR-181a and miR-1224. Studies have shown that miR-181a participates in inflammatory response by regulating B cell differentiation and T cell receptor signals. MiR-146 can inhibit inflammation and innate immune response by down-regulating various pro-inflammatory cytokines, but its specific mechanism

needs to be further studied. When inflammation exists, miR-1224 activates the immune response by down-regulating the expression of TNF- α. Epididymosomes also contain miR-29a, which enhances the expression of nuclear autoantigen sperm protein (NASP) and inhibits the proliferation of epididymal epithelial cells [63–68].

In addition, epididymosomes contain a small molecule: tRNA-derived small RNAs (tsRNA). Studies have shown that tsRNAs have the functions of regulating gene transcription, cell proliferation and apoptosis, and stress response [69, 70].
