**3. EVs in the male reproductive system**

The male reproductive system consists of internal organs - gonads (testis), reproductive ducts (epididymis, vas deferens, ejaculatory ducts, male urethra), and accessory glands (seminal vesicle, prostate, urethral bulbar gland) and external organs. Testes produce sperm and secrete male sex hormones. SPZ/sperm produced by the testis are first stored in the epididymis; during ejaculation, SPZ are excreted through the vas deferens, ejaculatory ducts, and urethra. Semen is the protect fluid around SPZ containing seminal plasma (SP), which is derived from testis (5%), epididymis and prostate (20%), seminal vesicles (65%), and seminal vesicles (65%). Seminal plasma lipids and/or EVs are rich in lipids, sugars, growth factors, TF and proteins, which play important roles in sperm survival, membrane integrity, maturation, motility, capacitation, acrosome reaction, and immune surveillance regulation [2, 33, 36]. Seminal plasma extracellular vesicles (Seminal plasma EVs, spEVs) are mainly derived from epididymis and prostate in the male reproductive tract, and a large number of them exist in the seminal plasma. Proteins in the seminal plasma are provided by spEVs [37]. According to the source, there are two main types of

spEVs: prostasomes and epididymosomes. A proteomic analysis showed that there are a total of 1474 proteins in seminal plasma-derived exosomes; bioinformatics analysis revealed that these proteins are involved in a variety of biological processes, such as cell growth and maintenance, metabolism, transport, energy pathways, and so on [38]. Human semen exosomes also contain a unique non-coding small RNA library, which may have a potential regulatory function to mediate fertilization by transmitting regulatory signals to the receptors to regulate the female reproductive tract [39].

### **3.1 EVs derived from the prostate**

The first EVs of the reproductive system found in humans are prostasomes, which were observed in human prostatic fluid and seminal plasma in 1978 [40]. Prostasomes are exosomes with a diameter of 30–500 nm, which are released from prostatic epithelial cells and are then transferred to the prostate duct. Prostasomes are characterized by a cholesterol-to-phospholipid ratio of 2:1, of which nearly 50% phospholipid is sphingomyelin, making its bilayer or multilayer lipoprotein plasma membrane very hard. This unique prostate membrane component allows the prostate to fuse with and transfer its contents to other cells [41, 42]. Prostasomes can transport substances such as sphingomyelin, cholesterol, and saturated glycolipids to sperm to reduce their membrane fluidity in order to prevent premature or spontaneous acrosome reaction and premature capacitation [43]. Previous studies have shown that prostasomes may inhibit capacitation and acrosome reaction mainly through cholesterol transfer [44, 45]. Once the appropriate time for acrosome reaction occurs, prostasomes also play important roles in the capacitation and induction of acrosome reactions as mediators of signal transmission to regulate the tyrosine phosphorylation pattern, which is necessary for sperm-oocyte interaction [46].

Sperm motility is affected by intracellular pH and <sup>2</sup> *Ca* <sup>+</sup> concentration. Prostasomes were determined to be associated with *Mg*<sup>2</sup><sup>+</sup> and <sup>2</sup> *Ca* <sup>+</sup> dependent ATPase activity when they were first identified, suggesting that they are related to sperm energy metabolism [47]. Annexins of prostasome can activate <sup>2</sup> *Ca* <sup>+</sup> channel and increase the level of <sup>2</sup> *Ca* <sup>+</sup> in sperm through carrying CD38 and RyR to sperm to stimulate the production of cyclic adenylate diphosphate, thus affecting sperm motility. After ejaculation, prostasomes interact with sperm and protect them from female reproductive tract acidic environment and regulate sperm motility through PH- dependent manner to maintain the ability of sperm fertilization and prepare for an encounter with oocytes [48, 49]. Prostasomes transfer proteins (such as galactose lectin 3 and CD48) can protect sperm from immune reaction by regulating immune response pathways, such as inhibition of complement pathway, lymphocyte proliferation, and phagocytosis of monocytes and neutrophils in the female reproductive tract [50].

Additionally, prostasomes have antioxidant properties. By interacting with polymorphonuclear neutrophils that produce ROS, prostasomes can reduce the production of ROS, protect sperm and improve sperm survival rate. Prostasomes also carry aminopeptidase N, a protein involved in regulating sperm motility which acts by regulating endogenous opioid peptides (such as enkephalin) [51, 52]. Prostasomes are known to contain a variety of coding and non-coding regulatory RNAs with potential regulatory functions [39]. Currently, there are few studies on nucleic acid cargos of prostasomes and their effects on the male reproductive system.
