The potential role of seminal extracellular vesicles as biomarkers of male fertility and sperm cryotolerance in livestock species

Abstract

Seminal extracellular vesicles are lipid bilayer membrane-bound vesicles that contain the important biomolecules of semen and function in intercellular communication in the reproductive tract. They are secreted by the male reproductive tract epithelium and the accessory sex glands particularly seminal vesicle and prostate. The number of extracellular vesicles in seminal plasma is greater than that in other body fluids, but are the least studied, especially in livestock species. In vitro studies on the influence of seminal extracellular vesicles on spermatozoa functions have revealed their potential role in improving semen quality and preservation. Their role as cryoprotectants in semen cryopreservation is a less developed area and has a good scope for research. SEVs are isolated from seminal plasma by various methods based on the principle of size and density gradient separation. Ultracentrifugation combined with density gradient ultracentrifugation is the most commonly used isolation method. Characterization is done based on their morphology, specific membrane markers (CD9and CD63), particle size and concentration. Many proteomic and transcriptomic studies have revealed compositional differences between the seminal extracellular vesicles of fertile and sub-fertile populations. This provides the hope of utilizing these vesicles as noninvasive diagnostic biomarkers of fertility. The ability of these vesicles to protect their cargo from phagocytosis and degradation makes them more relevant and reliable diagnostic biomarkers. This review discusses and summarizes the information from the studies available on seminal extracellular vesicles and their potential applications in improving semen quality and preservation. Also micro RNAs and protein cargo of SEVs show altered expression in pathological and infertility conditions. Hence their expression level in SEVs could be used as potential biomarkers for male fertility in livestock species.

Article highlights

Seminal extracellular vesicles regulate sperm maturation, motility, capacitation, acrosome reaction and successful fertilization.

SEVs from high fertile animal have the potential to improve the semen quality of animals with low fertility.

SEVs have the potential to predict fertility and cryotolerance of semen in livestock species..

Introduction

There have been continuous efforts in the livestock sector to identify relevant factors that regulate fertility in male animals. At the gamete level, the fertilization capacity of spermatozoa is largely determined by the components of seminal plasma and membrane-bound vesicles known as seminal extracellular vesicles (SEVs) [1]. SEVs are lipid bilayer nanovesicles with sizes ranging from 50 to 200 nm [2] that are secreted by the cells of the male reproductive tract epithelium, including Sertoli cells [3]; the testis, epididymis, and ampulla of the ductus deferens [4]; and accessory sexual glands such as the prostate and seminal vesicles [1]. These vesicles are well protected from degradation and contain the most important biomolecules such as signaling proteins, lipids, cytokines, enzymes, nucleic acids, DNA, and noncoding RNA (miRNA) [5, 6]. Due to their diverse composition, function, and target cells, various subtypes of SEVs have been isolated and characterized by different methods [7, 8]. The names of various SEVs have been derived according to their biogenesis, size, origin and biochemical characteristics. Based on their biogenesis (Fig. 1), these vesicles are referred to as exosomes (of endosomal origin) and microvesicles or microparticles (plasma membrane outbuds).

Fig. 1 [Images not available. See PDF.]

Generation of EVs: Inward budding of plasma membrane (PM) leads to the formation of early endosome which incorporates intracellular components and form multivesicular body (MVB). MVB has two fates—it either fuses with the PM and releases exosomes or it undergoes lysosomal pathway. Plasma membrane out buds detach from the cells and forms microvesicles. Apocrine secretion mechanism involves protrusion of apical bleb from PM containing different sized vesicles, it detaches, disintegrates and release vesicles in the lumen (Created with BioRender.com)

However, it has also been reported that they are also secreted through the apocrine pathway embedded within apical blebs [1]. Based on their size, EVs are classified as small (< 100 nm), medium (< 200 nm) or large (> 200 nm) [9]. Epididymosomes (50–250 nm) and prostasomes are the terms used according to their origin. Some of the few studies on livestock species have depicted their potential roles in sperm maturation, motility acquisition, fertilization capacity, capacitation modulation, acrosome reaction and viability maintenance during spermatozoa transport in the male and female tracts. They are also involved in immunomodulation of the female reproductive tract [10], fertilization and subsequent events, including early embryonic development and subsequent implantation [11, 12]. The effect of SEVs on these important reproductive events makes these nanoparticles even more interesting for research. Unwinding their potential functions will be helpful in developing strategies for fertility improvement and overcoming the constraints of existing assisted reproductive technologies, AI efficiency and semen cryopreservation. However, only a few reviews have addressed recent practical applications of SEVs in livestock species. This review summarizes the basic knowledge about SEVs, their isolation and characterization techniques, and their potential for improving the quality and cryotolerance capacity of semen. The potential use of SEVs as diagnostic biomarkers of male infertility and semen freezing resilience has also been discussed.

Isolation of SEVs

Seminal plasma is a complex fluid containing many bioactive molecules (sugars, oligosaccharides, glucans, lipids, inorganic ions, proteins and RNAs) that are present either in their free form or inside membrane-bound vesicles, i.e., SEVs [13]. These vesicles are heterogeneous in their composition and functional properties [14, 15]. There are many techniques available for the isolation of SEVs, as illustrated in Fig. 2, and these techniques are discussed briefly in this section. Their working principle, advantages and disadvantages are presented in Table 1. Differential Ultracentrifugation (DUC) is the most commonly used method for SEVs isolation [9]. It involves several rounds of centrifugation with increasing speed as the size of sedimented particles decreases at each centrifugation step. Particles are separated in order of their size and densities. In DUC, semen is first centrifuged at 800×g for 20 min at room temperature to remove spermatozoa, and the supernatant is then centrifuged at 2000×g for 20 min at 4 °C to eliminate cell debris and remaining sperm cells. The collected seminal plasma is stored at −80 °C until SEV isolation. For isolation, seminal plasma is then centrifuged at 16,000×g for 1 h at 4 °C, and the resulting supernatant is ultra centrifuged at 120,000 × g for 70 min at 4 °C to obtain the SEVs. SEV pellets are suspended in PBS and purified by repeated ultracentrifugation at 120,000×g for 70 min at 4 °C. Purified SEV pellets are resuspended in PBS and stored at −80 °C until further analysis [16].Parameters like speed and duration of ultracentrifugation should be adjusted according to the k factor of the rotor type and the sedimentation coefficient of the desired particles to be pelleted. This avoids differences in the results of the experiments done using different rotor types and improves the reproducibility of the isolation protocol [17]. Density Gradient Ultracentrifugation (DGUC) allows separation of EV subtypes of different densities. It uses iodixanol, sucrose or cesium chloride density gradients [18]. Size exclusion-based isolation is performed by ultrafiltration, hydrostatic filtration dialysis, and size-exclusion chromatography (SEC). Ultrafiltration is done by using membrane filters (polyethersulfone) with pore diameters of 0.1 µm, 0.22 µm or 0.45μmwhich separate particles larger than 100 nm, 220 nm and 450 nm respectively [19]. Microvesicles can be separated by filtration through 0.65 μm pore diameter microfilter followed by centrifugation at 10,000×g. Further filtration of the filtrate with 0.45, 0.22 and 0.1 μm filter followed by ultracentrifugation yield exosomes [20]. Hydrostatic filtration dialysis is performed by keeping the sample in a funnel attached to a dialysis membrane that is permeable to particles with molecular weight up to 1000 kDa. Thus, particles up to 1000 kDa are separated by the hydrostatic pressure of the fluid through the membrane. This step is followed by centrifugation at 40,000×g to sediment the extracellular vesicles [21]. In size exclusion chromatography (SEC) or gel filtration, sample is added to a stationary phase column filled with a porous material including crosslinked dextrans (Sephadex), agarose (Sepharose), allyl dextran (Sephacryl). Molecules smaller than the pore size are trapped within the porous beads, so they are eluted slowly, while large molecules are eluted at a faster rate [20]. Several commercially available SEC columns include qEV Size Exclusion Columns (Izon Science Ltd., United Kingdom), Sephacryl S-400 (GE Healthcare, United Kingdom), Sepharose CL- 2B (30 mL; GE Healthcare, Sweden) [22]. The precipitation method involves adding semen samples to water-excluding polymers such as polyethylene glycol (PEG) [23], lectins [24], the Vn96 polypeptide and charge-based precipitation using positively charged molecules such as protamine [25, 26]. Polymer (PEG) solution is mixed with the sample and incubated at 4 °C for twelve hours. These polymers decrease the solubility of EVs in the solution. This is followed by low-speed centrifugation (1500×g) which aggregates EVs out of the solution [27]. The protein organic solvent precipitation (PROSPR) method [28] and the ExoQuick, ExoSpin and Total Exosome Isolation reagents (Thermo Fisher) are commercially available kits based on precipitation methods [29]. The isolation of SEVs by PEG precipitation is an effective, reliable and fast method for use in academic laboratories, clinics and large samples [30, 31]. Biochemical capture techniques are immunoaffinity-based isolation methods that use antibodies against specific markers on the surface of EVs, such as CD63 and CD9. Antibodies are conjugated either to the surface through which the sample passes or to submicron-sized antibody beads [32, 33]. This method is most commonly used with other isolation methods to increase the purity of obtained SEVs [34]. Advanced methods include microfluidic-based isolation methods which use both the physical and biochemical properties of EVs at the microscale and are ultrasensitive [35]. The devices used in this technique are nano-filters, nanoarrays and nanoporous membranes that follow size-based isolation [36]. Biochemical-based isolation methods include the integration of micrometric magnetic beads, droplet microfluidics, antibody-coating within the microfluidic channels of the device that capture EVs [37]. Microfluidic chips and Exochip are immunoaffinity-based EV isolation methods [38]. The efficiency of these devices can be enhanced by the application of electrophoresis [39], pressure or acoustic waves [40]. The choice of method for isolation of SEVs depends on the availability of equipments and their downstream applications. Generally, two or more methods are used in combination to gain maximum purity of SEVs. DUC combined with DGUC has been found to be the most suitable method for the isolation of SEVs [41, 42].

Fig. 2 [Images not available. See PDF.]

Schematic representation of common methods of EVs isolation (Created with BioRender.com)

Table 1. Common isolation methods of SEVs in livestock species

Isolation method	Working principle	Advantages	Disadvantages	Species	References
Differential Ultracentrifugation (DUC)	Size and buoyant density of particles	Smaller size EVs can be separated Most widely used method	Low purity, poor reproducibility with different rotors, disruption of vesicle surface, requirement of expensive equipment	Bovine, Porcine, Buffalo, Canine, Feline	[14–16, 43–51]
Density Gradient Ultracentrifugation	Size, mass and density	Different subtypes of EVs can be isolated	Time consuming, require expensive equipments	Ram, Porcine	[7, 41, 48]
Ultrafiltration	Size of particles	Makes EVs isolation easy when combined with UC and other methods	Less yield and purity, cause EVs rupture	Used in combination with UC in porcine	[45, 46]
Size exclusion chromatography	Hydrodynamic radius of particles	Good integrity of EVs is maintained, fast, good purity	Poor specificity for isolating different subtypes of EVs	Rabbit, Pig	[13, 47, 52–54]
Polymer precipitation (Polyethylene glycol)	Solubility of particles in medium	Easy, fast, affordable, suitable for large volume samples	Less purity	Human, Not used in livestock species	[31]
Immunoaffinity	Surface marker proteins	High specificity and purity	Specific antibodies are required, expensive	Not used in livestock species	[32, 33]
Microfluidic devices	Combined physical and biochemical properties of particles	Rapid, high purity	Expensive and non- standardization of protocols	Not used for SEVs in livestock species	[35]

Characterization of SEVs

After isolation, SEVs are to be characterized to distinguish them from other nanoparticles and their diverse subtypes present in seminal plasma. Some of the techniques used for the characterization of SEVs include transmission electron microscopy (TEM), Scanning Electron Microscopy (SEM), nanoparticle tracking analysis (NTA), western blotting [41], dynamic light scattering (DLS) [16] and flow cytometry [14]. TEM, preferably cryo-TEM, is used to observe the morphology of SEVs. NTA tracks the Brownian motion of each EV based on its light scattering and detects its velocity, which is correlated with its size. Thus, it is used to determine the size and concentration of EVs. DLS is also based on the principle of light scattering and is used for measuring the size of particles. Western blotting is used to detect specific marker membrane proteins (CD63) and the protein composition of the EVs (Alix, TSG101). Antibodies used as EV biomarkers include anti-heat shock protein 70, anti-tetraspanin cell surface protein CD9 and anti-ALIX protein [13]. Flow cytometry technique is based on light scattering by particles and is a powerful tool for the simultaneous analysis of size, shape, concentration and specific markers. Flow cytometers are suitable for quick analysis of a large number of vesicles [55]. Proteomic composition is decoded by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) or matrix-assisted laser desorption/ionization–TOF mass spectrometry, and the identified proteins are finally quantified using sequential window acquisition of all theoretical mass spectra (SWATH–MS) [54].

Most of the characterization approaches for SEVs are based on their membrane composition and transmembrane proteins, which are specific membrane markers [9]. The membrane of SEVs is rich in cholesterol and sphingomyelin [56]. Specific marker proteins present in SEVs can be be used to identify  from coisolated particles as well as from other subtypes of vesicles. At present, there are very few studies on the characterization of SEVs based on specific marker proteins. More studies in this area are needed to characterize all the subtypes of SEVs present in seminal plasma. The International Society for Extracellular Vesicles (ISEV) recommends analyzing transmembrane proteins such as CD9, CD63, and CD81 to characterize isolated SEVs [9]. CD44 is a specific marker for epididymosomes, and CD9 is a specific marker for cauda epididymosomes in boars [57]. The differential expression of tetraspanins CD81/CD63 is greater in exosomes, and that of CD9/CD63 is greater in microvesicles of boar SEVs, as characterized by flow cytometry [6, 14, 16]. Dipeptidyl peptidase IV/CD26 is a specific surface protein marker in the seminal plasma prostasome of horses [58]. Annexin A1 is a specific marker for microvesicles [59]. More research into the development of strategies to characterize SEVs would be helpful in understanding the actual composition, function and target cell of a specific subtype. This would be helpful in utilizing these vesicles for various practical purposes in assisted reproductive technologies and for therapeutic purposes for infertility.

Seminal extracellular vesicles as a tool for improving semen quality and preservation

Seminal extracellular vesicles contain biomolecules that are important for spermatozoa maturation, motility acquisition, transit in the female tract, fertilization and the determination of overall semen quality [1]. The small miRNAs present in SEVs, such as ssc-miR-10b, are directly related to fertility [60] and are also involved in immunomodulation of the female reproductive tract [10]. Many studies have confirmed the improvement in poor semen quality after exposure to isolated SEVs of good semen, suggesting the scope of SEVs to improve seminal quality. This is the result of the complex interaction between SEVs and spermatozoa which includes binding, fusion, content transfer and detachment of SEVs. Content transfer by SEVs include both loading and removal of molecules from spermatozoa. The mechanism of this interaction is not clear but believed to be due to direct membrane fusion of SEVs with spermatozoa or by formation of transient fusion pores in the membrane [1]. This is the possible mechanism by which SEVs regulate various physiological functions of spermatozoa.

Table 2 summarizes the important in vitro studies on the influence of SEVs on sperm motility, in vitro fertilization rates, preservability and post-thaw quality in livestock species. These studies proved that SEVs can transfer their contents to spermatozoa and can affect their physiological functions upon coincubation. SEVs isolated from proven highly fertile bull semen co-incubated with spermatozoa of low fertility improved in vitro fertilization rate and progressive motility of spermatozoa. The incorporation of vesicles is maintained even after cryopreservation, facilitating the use of treated semen for in vivo or in vitro fertilization in the future [43]. This approach could open the way for maintaining low-fertility breeding bulls with high genetic value. Another study on boar reported that SEVs can maintain sperm motility in vitro even after 10 h of coincubation [61], which may be mediated by ATP transfer via SEVs to the midpiece [52]. During the preservation of boar sperm in the liquid state at 17 °C, the addition of SEVs to the diluent increased motility, viability, plasma membrane integrity, and total antioxidant capacity and reduced the malondialdehyde content of boar spermatozoa. This could be due to the transfer of the spermadhesin, AWN and PSP-1 proteins, which act as capacitation inhibitors, to the sperm membrane to maintain sperm function by inhibiting premature capacitation during long-term liquid storage [61]. This finding highlights the potential of using SEVs for the preservation of semen. Most of the recent studies are consistent with the findings that SEVs can improve sperm quality in terms of reducing oxidative stress and maintaining plasma membrane integrity, sperm fertilization capacity [61], maturation acquisition and post thaw motility [51, 62]. Contrary to this, few studies have reported inhibitory effect of SEVs on sperm capacitation, the acrosome reaction, spermatozoa ATP production and the binding capacity of sperm to the zona pellucida when used in IVF medium, resulting in low IVF output [39, 53, 56]. Other studies have shown positive effect of SEVs on acrosome integrity, capacitation, acrosome reaction and in vitro fertility [47, 63]. These contradictory results might be due to the different isolation techniques, contamination of the working conditions by other proteins and compositional differences in the SEVs. The improved in vitro fertilization rate obtained in the study on bulls (Table 2) might be because the comparison was with low-quality spermatozoa, and coincubation was performed before the IVF procedure and not in the IVF medium, as in another study in boars [53]. However, further studies are needed to understand which molecules of SEVs are involved in improving the quality and preservation of semen in livestock species.

Table 2. Species specific studies on influence of coincubation of SEVs with spermatozoa- Findings and possible factors involved

Species	Procedure involved	Effective dose and time	Result	Possible factors Involved	References
Bull	Coincubation of low-quality spermatozoa with high quality SEVs	400 × 106 EVs/mL for 1 h	Improved IVF rate from 19.48% to 41.41% and blastocyst rate (10.32% to 22.17%)	P25b, GLI pathogenesis related 1 like 1, hyaluronan-hydrolyzing protein and sperm adhesion molecule 1, hyaluronidases	[43]
Boar	SEVs added in semen diluent for preservation at 17oCfor 10 days	Exo-0 = no exosomes Exo-1 = 2 × 1010 particles/mL Exo-4 = 8 × 1010 particles/mL Exo-16 = 32 × 1010 particles/mL Time of liquid storage = 10 days	Improved sperm motility on day 10 Exo-0 = 30.25 ± 2.35 Exo-1 = 49.50 ± 1.50 Exo-4 = 56.27 ± 1.75 Exo-16 = 56.27 ± 1.75 Improved PM integrity on day 10 Exo-0 = 35.20 ± 2.88 Exo-1 = 55.30 ± 3.39 Exo-4 = 66.10 ± 3.94 Exo-16 = 67.96 ± 3.74 Increased antioxidant capacity activity & decreased MDA content (Exo-4 found as optimal concentration) Inhibit premature capacitation of spermatozoa (highest with diluent containing Exo-16)	Exosomes binding with the sperm surface AWN and PSP-1 protein (decapacitation factors) transferred by exosomes (Exo-4 and Exo-16) in the diluent	[52, 61]
Boar	Addition of SEVs in IVF medium	SEVs in IVF medium added at 0.1 mg/ml and 0.2 mg/ml Concentrationdid not affect these results	Impaired IVF outcomes Reduced fertilization efficiency (45% to 15%) and reduced penetration rate of spermatozoa to zona (60% to 20%) No effect on capacitation and acrosome integrity Reduced sperm metabolism	Reduced sperm ability to bind to the ZP PSP-I and PSP-II protein	[53]
Boar	Coincubation	E1 = 300 μg/mL E2 = 600 μg/mL (SEVs protein concentration) E0 = no SEVs	Inhibit acrosome reaction and in vitro fertility Decreased blastocyst rates E0 = 21.41 ± 3.9% E1 = 14.69 ± 2.7% E2 = 13.5 ± 4.9%	EZRIN protein	[41]
Boar	Coincubation	SEVs: Sperm 2:1 protein ratio for 2 h	Improved Acrosome Reaction (9 ± 2% to 18 ± 4%)	Hydrolytic enzymes and Calcium by the SEVs	[47]
Cat	Coincubation 1.Immature (caput) spermatozoa with epididymal EVs 2.Frozen-thawed cauda spermatozoa with epididymal EVs (epididymosomes)	4 μg/μL total SEVs protein in medium for 75 min at38°C	Improved IVF rate post insemination At 17 h = Less distance between two pronuclei in the fertilized oocyte (16.7 μm vs 1.2 μm) Increased morula-blastocyst rate On day 3 = 50% vs 81.3% On day 7 = 16.7% vs 89.3% (Control vs EV treated sperm) At 3 h post-thaw, improved post-thaw motility (approx. 60% to 110%) and forward progressive motility (from 50 to 100%) Motility at time 0 normalized to 100% (control and treated samples both)	Improved sperm centrosomal maturation and function resulted in faster pronuclear syngamy NUMA1 protein Sorbitol dehydrogenase, interleukin binding factors 2 and 3, hexokinase 1 (sperm motility); ELSPBP1 (removal of defective sperms); MAPK, thioredoxin (antioxidant)	[51]

One study on human sperm cryopreservation revealed the protective effects of SEVs in minimizing sperm injuries during cryopreservation [64]. Treatment of spermatozoa with exosomes during the freezing procedure has been found to be effective in improving the post thaw quality of canine [65] and porcine semen [48, 61]. Exosomes derived from cells other than seminal plasma have also been found to be effective in minimizing cryoinjury to sperm [66]. All these studies in animals have shown the application of seminal extracellular vesicles in in-vitro preservation or post thaw improvement of semen quality. Mokarizadeh et al. reported decreased expression of mitochondrial ROS modulator gene in frozen/thawed rat spermatozoa treated with mesenchymal stem cell derived extracellular vesicles, which might be the reason for their increased antioxidant capacity during freezing [67]. Some studies have shown the regenerative potential of extracellular vesicles which plays an important role in repairing damaged membrane during sperm freezing [66]. However, no study has investigated the role of SEVs in semen cryopreservation in animals indicating a need for further studies in this area to unfold the real mechanisms that are involved in sperm cryoprotection by the SEVs during the cryostorage.

Liposomes, or artificial vesicles, are nanovesicles with lipid bilayers that are artificially produced and loaded with antioxidants, lipid-related compounds, cryoprotectants or any molecule of interest. They have been reported to improve the fertility of semen in buffalo, ovine, porcine, equine and bovine, as reviewed by Saadeldin et al. [66]. Biohybrid SEVs are prepared by fusing liposomes loaded with the needed biomolecules with native SEVs. The advantage of using biohybrid SEVs over liposomes in semen extenders is that they retain the properties of SEVs, such as membrane receptors and phagocytosis resistance, while maintaining the transfer of suitable molecules of interest to target cells [68]. These are some of the alternative approaches to SEVs which are being explored. Furthermore, therapeutic potential of SEVs for infertility is another aspect which needs to be explored. MiRNAs in the vesicles are capable of regulating the protein levels of target cells makes them important sources for developing therapeutic strategies for improving fertility [16]. For example, miR-27b in humans has potential for treating infertility [69]. However, little is known about the therapeutic potential  of the SEVs in livestock species.

SEVs—evolving as potential fertility prediction and sperm cryotolerance markers

Male fertility is the key determinant of the livestock breeding industry and is generally predicted on the basis of the results of artificial insemination, which is a time-consuming approach. Identifying parameters that can predict male fertility, will be helpful for selecting good-quality semen for insemination and for genetic improvement. Since SEVs critically contribute to spermatozoa function, their dysregulation causes subfertility and infertility [70]. This suggests that any physiological or pathological change can alter the composition of SEVs. It has been shown that the expression profile of the components (miRNA or protein) of these vesicles differs between semen of differential fertility and quality. These findings provide the basis for the use of SEVs as novel non invasive fertility prediction markers. A few proteomic and transcriptomic studies have been performed to identify reliable biomarkers for semen fertility.

Proteomic studies on buffalo and chicken SEVs have revealed aquaporin-7 (AQP7), ATP2B4, and testicular expression protein 101 (TEX101)could emerge as potential biomarkers of sperm fertility [49]. These proteins can be transferred from SEVs to spermatozoa and vice versa, thus their expression levels in SEVs reflects semen quality [71]. AQP7 is a protein expressed in spermatozoa and is positively correlated with its motility and fertility. It is involved in energy production pathways, thus participating in sperm capacitation and fertilization. Low expression of AQP7 in SEVs has been found in low motility group, suggestive of a potential fertility biomarker [49]. TEX101 is expressed in bull, mouse and human seminal plasma exosomes and is involved in initiating the acrosome reaction and sperm-oocyte fusion. The decreased expression levels of TEX101 in seminal plasma exosomes has been found to be related with subfertility having the potential to act as biomarker of male fertility in future [49].

Table 3 summarizes the differentially expressed species-specific miRNAs that can be used as semen fertility markers. Most transcriptomic studies have been performed in boars, and a few have been conducted in rams, bulls and rabbits, among other livestock species. These studies have revealed that some miRNAs related to reproduction are differentially expressed between good- and poor-quality semen. In rabbit [13], boar [72] and man [73], miR-184 is differentially expressed and downregulated in the fertile group compared to the sub-fertile group. In boar, high expression of miR-184 has found to be correlated with low quality semen and less litter size [74]. In man, abundance of miR-184 in SEVs has been found in asthenozoospermic semen [73]. Thus, it could be a potential biomarker for predicting male fertility and semen quality in these species. Differentially expressed miRNAs in rabbit bucks such as, miR-34b/c and miR-449a/b/c are essential for normal spermatogenesis but unnecessary for fertilization and preimplantation development, therefore their abundance in SEVs has been found to be correlated with subfertility [75]. In bulls, miR-122, which influences spermatocyte development and maturation, was found to be upregulated in infertile bulls [76, 77]. Several miRNAs, including miR-222, in boar SEVs are differentially expressed in animals with different sperm motility [2]. Another study on boar seminal plasma exosomes isolated from two boar breeds with different semen motility identified six miRNAs (miR-122-5p, miR-486, miR-451, miR-345-3p, miR-362, and miR-500-5p) with different expression levels that can serve as potential biomarkers for boar semen motility [45].These differentially expressed miRNAs influences pathways that regulate semen motility. MiRNA, miR-486 upregulation in bulls and boars reflects low motility semen [45]. Studies have shown that miRNA expression of SEVs is a potential diagnostic marker of many male reproductive tract pathologies [35, 78]. One such study has been done in bull to reflect the effect of heat stress on semen quality. Heat stress negatively affects semen quality and thus impairs male fertility [44]. miR-126-5p andmiR-23b-5p show downregulation in heat stressed bull semen when compared with normal semen. These SEVs miRNAs functions to maintain spermatozoa in a low metabolic state with in the epididymis. Their dysfunction results in poor quality spermatozoa [44].

Table 3. Species-specific studies about SEVs components as potential fertility prediction and sperm cryotolerance markers

Species	Differentially expressed protein/miRNA	Expression level related to particular fertility parameter	References
Buffalo	AQP7, ATP2B4	Low expression in low motility group	[49]
	PPEF1, ST13, TXNDC8, SPACA1 and GSTM2	Down regulated in low motility group
	LRRC37	Upregulated in low motility group
	ADAM2, ADAM3 and ADAM32	Downregulated in low motility SEVs
Chicken	HSP90AA1	Higher expression in fertile roosters than sub-fertile male	[7, 79]
Rabbit, boar, man	miR-184	Downregulated in fertile male	[14, 72, 73]
Bovine	miR-34b	Downregulated in fertile male	[44, 77, 80]
	miR-486	Upregulated in low motility semen
	miR-23b-5p, miR-126-5p	Downregulated in heat stressed bulls
	miR-122	Upregulated in infertile bulls
Rabbit	miR-190b-5p, miR-193b-5p, let-7b-3p, miR-378-3p, miR-190b-5	Upregulated in fertile buck	[13]
	miR-7a-5p, miR-33a-5p, miR-449a-5p, miR-146a-5p	Down regulated in sub-fertile buck
Boar	miR-222, miR-122–5p, miR-486, miR-451 miR-345–3p, miR-362 miR-500–5p	Upregulated in low sperm motility group Downregulated in low sperm motility group	[2, 45]
Boar	ssc-miR-130a ssc-miR-9	Abundant in low freezability semen	[46]

Considering the widespread application of semen cryopreservation in the livestock industry and inter- or intra-male variations in semen freezing resilience, biomarkers for identifying semen with good cryotolerance are needed. There are many studies on seminal plasma and spermatozoa components acting as biomarkers for semen freezability [70, 81–83], but to our knowledge only one study on SEVs have been conducted in livestock species by Pedrosa et al. [46]. Since seminal extracellular vesicle biomolecules are better protected from degradation than free fluid biomolecules, they can serve as relevant biomarkers of function or dysfunction or cryotolerance [84]. The differential expression of SEVs cargo in different freezability semen groups could make them good freezability markers. In boar semen miRNA groups (ssc-miR-130a and ssc-miR-9) which are related to the sperm plasma membrane integrity, maturation process and energy production pathways of spermatozoa have been found to be abundant in SEVs of low freezability semen hence can be used to predict boar semen cryotolerance. It has now become necessary to exclude freezing of low freezability semen, thus eliminating low freezable males on the basis of a reliable freezability marker could in part reduce the costs of maintenance, infertility in the herd and further reduce its transmission down the progeny. Further selective characterization of extracellular vesicles in semen could aid in the development of noninvasive biomarkers of semen fertility and cryotolerance.

Conclusion

SEVs are lipid bilayer nano vesicles with a size range of 50–200 nm, present in the seminal plasma as the secretory product of male reproductive tract epithelia and accessory sex glands. Isolation and characterization of SEVs is the first step for their in vitro studies. Ultracentrifugation combined with density gradient ultracentrifugation is the most commonly used isolation technique so far. Novel methodologies such as PEG precipitation, size-exclusion chromatography, commercial kits and advanced microfluidics are rapid, efficient, inexpensive and are suitable for small scale sample volumes. More studies for standardization of SEVs isolation protocols should be done for accurate, reproducible and consistent results. Characterization is commonly done by electron microscopy, dynamic light scattering, nanoparticle tracking analysis and western blotting. SEVs biomolecules (miRNA or proteins) have important functions in spermatozoa maturation, fertilization and determination of overall semen quality. Studies have demonstrated their beneficial role in improving semenquality, preservability and post-thaw sperm quality. Further studies are needed to know the SEVs components involved in influencing fertility, so that a particular subtype of SEVs can be used for a specific function. Hybrid nano-vesicles are artificially fabricated by fusion of SEVs with liposomes loaded with desired biomolecules. They have promising scope to be explored in semen cryopreservation and improvement of assisted reproductive technologies. Potential applications of SEVs as diagnostic marker of fertility and semen cryopreservability have also been studied based on altered expression of their cargo as per the semen of differential quality. More such studies should be encouraged in future to establish the differentially expressed cargo as biomarkers for prediction of male fertility.

Acknowledgements

Not applicable.

Author contributions

F. A. L. conceived the concept, S. R. and F. A. L. performed the literature search and data analysis, S. R. wrote the original draft and prepared Figs. 1–2, J. B. F. S., G. R. B. and F. A. L. critically revised the paper. All authors reviewed the manuscript.

Funding

The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval

Not applicable.

Competing interests

The authors declare no competing interests.

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