2.1 Prostasomes

Different research groups have investigated the interactions of prostasomes with sperm cells. For example, it was discovered that the prostasomes found in the seminal plasma of mammals have a greater specificity for sperm than for other cells such as lymphocytes (Kumar et al., Reference Kumar, Pandita, Ganguly, Soren and Pagrut2018). In this sense, it should be mentioned that the sperm surface has a slightly lower net negative charge than the prostasome one (Ronquist et al., Reference Ronquist, Nilsson and Hjertën1990), which together with the probable existence of a binding protein mechanism, similar to that described by Al-Dossary et al., (Reference Al-Dossary, Bathala, Caplan and Martin-DeLeon2015), could explain their specificity and ease of merging with each other. However, this has not yet been elucidated (Al-Dossary et al., Reference Al-Dossary, Bathala, Caplan and Martin-DeLeon2015). Prostasomes contain divalent cations of calcium (Ca²⁺), zinc (Zn²⁺) and magnesium (Mg²⁺) and, as mentioned, their particular membrane is rich in sphingomyelin, also containing a 2:1 ratio of cholesterol/phospholipids (Kumar et al., Reference Kumar, Pandita, Ganguly, Soren and Pagrut2018). The Ca²⁺ cation participates together with bicarbonate in sperm capacitation, stimulating the soluble enzyme adenylate cyclase, which when activated generates an increase in the synthesis of cyclic AMP that activates protein kinase A, triggering the phosphorylation of tyrosines of various proteins sperm cells (Visconti et al., Reference Visconti, Moore, Bailey, Leclerc, Connors, Pan, Olds-Clarke and Kopf1995; Litvin et al., Reference Litvin, Kamenetsky, Zarifyan, Buck and Levin2003). Ca²⁺ is also the trigger for the acrosomal reaction, a process that allows sperm to penetrate the outer coatings of the oocyte (Visconti et al., Reference Visconti, Moore, Bailey, Leclerc, Connors, Pan, Olds-Clarke and Kopf1995; Wong et al., Reference Wong, Flik, Groenen, Swinkels, Thomas, Copius-Peereboom, Merkus and Steegers-Theunissen2001; Flesch et al., Reference Flesch, Wijnand, Van de Lest, Colenbrander, Van Golde and Gadella2001; Chávez et al., Reference Chávez, De Blas, De La Vega-Beltrán, Nishigaki, Chirinos, González-González, Larrea, Solís, Darszon and Treviño2011). The Mg²⁺ cation acts as a cofactor of many enzyme systems and has been related to the modulation of sperm motility (Wong et al., Reference Wong, Flik, Groenen, Swinkels, Thomas, Copius-Peereboom, Merkus and Steegers-Theunissen2001). Similarly, the Zn²⁺ cation acts as a cofactor for many metalloenzymes, and has been associated with different processes related to DNA, such as stabilization and decondensation of sperm chromatin (Kvist, Reference Kvist1980; Kvist et al., Reference Kvist, Kjellberg, Björndahl, Soufir and Arver1990), and expression of genes that encode for steroid receptors, whose transcription factors include those that contain zinc fingers (Mackeh et al., Reference Mackeh, Marr, Fadda and Kino2018). It is also involved in the activity of acrosin, an important hydrolytic enzyme present in the acrosome (Steven et al., Reference Steven, Griffin and Chantler1982). As far as sphingomyelin is concerned, it has been observed that it slows down the efflux of sterols in human spermatozoa, therefore preventing premature capacitation and acrosomal reaction (Cross, Reference Cross2000). In pigs, however, it inhibits the efflux of cholesterol and fluidity of the apical plasma membrane of the sperm head (manifestations of sperm capacitation), but not the acrosomal reaction (Piehl et al., Reference Piehl, Fischman, Hellman, Cisale and Miranda2013), suggesting that the effect of this sphingolipid varies between sperm of different species.

One of the first proteins described in prostasomes is the Mg²⁺ and Ca²⁺-dependent ATPase, which can increase the availability of ATP for sperm, which is necessary for motility (Ronquist et al., Reference Ronquist, Brody, Gottfries and Stegmayr1978). Prostasomes also contain the macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine classified as a ‘moonlighting’ protein, which was purported to be associated with the regulation of the innate immune response of the vagina to the presence of sperm proteins; nevertheless, this is yet to be experimentally confirmed (Frenette et al., Reference Frenette, Légaré, Saez and Sullivan2005).

Additionally, prostasomes have been described as containing paternal chromosomal DNA fragments, which transfer to sperm (Ronquist et al., Reference Ronquist, Ronquist, Carlsson and Larsson2009, Reference Ronquist, Larsson, Ronquist, Isaksson, Hreinsson, Carlsson and Stavreus-Evers2011). It has been proposed that this process is performed to guide homology-directed repair (HDR) mechanisms in the embryo in case of sperm DNA damage caused, for example, by oxidative stress, which has not been confirmed experimentally (Ronquist et al., Reference Ronquist, Larsson, Ronquist, Isaksson, Hreinsson, Carlsson and Stavreus-Evers2011). Conversely, human prostasomes were reported to exert an antibiotic effect against Bacillus megaterium, because these EVs contain large amounts of chromogranin B, a protein that permeabilizes bacterial walls and membranes, therefore protecting sperm from possible attack by some microorganisms (Carlsson et al., Reference Carlsson, Nilsson, Larsson, Stridsberg, Sahlén and Ronquist2003). Figure 1 and Table 1 show the contents and effects of prostasomes on sperm.

Figure 1.

Prostasomes. The figure shows the environment of the prostasomes and possible subvesicular localization of their components (enlarged images in digital format show the molecules in detail). Created with BioRender.com

Table 1.

Sperm-related EVs

* Experimentally not proven or mechanism not known.

2.2 Epididymosomes

Sperm motility is a process with great energy expenditure. Zn²⁺ prevents sperm from wasting energy during their stay in the testes, by preventing the thiol groups of outer dense fibres (ODFs) from oxidizing and therefore forming disulphide bonds, necessary for proper motility. When sperm travel through the epididymis, epididymosomes enter the scene, delivering MIF to these gametes, which is responsible for removing Zn²⁺ from ODFs (Frenette et al., Reference Frenette, Légaré, Saez and Sullivan2005). However, as for Zn²⁺, MIF is a repressor of premature sperm motility, as it also reduces the thiol groups of ODFs, preventing the formation of disulphide bonds. Therefore it must also be eliminated at some point in order for sperm to become fully competent in the female environment, although the mechanisms involved in that removal are still unknown (Eickhoff et al., Reference Eickhoff, Baldauf, Koyro, Wennemuth, Suga, Seitz, Henkel and Meinhardt2004; Frenette et al., Reference Frenette, Légaré, Saez and Sullivan2005).

Conversely, while sperm travel through the epididymis during their maturation process, they receive dicarbonyl/l-xylulose reductase (DCXR) from epididymosomes, another moonlighting protein with various functions (Ebert et al., Reference Ebert, Kisiela and Maser2015). DCXR bound to the outer acrosomal membrane can modulate the primary adhesion of sperm to glycoproteins in the zona pellucida of oocytes during the fertilization process, presenting specificity at the species level (Boué et al., Reference Boué, Blais and Sullivan1996). It has been described under the name P34H in humans, P26h in hamsters and P25b in cattle, and it has also been found in mice and pigs (Tumova et al., Reference Tumova, Zigo, Sutovsky, Sedmikova and Postlerova2021). Glioma pathogenesis-related1-like protein 1 (GliPr1L1) is another protein that is delivered to sperm via epididymosomes. When blocked by anti-GliPr1L1 antibodies, the percentage of sperm that bind to the zona pellucida of oocytes is reduced, demonstrating its relevance in this process (Caballero et al., Reference Caballero, Frenette, D’Amours, Belleannée, Lacroix-Pepin, Robert and Sullivan2012). However, the molecular mechanisms through which this protein contributes to the sperm binding to the zona pellucida have not yet been elucidated (Yudin et al., Reference Yudin, Li, Robertson, Tollner, Cherr and Overstreet2002; Caballero et al., Reference Caballero, Frenette, D’Amours, Belleannée, Lacroix-Pepin, Robert and Sullivan2012).

In cattle (and humans; Frenette et al., Reference Frenette, Thabet and Sullivan2006), epididymosomes have been seen to contain two enzymes involved in the synthesis of fructose via the polyol pathway: aldose reductase 1 B5 (AKR1B5) and sorbitol dehydrogenase (SDH) (Frenette et al., Reference Frenette, Lessard and Sullivan2004). AKR1B5 reduces glucose generating sorbitol, a polyol that is not very permeable to the plasma membrane, through a reaction is optimal at pH 6.5, which can be found throughout the caput and corpus of the epididymis (Burg, Reference Burg1995; Frenette et al., Reference Frenette, Lessard and Sullivan2004). Then, sorbitol is oxidized by SDH into fructose, the main carbohydrate used by sperm to produce energy through glycolysis, which is also more permeable to the membrane than sorbitol (Cao et al., Reference Cao, Aghajanian, Haig-Ladewig and Gerton2009). SDH optimally generates fructose at pH 7.2, which can be found in the cauda of the epididymis and in semen (Frenette et al., Reference Frenette, Lessard and Sullivan2004, Reference Frenette, Thabet and Sullivan2006). However, it has not yet been determined whether epididymosomes can deliver both enzymes to sperm. In this sense, AKR1B5 is associated with this type of EVs in cattle and animals with a developed epididymis, it could modulate a mechanism to decrease the energy metabolism of sperm during their journey through the caput and corpus of the epididymis by transforming glucose into sorbitol to limit the entry of carbohydrates into sperm, in addition to generating a hyperosmotic milieu that can improve the survival and storage of these gametes (Crichton et al., Reference Crichton, Hinton, Pallone and Hammerstedt1994; Frenette et al., Reference Frenette, Lessard and Sullivan2004). Then, during the ejaculation process, SDH associated to epididymosomes transforms sorbitol into fructose, which is capable of crossing the sperm membrane to serve as an energy source that supplies the subsequent sperm processes that culminate in fertilization (Frenette et al., Reference Frenette, Thabet and Sullivan2006).

Moreover, miRNA transfer from epididymosomes to sperm has been described in mice, establishing that epididymosomes and sperm share 82% of the miRNA profile. It is worth mentioning miR-30, miR-465, miR-466, miR-467, and miR-669, which could be involved in the translational regulation in embryos during their early stages and in epigenetic inheritance processes (Reilly et al., Reference Reilly, McLaughlin, Stanger, Anderson, Hutcheon, Church, Mihalas, Tyagi, Holt, Eamens and Nixon2016).

As mentioned above, there are two subpopulations of epididymosomes, one being positive for CD9 and CD26 tetraspanins, and the other being positive for epididymal sperm binding protein 1 (ELSPBP1; Sullivan, Reference Sullivan2015). CD9⁺ epididymosomes are 10–100 nm in diameter and contain high levels of proteins such as MIF, DCXR, GliPr1L1 and AKR1B5 (Sullivan, Reference Sullivan2015). Tests with anti-CD9 and anti-CD26 antibodies indicated that the blockade of both tetraspanins prevents the fusion of CD9⁺ epididymosomes with sperm, which implicates both tetraspanins in the fusogenic process (Caballero et al., Reference Caballero, Frenette, Belleannée and Sullivan2013). Furthermore, CD9⁺ epididymosomes were described as having a preference for living and functional sperm (Caballero et al., Reference Caballero, Frenette, Belleannée and Sullivan2013). Conversely, in cattle, it has been observed that the ELSPBP1⁺-epididymosome subpopulation has greater affinity for dead spermatozoa during their journey through the caput and cauda of the epididymis, which is reinforced by the presence of the Zn²⁺ cation (D’Amours et al., Reference D’Amours, Frenette, Bordeleau, Allard, Leclerc, Blondin and Sullivan2012). It should also be noted that such EVs do not fuse with dead sperm in vitro (D’Amours et al., Reference D’Amours, Frenette, Bordeleau, Allard, Leclerc, Blondin and Sullivan2012). The function of ELSPBP1 has not yet been determined nor have the mechanisms underlying the specificity of ELSPBP1⁺ EVs for dead male gametes in vivo (D’Amours et al., Reference D’Amours, Frenette, Caron, Belleannée, Guillemette and Sullivan2016). See Figure 2 and Table 1 for a description of the contents and effects of epididymosomes on sperm.

Figure 2.

Epididymosomes and EVs released by Sertoli cells. The figure shows the environment of the epididymosomes and EVs released by Sertoli cells, and possible subvesicular localization of their components (enlarged images in digital format show the molecules in detail). Created with BioRender.com

2.3 Oviductosomes, uterosomes, vaginosomes and EVs released by Sertoli cells

In mice and cattle, it has been seen that when sperm reach the female reproductive tract they interact with oviductosomes, which are capable of transferring proteins such as plasma membrane calcium ATPase 4 (PMCA4), which is the main Ca²⁺ flow pump in these gametes (Al-Dossary et al., Reference Al-Dossary, Strehler and Martin-DeLeon2013). Another study that delved into the interactions of oviductal fluid proteins with bovine spermatozoa (Lamy et al., Reference Lamy, Nogues, Combes-Soia, Tsikis, Labas, Mermillod, Druart and Saint-Dizier2018) identified 21 probable oviductosomal proteins that interact with spermatozoa. These include annexins A1 (ANXA1) and A2 (ANXA2), non-muscle myosin heavy chain IIa (MYH9) and IIc (MYH14), heat shock 70 kDa protein 5 (HSPA5), and oviduct-specific glycoprotein (OVGP1). These proteins are involved in the modulation of bovine sperm capacitation, motility and viability, as was observed in vitro and in silico (King and Killian, Reference King and Killian1994; Abe et al., Reference Abe, Sendai, Satoh and Hoshi1995; Almiñana et al., Reference Almiñana, Corbin, Tsikis, Alcântara-Neto, Labas, Reynaud, Galio, Uzbekov, Garanina, Druart and Mermillod2017; Lamy et al., Reference Lamy, Nogues, Combes-Soia, Tsikis, Labas, Mermillod, Druart and Saint-Dizier2018). It was described that murine uterosomes, (as well as murine, human and bovine prostasomes, epididymosomes and oviductosomes) can deliver proteins such as PMCA4 and sperm adhesion molecule 1 (SPAM1) to spermatozoa, with the expression levels of both proteins being dependent on hormonal stimuli throughout the estrous or menstrual cycle (Martin-DeLeon, Reference Martin-DeLeon2016). SPAM1 is a hyaluronidase anchored to glycosylphosphatidylinositol (GPI) on the sperm surface and in the inner membrane of the acrosome, which participates in processes such as the acrosomal reaction, binding and degradation of hyaluronic acid from the matrix of cumulus cells and binding to the zona pellucida (Griffiths et al., Reference Griffiths, Galileo, Reese and Martin-DeLeon2008). Other less studied EVs of the female reproductive tract such as vaginosomes and EVs from the endometrium (endometriosomes) have been shown to modulate different processes in sperm in vitro, such as capacitation and acrosomal reaction, because they contain proteins such as PMCA4 and SPAM1 (Franchi et al., Reference Franchi, Cubilla, Guidobaldi, Bravo and Giojalas2016; Fereshteh et al., Reference Fereshteh, Bathala, Galileo and Martin-DeLeon2019).

Moreover, it is important to mention that the mechanism involved in the fusogenic process for the delivery of the molecular cargo to spermatozoa by oviductosomes has been proposed to be mediated by tetraspanin CD9, which serves as a scaffolding protein binding to adhesion proteins such as α_vβ₃ and α₅β₁ integrins, and whose ligands are vitronectin and fibronectin, respectively (Al-Dossary et al., Reference Al-Dossary, Bathala, Caplan and Martin-DeLeon2015). CD9, the two integrins and their respective ligands are present in oviductosomes, as well as throughout the head and mid-piece of sperm. These are places where the receptor–ligand interaction should take place and give way to the fusogenic process and the subsequent delivery of the different molecules from EVs to sperm. Although it was confirmed by confocal microscopy that epididymosomes and uterosomes also interact with the head and mid-piece of murine sperm, this has not been shown to occur through the same mechanism (Griffiths et al., Reference Griffiths, Galileo, Reese and Martin-DeLeon2008).

Finally, the EVs released by Sertoli cells were reported to contain proteins involved in the conditioning of the testicular environment to give way to spermatogenesis, highlighting that the protein profiles of these EVs respond in vitro to testosterone and FSH stimuli (Mancuso et al., Reference Mancuso, Calvitti, Milardi, Grande, Falabella, Arato, Giovagnoli, Vincenzoni, Mancini, Nastruzzi, Bodo, Baroni, Castagnola, Marana, Pontecorvi, Calafiore and Luca2018). Figure 2, Figure 3 and Table 1 show the contents and effects of oviductosomes, uterosomes, vaginosomes and EVs released by Sertoli cells on sperm.

Figure 3.

Oviductosomes, vaginosomes, uterosomes and oocytosomes. The figure shows the environment of the oviductosomes, vaginosomes, uterosomes and oocytosomes, and possible subvesicular localization of their components (enlarged images in digital format show the molecules in detail). Created with BioRender.com

2.4 New perspectives

The properties exhibited by the EVs mentioned in section 2 can be exploited to enhance the efficacy of ARTs from the sperm factor, with the most reasonable being to use their vesicular content, such as cations, carbohydrates, lipids, proteins or nucleic acids, to intervene in important processes such as capacitation, acrosomal reaction and/or binding to the zona pellucida. However, slightly broader perspectives can be proposed for its application. For example, as mentioned above, prostasomes have affinity for sperm and show an intrinsic ability to transfer chromosomal DNA molecules to them. This can be of great interest to improve sperm-mediated gene transfer (SMGT), therefore allowing animal transgenesis in different species without the need for expensive equipment, and also to reduce the percentage of embryos with mosaicism (Smith and Spadafora, Reference Smith and Spadafora2005; Canovas et al., Reference Canovas, Gutierrez-Adan and Gadea2010).

Human prostasomes contain large amounts of chromogranin B, which, in addition to the specificity and low immunogenicity of EVs in general (Kim et al., Reference Kim, Yang, Oh, Hong, Seo and Jang2017), offer an interesting alternative as the delivery vehicle for the antibiotic compound to combat resistant infections. However, further clinical trials are needed to validate this approach. Conversely, the high sphingomyelin and cholesterol content of proteasomal membranes that is transferred to sperm membranes could make sperm cryopreservation techniques more efficient by stabilizing their membranes, therefore reducing cryocapacitation, risk of biological contaminants, as for using egg yolk, and cryodamage that compromises sperm viability (Singh et al., Reference Singh, Kumar and Atreja2014). This makes these EVs a relatively inexpensive and accessible alternative to increase the percentage of competent sperm post-thaw. As for the DCXR protein in the spermatozoa, transferred by the epididymosomes, this can be used as a marker of epididymal maturation, to select the most suitable spermatozoa for different ARTs. Similarly, the subpopulation of ELSPBP1⁺ epididymosomes, which have affinity for dead sperm in vivo, could help to improve the selection of sperm to be used in intracytoplasmic sperm injection (ICSI), especially when such gametes come from individuals with fertility problems due to low or no sperm motility. Finally, EVs from Sertoli cells could provide a range of different biomarkers of infertility related to hormones such as FSH and testosterone, as hormonal stimuli modify the protein profiles of these EVs (Mancuso et al., Reference Mancuso, Calvitti, Milardi, Grande, Falabella, Arato, Giovagnoli, Vincenzoni, Mancini, Nastruzzi, Bodo, Baroni, Castagnola, Marana, Pontecorvi, Calafiore and Luca2018).

3.1 Folliculosomes

In folliculosomes, the main biomolecules that have been studied are miRNAs. These small ‘pieces’ of ribonucleic acid have attracted the interest of different research groups due to their ability to intervene in the expression process of different genes. Either by complementing themselves with the 3′-untranslated region (3′-UTR) of an mRNA for its subsequent degradation (decreasing its expression), or by complementing itself with an endogenous miRNA from the target cell, which prevents the translation of an mRNA of interest, avoiding the degradation of the latter (increasing its expression) (Figure 4). In silico analyses predicted that folliculosomal miRNAs can modulate mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK), transforming growth factor beta (TGFβ) and canonical WNT signalling pathways (da Silveira et al., Reference da Silveira, Veeramachaneni, Winger, Carnevale and Bouma2012; Sohel et al., Reference Sohel, Salilew-Wondim, Hölker, Rings, Schellander and Tesfaye2013b). In follicular cells, the activation of the MAPK/extracellular signal-regulated kinases (MAPK/ERK) signalling pathway depends on hormones such as FSH and LH, which trigger a molecular mechanism that culminates in the activation of the ERK protein. This serine/threonine kinase is capable of translocating to the nucleus and from there regulate the transcriptional processes of various genes through phosphorylating transcription factors, which ultimately stimulates the proliferation of granulosa cells and the expansion of cumulus cells (Orton et al., Reference Orton, Sturm, Vyshemirsky, Calder, Gilbert and Kolch2005; Santonocito et al., Reference Santonocito, Vento, Guglielmino, Battaglia, Wahlgren, Ragusa, Barbagallo, Borzì, Rizzari, Maugeri, Scollo, Tatone, Valadi, Purrello and Di Pietro2014). However, the activation of MAPK in cumulus cells also requires factors of the TGFβ family, such as bone morphogenetic protein (BMP), which are produced by the oocyte and are involved in the formation of complexes R-SMAD/coSMAD that are translocated to the cell nucleus to act as transcription factors of multiple genes, therefore modulating the proliferation, differentiation and homeostasis of follicular cells (Lyons and Prasad, Reference Lyons and Prasad2012; Santonocito et al., Reference Santonocito, Vento, Guglielmino, Battaglia, Wahlgren, Ragusa, Barbagallo, Borzì, Rizzari, Maugeri, Scollo, Tatone, Valadi, Purrello and Di Pietro2014). In addition, the canonical WNT pathway culminates in blocking the protein complex involved in the degradation of β-catenin, which translocates to the cell nucleus and acts as a transcription factor for multiple genes involved in the growth and development of follicular cells (Figure 4) (Santonocito et al., Reference Santonocito, Vento, Guglielmino, Battaglia, Wahlgren, Ragusa, Barbagallo, Borzì, Rizzari, Maugeri, Scollo, Tatone, Valadi, Purrello and Di Pietro2014; Nusse and Clevers, Reference Nusse and Clevers2017).

Figure 5.

Folliculosomes and oviductosomes. Effects of folliculosomes (light blue) on oocytes (upper region) and oviductosomes (pink) on cumulus cells (lower region). Signalling pathways involving folliculosomal or oviductosomal content are represented as different colours depending on the species in which they have been studied, as indicated in the legend at the top left. Arrows (→) represent activation, stimulation or effects. Truncated lines (—|) represent inhibition or suppression. Different coloured rectangles symbolize proteins or signalling pathways, while orange rectangles symbolize effects. Ovals symbolize genes (enlarged images in digital format show the molecules in detail). Ac, acetylation. Created with BioRender.com

In 2012, da Silveira and colleagues reported the profiles of miRNAs and folliculosomal proteins in young and old mares. Specifically, the transcriptomic analysis of these vesicles revealed that 120 miRNAs were differentially expressed according to the age of the animals, highlighting that miR-181a, miR-375 and miR-513a-3p were more abundant in old mares. In silico analyses predicted that these miRNAs target TGFβ-pathway genes, coinciding with the reduction in fertility at older ages, probably due to abnormal folliculogenesis and oogenesis (da Silveira et al., Reference da Silveira, Veeramachaneni, Winger, Carnevale and Bouma2012). Regarding the proteomic analysis in the folliculosomes, the presence of the alpha chain of inhibin (INHA) was detected. This hormone suppresses FSH secretion during the selection of the dominant follicle through a molecular mechanism that has not yet been clarified (Chen et al., Reference Chen, Wang, Lin, Chang, Chuang and Ying2006; da Silveira et al., Reference da Silveira, Veeramachaneni, Winger, Carnevale and Bouma2012). In the horse, follicular sEVs were described to be able to modulate the growth and development of the follicle and the oocyte throughout the oestrous cycle. These follicular sEVs intervene in the TGFβ/BMP pathway of granulosa cells through miRNAs that target the transcripts of activin A receptor, type I (ACVR1) and DNA-binding protein inhibitor 2 (ID2) proteins (da Silveira et al., Reference da Silveira, Carnevale, Winger and Bouma2014). Furthermore, in cattle, it has been described that folliculosomes support the proliferation of granulosa cells by regulating Src, MAPK and PI3K pathways, highlighting that EVs from small follicles have a more marked effect than those from the large ones and that granulosa cells, regardless of the size of their follicle of origin, have a preferential uptake of small follicle folliculosomes (Hung et al., Reference Hung, Navakanitworakul, Khan, Zhang, Davis, McGinnis and Christenson2017). Also in cattle, it has been reported that folliculosomes contain a dynamic miRNA profile throughout oogenesis, some of which may intervene in both the canonical WNT pathway of follicular cells and purine metabolism, protein ubiquitination and cAMP-mediated signalling pathways (Sohel et al., Reference Sohel, Hoelker, Noferesti, Salilew-Wondim, Tholen, Looft, Rings, Uddin, Spencer, Schellander and Tesfaye2013a, Reference Sohel, Salilew-Wondim, Hölker, Rings, Schellander and Tesfaye2013b). In humans, it has been reported that the miRNAs present in folliculosomes and secreted by granulosa and cumulus cells can interfere with the WTN, MAPK, ErbB and TGFβ/BMP signalling pathways, therefore modulating follicular development (Santonocito et al., Reference Santonocito, Vento, Guglielmino, Battaglia, Wahlgren, Ragusa, Barbagallo, Borzì, Rizzari, Maugeri, Scollo, Tatone, Valadi, Purrello and Di Pietro2014). In porcine (Pan et al., Reference Pan, Toms, Shen and Li2015), bovine (Sohel et al., Reference Sohel, Hoelker, Noferesti, Salilew-Wondim, Tholen, Looft, Rings, Uddin, Spencer, Schellander and Tesfaye2013a) and equine (da Silveira et al., Reference da Silveira, Veeramachaneni, Winger, Carnevale and Bouma2012), the levels of folliculosomal miR-378 were found to decrease as the oocyte matured. This miRNA was proposed to modulate cumulus cell expansion and oocyte maturation through the translational disruption of proteins involved in these processes, such as HAS2 and PTGS2, and CX43, ADAMTS1 and PGR, respectively (Pan et al., Reference Pan, Toms, Shen and Li2015). In addition, miR-378 targets the Cyp19A1 gene that codes for aromatase, a key enzyme in steroidogenesis. Such translational disruption inhibits oestradiol synthesis in cumulus cells, which suggests that this miRNA modulates the development of cumulus–oocyte complexes (COCs) (Pan et al., Reference Pan, Toms, Shen and Li2015). In cattle and mice, another way in which folliculosomes, especially those from small antral follicles, support the expansion of cumulus cells is via the upward expression of genes involved in this process, such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3) and tumour necrosis factor alpha-induced protein 6 (Tnfaip6) (Hung et al., Reference Hung, Hong, Christenson and McGinnis2015). The explanation about why folliculosomes from smaller follicles have greater effects on the expansion of cumulus cells has come from the work of Navakanitworakul et al. (Reference Navakanitworakul, Hung, Gunewardena, Davis, Chotigeat and Christenson2016). These authors determined that while large follicles contain fewer folliculosomes than their small counterparts and that their borne miRNAs mainly modulate inflammatory response pathways, the miRNAs of small follicle folliculosomes mostly regulate the pathways of cell proliferation (Navakanitworakul et al., Reference Navakanitworakul, Hung, Gunewardena, Davis, Chotigeat and Christenson2016). In contrast, pig folliculosomes from 2 to 5 mm (small) follicles do not have a significant effect on the expansion of cumulus cells in vitro, despite upregulating the expression of Has2 and Tnfaip6, both are involved in that process when added together with FSH to the culture medium (Matsuno et al., Reference Matsuno, Kanke, Maruyama, Fujii, Naito and Sugiura2019). A recent study, also in pigs, found that miR-92a modulated the formation and diameter of the follicular antrum and the acetylation levels of the H4K12 protein in the oocyte, which is associated with correct oocyte development (Inoue et al., Reference Inoue, Munakata, Shinozawa, Kawahara-Miki, Shirasuna and Iwata2020). According to this, it is worth mentioning that miR-92a together with miR-17b and miR-145b contribute to the organization of oocyte chromatin (Inoue et al., Reference Inoue, Munakata, Shinozawa, Kawahara-Miki, Shirasuna and Iwata2020). Regarding the folliculosomes from recently ovulated corpora lutea, which contain low levels of progesterone, they present a high content of miRNAs that could upwardly modulate genes involved in the MAPK and TGFβ pathways in cumulus cells, therefore supporting oocyte development (de Ávila et al., Reference de Ávila, Bridi, Andrade, del Collado, Sangalli, Nociti, da Silva Junior, Bastien, Robert, Meirelles, Perecin and da Silveira2020).

The aforementioned studies showed the dynamics of the content of folliculosomes throughout the oestrous cycle and how they can modulate different pathways in granulosa and cumulus cells through their borne miRNAs. However, there are other factors that can modify the characteristics and content of folliculosomes. It has been reported, for example, that mastitis reduces the diameter of folliculosomes in bovine (Santos et al., Reference Santos, Bottino, Santos, Simões, Souza, Ferreira, da Silveira, Ávila, Bride and Sales2018), and that the metabolic state influences the miRNA profile of these EVs in cattle and humans (Hailay et al., Reference Hailay, Hoelker, Poirier, Gebremedhn, Rings, Saeed-Zidane, Salilew-Wondim, Dauben, Tholen, Neuhoff, Schellander and Tesfaye2019; Martinez et al., Reference Martinez, Baccarelli, Liang, Dioni, Mansur, Adir, Bollati, Racowsky, Hauser and Machtinger2019). Exposure to oxidative stress conditions such as hyperthermia has also been shown to modulate the folliculosomal content, to protect the oocyte from possible damage caused by non-physiological conditions (Saeed-Zidane et al., Reference Saeed-Zidane, Linden, Salilew-Wondim, Held, Neuhoff, Tholen, Hoelker, Schellander and Tesfaye2017; Morales Dalanezi et al., Reference Morales Dalanezi, Mogollon Garcia, de Andrade Ferrazza, Fagali Franchi, Kubo Fontes, de Souza Castilho, Gouveia Nogueira, Dos Santos Schmidt, Sartori and Pinheiro Ferreira2019). However, it has been observed that folliculosomes produced under non-stressful conditions have the intrinsic ability to protect oocytes from the stress caused in extreme circumstances, such as cryopreservation (Ferraz et al., Reference Ferraz, Fujihara, Nagashima, Noonan, Inoue-Murayama and Songsasen2020) or hyperthermia (Rodrigues et al., Reference Rodrigues, Tuna, Alli, Tribulo, Hansen, Koh and Paula-Lopes2019). Figure 4, Figure 5 and Table 2 summarize the contents and effects of folliculosomes on the oocyte and follicular cells.

Table 2.

Follicle cell- and oocyte-related EVs

* Experimentally not proven or mechanism not known.

3.2 Oviductosomes and other EVs

As mentioned above, the dog oocyte completes its meiotic maturation in the oviduct, that is why the role of oviductosomes in canine oogenesis has been studied. It was reported that, in vitro, oviductosomes improve the concentration, viability and proliferation rate of cumulus cells and also reduces the accumulation of reactive oxygen species (ROS) and the rate of apoptosis, through regulating genes involved in the EGFR/MAPK signalling pathways (Egfr, Pka, Tace/Adam17, Mapk1/3, Mapk14, Ptgs2, Tnfaip6, Gdf9 and Bmp15; Lee et al., Reference Lee, Oh, Kim and Lee2020a, Reference Lee, Oh, Kim and Lee2020b). It is important to note that, at least in dogs, it was confirmed that cumulus cells and oocytes absorbed oviductosomes (Lee et al., Reference Lee, Oh, Kim and Lee2020b).

Similarly, the effect of EVs from other cell types on the follicular environment was investigated. EVs secreted by human adipose tissue derived from mesenchymal stem cells has been showed to counteract primary ovarian insufficiency (POI) in mouse models. Increasing the number of ovarian follicles and normalizing the levels of oestradiol, FSH and AMH (Huang et al., Reference Huang, Lu, Ding, Zou, Wang and Li2018). In addition, such EVs showed the increment of the proliferation rate of granulosa cells, also reducing apoptosis through the modulation of genes involved in the SMAD signalling pathway (Huang et al., Reference Huang, Lu, Ding, Zou, Wang and Li2018). Comparable effects were observed with EVs from human umbilical cord mesenchymal stem cells, as they restored ovarian function in a POI murine model, promoting the proliferation of granulosa cells and reducing the concentration of ROS via inhibiting the expression of Sirt7 through the delivery of miR-17-5p (Ding et al., Reference Ding, Zhu, Shen, Lu, Zou, Huang, Li and Huang2020). See Figure 5 and Table 2 for a description of the contents and effects of oviductosomes on cumulus cells.

3.3 New perspectives

Although spermatosomes do not appear to be present in mammals, it has been shown that they can be synthesized in vitro (Atif et al., Reference Atif, Salam, Ahmad, Hasan, Jamal, Sudhanshu, Azevedo and Owais2008). However, due to the nature of their membrane proteins (Tumova et al., Reference Tumova, Zigo, Sutovsky, Sedmikova and Postlerova2021) and the fact that oocytes are physiologically adapted to the presence of spermatozoa, they become an attractive vehicle with high affinity and low cytotoxicity to guide different molecules such as miRNAs, mRNAs or proteins towards COCs to enhance their maturation in vitro. For example, synthetic miRNAs that decrease Fatty Acid Binding Protein 3 (FABP3) synthesis in cumulus cells of COCs in vitro, could be delivered through spermatosomes to reduce excessive lipid accumulation in the oocyte, which is detrimental to these gametes (del Collado et al., Reference del Collado, da Silveira, Sangalli, Andrade, da Silva Sousa and Silva2017). Conversely, many of the folliculosomal miRNAs can be used as biomarkers (Yang et al., Reference Yang, Liu and Huang2017) or therapeutic targets for different conditions. In particular, miR-132 and miR-320 levels could be used to diagnose polycystic ovary syndrome and therapeutic targets for that condition (and its follow-up), as indicated by Sang et al. (Reference Sang, Yao, Wang, Feng, Wang, Zhao, Xing, Jin, He, Wu and Wang2013). Another interesting target is the folliculosomal miR-378, which can be blocked in COCs to significantly improve the rate of MII oocytes in vitro, as it is likely that many aspirated oocytes are in an immature state in which the concentration of this miRNA is high (Pan et al., Reference Pan, Toms, Shen and Li2015). While there are not studies about the effect of oviductosomes on COCs in other domestic mammals, the possibility exists that these EVs could have a positive effect on in vitro oocyte maturation. This possibility should not be dismissed due to the existing differences in canine oogenesis relative to other mammals. Finally, just as applications to improve ARTs from the oocyte point of view may arise using oviductosomes, it is likely that the in-depth study of the other biomolecules contained in the folliculosomes, such as lipids and proteins, may open new possibilities. In this context, not discarding of course the content of EVs from other cell types, for example, from mesenchymal stem cells derived from adipose tissue or umbilical cord.

4.1 Folliculosomes and oviductosomes

The previous section discussed many of the positive effects that folliculosomes can have on folliculogenesis and oogenesis. However, a study carried out by da Silveira et al. (Reference da Silveira, Andrade, Del Collado, Sampaio, Sangalli, Silva, Pinaffi, Jardim, Cesar, Nogueira, Cesar, Coutinho, Pereira, Perecin and Meirelles2017) showed that the benefits of such EVs can even go beyond early embryonic development. Indeed, these authors reported that the addition of folliculosomes to the in vitro maturation medium of oocytes and bovine embryo culture with a maximum of four cells modulates the expression of genes related to metabolism and development by increasing the content of different miRNAs, such as bta-miR-631, and inducing changes in the global levels of DNA methylation and hydroxymethylation (da Silveira et al., Reference da Silveira, Andrade, Del Collado, Sampaio, Sangalli, Silva, Pinaffi, Jardim, Cesar, Nogueira, Cesar, Coutinho, Pereira, Perecin and Meirelles2017). It is likely that the positive effect of these EVs on early embryos is a reflection that folliculosomes are present in the oviduct after ovulation and during fertilization. Nevertheless, the stellar EVs in this scene are undoubtedly the oviductosomes. As mentioned previously, oviductosomes are an important component during the early days of embryonic development after conception. It was reported that culturing bovine embryos in medium conditioned by bovine oviduct epithelial cells (BOEC) and in medium supplemented with fresh and thawed oviductosomes (isolated from the conditioned medium) increased the survival of post-vitrification blastocysts and the number of trophectoderm cells, supporting the idea that oviductosomes are the main cause of the beneficial effect of conditioned medium (Lopera-Vásquez et al., Reference Lopera-Vásquez, Hamdi, Fernandez-Fuertes, Maillo, Beltrán-Breña, Calle, Redruello, López-Martín, Gutierrez-Adán, Yañez-Mó, Ramirez and Rizos2016). These results are consistent with those obtained in another study carried out by the same group, in which supplementing the culture medium of preimplantation bovine embryos with oviductosomes secreted in vivo by the oviductal isthmus during the early luteal phase was found to improve embryo survival after vitrification (Lopera-Vásquez et al., Reference Lopera-Vasquez, Hamdi, Maillo, Gutierrez-Adan, Bermejo-Alvarez, Ramírez, Yáñez-Mó and Rizos2017). The beneficial effects on embryo survival in both works could be orchestrated to some degree by genes that are upregulated in embryos exposed to oviductosomes. Among them are the Aqp3 gene, which encodes for the Aquaporin 3 channel involved in the rapid transport of water and cryoprotectants across the plasma membrane, something that could be vital during vitrification/warming procedures. Other genes are Dnmt3A, which encodes for DNA (cytosine-5)-methyltransferase, essential during the epigenetic modifications necessary for correct embryonic development, such as the methylation of the second X chromosome; and Snrpn, which encodes for Small Nuclear Ribonucleoprotein-associated Protein N, a protein that has an important role in mRNA maturation, specifically during alternative splicing (Lopera-Vásquez et al., Reference Lopera-Vásquez, Hamdi, Fernandez-Fuertes, Maillo, Beltrán-Breña, Calle, Redruello, López-Martín, Gutierrez-Adán, Yañez-Mó, Ramirez and Rizos2016; Lopera-Vásquez et al., Reference Lopera-Vasquez, Hamdi, Maillo, Gutierrez-Adan, Bermejo-Alvarez, Ramírez, Yáñez-Mó and Rizos2017). In another study that compared the effects of fresh and post-thawed oviductosomes on the development and quality of bovine embryos, EVs that underwent storage at −80°C were found to increase the rate of blastocyst formation, their hatching and the number of total cells in in vitro produced bovine embryos, with respect to the presence of fresh EVs or the control without EVs (Almiñana et al., Reference Almiñana, Corbin, Tsikis, Alcântara-Neto, Labas, Reynaud, Galio, Uzbekov, Garanina, Druart and Mermillod2017). In the same work, differences were reported at the proteomic level in oviductosomes secreted by cells grown in vitro compared with those secreted in vivo, observing that the latter have a higher content of proteins that modulate the sperm–oocyte interaction and fertilization, and the development, quality, and implantation of bovine embryos generated in vitro (Almiñana et al., Reference Almiñana, Corbin, Tsikis, Alcântara-Neto, Labas, Reynaud, Galio, Uzbekov, Garanina, Druart and Mermillod2017). Such differences at the proteomic level could be related to the lack of cross-communication between oviductal cells cultured in vitro and other cell types. This contrasts with the in vivo scenario, in which cells are exposed to different hormonal concentrations throughout the oestrous or menstrual cycle. The latter was confirmed by another study carried out by the same group, in which the content dynamics of oviductosomes were analyzed throughout the bovine oestrous cycle. Significant changes were found in the composition of proteins, mRNA and non-coding RNA (such as miRNAs) (Almiñana et al., Reference Almiñana, Tsikis, Labas, Uzbekov, da Silveira, Bauersachs and Mermillod2018), evidencing the need of considering hormonal stimuli in oviductal cells cultured in vitro as an important factor that modulates the content of their EVs and, therefore, the effects that these may have on embryos. The latter cannot be ignored when it comes to purifying oviductosomes from samples obtained in slaughterhouses, making it necessary to consider the moment of the oestrous cycle in which the oviductal cells were found. In effect, this may be required to obtain the desired and reproducible results with respect to the effect of such EVs on the embryo. Another factor that can modulate the content of oviductosomes is the presence of zygotes in the oviduct, as shown by the results obtained by Qu et al. (Reference Qu, Zhao, Wang, Zhang, Li, Fan and Liu2019). These authors described that the supplementation of murine embryo culture medium with oviductosomes from pregnant females upregulates the expression of the Bcl2 (anti-apoptotic) and Oct4 (pluripotency) genes, and downregulates that of Bax (pro-apoptotic). This leads to the improvement in embryonic quality, blastocyst formation and birth rates according to the addition of oviductosomes from non-pregnant females (Qu et al., Reference Qu, Zhao, Wang, Zhang, Li, Fan and Liu2019). Conversely, in pigs it was shown that oviductosomes can modulate the hardening of the zona pellucida and reduce polyspermia during in vitro fertilization (IVF) (Alcântara-Neto et al., Reference Alcântara-Neto, Fernandez-Rufete, Corbin, Tsikis, Uzbekov, Garanina, Coy, Almiñana and Mermillod2020). The molecules involved in this mechanism still remain unknown, but MYH9 and OVGP1 proteins, both present in oviductosomes, have been purported to modify the structure of the zona pellucida once the oocyte was fertilized to avoid polyspermia (Alcântara-Neto et al., Reference Alcântara-Neto, Fernandez-Rufete, Corbin, Tsikis, Uzbekov, Garanina, Coy, Almiñana and Mermillod2020). Figure 6 and Table 3 show the contents and effects of folliculosomes and oviductosomes on the embryos.

Table 3.

Embryo-related EVs

* Experimentally not proven or mechanism not known.

4.2 Uterosomes and endometriosomes

Uterosomes were demonstrated to be another important actor during in vivo and in vitro embryogenesis. In 2014, these EVs were described to be implicated in the transfer of endogenous beta retrovirus Jaagsiekte (enJSRVs) RNA from uterine cells to the conceptus (Burns et al., Reference Burns, Brooks, Wildung, Navakanitworakul, Christenson and Spencer2014). Later, another work showed that uterosomes secreted throughout the estrous cycle and during the first months of pregnancy stimulated embryo trophectoderm cells to proliferate and secrete interferon tau (IFN-τ). This protein is crucial during the first month of pregnancy in ruminants, as it coordinates the production of progesterone and regulates genes involved in the transport of nutrients to the embryo, among other functions (Bazer et al., Reference Bazer, Ying, Wang, Dunlap, Zhou, Johnson and Wu2015; Ruiz-González et al., Reference Ruiz-González, Xu, Wang, Burghardt, Dunlap and Bazer2015). The analysis of the content of uterosomes identified the presence of mRNA of genes that encode for heat shock protein 70 (Hspa1A; Hsp70), interleukins, interferon regulatory factors (IRF) and the envelope protein of the retrovirus in JSRVs (Burns et al., Reference Burns, Brooks, Wildung, Navakanitworakul, Christenson and Spencer2014; Ruiz-González et al., Reference Ruiz-González, Xu, Wang, Burghardt, Dunlap and Bazer2015). These proteins regulate the proliferation and secretion of IFN-τ via their interaction with the Toll-like receptors (TLR) of the trophectoderm cells, which demonstrates the importance of the communication of the uterus with the embryo mediated by uterosomes (Ruiz-González et al., Reference Ruiz-González, Xu, Wang, Burghardt, Dunlap and Bazer2015). Similar results were observed when adding early luteal phase uterosomes to the culture medium of bovine embryos, which increases the expression of genes involved in embryonic growth and development, such as acrogranin and Ifn-τ, and decreases that of Bax and Bip (rough endoplasmic reticulum stress marker). This leads to the improvement of cleavage rate, blastocyst formation and number of blastomeres and reduces the apoptosis rate, with respect to embryos grown in the absence of these EVs (Qiao et al., Reference Qiao, Ge, Ma, Zhang, Zuo, Wang, Zhang and Wang2018). In mice, uterosomes also modulate embryonic development positively and reduce apoptosis, via increasing the expression of genes such as Bcl2 and Oct4, and reducing that of Bax (Lv et al., Reference Lv, Yu, Wang, Yi, Zeng and Xiao2018). These effects could be regulated to a certain degree by miR-21 present in uterosomes, which increases its expression as the embryo develops, and has also been implicated in the modulation of anti-apoptotic processes (Lv et al., Reference Lv, Yu, Wang, Yi, Zeng and Xiao2018). Although more studies are needed to understand the mechanisms through which this particular miRNA acts.

Conversely, endometriosomes, which can be considered a specific type of uterosomes, were also demonstrated to exert interesting effects on embryonic development. It was observed that the system of co-culture of murine embryos with endometrial cells results in a higher rate of blastocyst formation than the co-culture with oviductal cells (Desai et al., Reference Desai, Kennard, Kniss and Friedman1994), which could be orchestrated mainly by the EVs released into the culture medium (Lopera-Vásquez et al., Reference Lopera-Vásquez, Hamdi, Fernandez-Fuertes, Maillo, Beltrán-Breña, Calle, Redruello, López-Martín, Gutierrez-Adán, Yañez-Mó, Ramirez and Rizos2016). The factors secreted by oviductal cells could be beneficial for the production of embryos in vitro, being an important component of the natural environment of the embryo during its first days of development (Lopera-Vásquez et al., Reference Lopera-Vásquez, Hamdi, Fernandez-Fuertes, Maillo, Beltrán-Breña, Calle, Redruello, López-Martín, Gutierrez-Adán, Yañez-Mó, Ramirez and Rizos2016). Some studies have demonstrated that they could also release anti-implantation and anti-hatching molecules, as during the embryo journey along the oviduct, implantation or premature hatching must be avoided to prevent ectopic gestation or abnormal development (Bongso et al., Reference Bongso, Ng and Ratnam1990; Pauerstein et al., Reference Pauerstein, Eddy, Koong and Moore1990). The confirmation of the presence of anti-implantation and anti-hatching molecules in oviductosomes would explain to some extent the observations of Lopera-Vásquez et al. (Reference Lopera-Vásquez, Hamdi, Fernandez-Fuertes, Maillo, Beltrán-Breña, Calle, Redruello, López-Martín, Gutierrez-Adán, Yañez-Mó, Ramirez and Rizos2016), who reported a reduction in the blastocyst formation rate on day 9 when oviductosomes were added to the embryo culture medium. For its part, the positive effect of endometriosomes were reported in multiple studies. In humans, these EVs contain miRNAs relevant to embryo implantation, including hsa-miR-200c, hsa-miR-17 and hsa-miR-106a (Ng et al., Reference Ng, Rome, Jalabert, Forterre, Singh, Hincks and Salamonsen2013). In another study, in which the effects of the presence of human endometriosomes on murine cultured embryos were analyzed, an increase in embryonic adhesion and an upregulation of genes involved in this process, such as Itgb3, Itga7 and Cdh5, were observed, suggesting hsa-miR-30d (present in these EVs) to be the main component responsible for such effects (Vilella et al., Reference Vilella, Moreno-Moya, Balaguer, Grasso, Herrero, Martínez, Marcilla and Simón2015). Similarly, murine embryonic development has been favoured by the EVs released by human mesenchymal endometrial cells, increasing the number of blastomeres and the hatching rate of blastocysts (Blázquez et al., Reference Blázquez, Sánchez-Margallo, Álvarez, Matilla, Hernández, Marinaro, Gómez-Serrano, Jorge, Casado and Macías-García2018). In addition, endometriosomes were seen to stimulate embryos to release pro-angiogenic factors such as VEGF and PDGF-AA, which could modulate endometrial angiogenesis, vascularization, differentiation and tissue remodelling (Blázquez et al., Reference Blázquez, Sánchez-Margallo, Álvarez, Matilla, Hernández, Marinaro, Gómez-Serrano, Jorge, Casado and Macías-García2018). Endometriosomes are also involved in the response to oxidative stress in murine embryos grown in vitro, as they contain at least 23 proteins involved in this process (Marinaro et al., Reference Marinaro, Macías-García, Sánchez-Margallo, Blázquez, Álvarez, Matilla, Hernández, Gómez-Serrano, Jorge, Vázquez, González-Fernández, Pericuesta, Gutiérrez-Adán and Casado2019). Moreover, as in oviductal cells, endometrial cells respond to different stimuli that affect their secretion rate and content. In sheep, for example, progesterone was reported to regulate 1611 genes and nine miRNAs in endometrial cells and increase the rate of secretion of endometriosomes (Burns et al., Reference Burns, Brooks, O’Neil, Hagen, Behura and Spencer2018). Another factor that was shown to modulate the secretion rate of endometriosomes was the concentration of O₂. In effect, under hypoxia conditions (O₂ at 1%), bovine endometrial cells release more EVs and their content in proteins involved in embryo development is higher (Koh et al., Reference Koh, Peiris, Vaswani, Reed, Rice, Salomon and Mitchell2016). See Figure 7 and Table 3 for a description of the contents and effects of uterosomes and endometriosomes on embryos.

Figure 7.

Uterosomes and endometriosomes. The figure shows the environment of the uterosomes and endometriosomes, and possible subvesicular localization of their components (enlarged images in digital format show the molecules in detail). Created with BioRender.com

4.3 EVs released by embryos

Regarding the EVs released by embryos, it has been reported that they can modulate their environment in vivo by delivering factors that promote angiogenesis and tissue remodelling to the different uterine cells. In this sense, it was described that, in pigs, EVs released by trophectoderm cells contain miRNAs such as miR-126–5P, miR-296–5P, miR-16 and miR-17–5P, which can stimulate angiogenesis in uterine cells (Bidarimath et al., Reference Bidarimath, Khalaj, Kridli, Kan, Koti and Tayade2017). EVs from good quality bovine embryos had also been shown to modify gene expression in oviductal cells, acting specifically on the expression of genes induced by IFN-τ such as Oas1y, Mx1 and Isg15 (Dissanayake et al., Reference Dissanayake, Nõmm, Lättekivi, Ord, Ressaissi, Godakumara, Reshi, Viil, Jääger, Velthut-Meikas, Salumets, Jaakma and Fazeli2021).

Saadeldin et al. (Reference Saadeldin, Kim, Choi and Lee2014) described that EVs released by parthenotes co-cultured with SCNT-generated porcine embryos contained mRNAs of genes involved in cell pluripotency and proliferation, such as Oct4, Sox2, Klf4, Nanog and c-Myc, which are captured by SCNT embryos and improve the rate of blastocyst formation (Saadeldin et al., Reference Saadeldin, Kim, Choi and Lee2014). Another study that investigated the EVs released by human embryos also reported that they contained transcripts of Oct4 and Nanog genes (Giacomini et al., Reference Giacomini, Vago, Sanchez, Podini, Zarovni, Murdica, Rizzo, Bortolotti, Candiani and Viganò2017). Similarly, the blastocoel fluid was reported to contain EVs and different miRNAs that could modulate critical signalling pathways involved in embryo development, such as pluripotency, cell reprogramming, epigenetic modifications, intercellular communication, cell adhesion and cell fate. This reveals the dynamic communication mediated by EVs between the different cells of the same embryo (Battaglia et al., Reference Battaglia, Palini, Vento, La Ferlita, Lo Faro, Caroppo, Borzì, Falzone, Barbagallo, Ragusa, Scalia, D’Amato, Scollo, Musumeci, Purrello, Gravotta and Di Pietro2019). Conversely, it has been observed that the quality of the embryo influences the content of the EVs that they release (Dissanayake et al., Reference Dissanayake, Nõmm, Lättekivi, Ord, Ressaissi, Godakumara, Reshi, Viil, Jääger, Velthut-Meikas, Salumets, Jaakma and Fazeli2021). In addition, it is likely that the vesicles secreted by embryos generated by different techniques, e.g. IVF, ICSI, SCNT or parthenogenetic activation (PA), have also differences in their content. According to this, it is worth mentioning that EVs secreted by IVF-generated bovine embryos differ morphologically from those released by bovine parthenotes, and that the rate of EV secretion from the embryos is higher than that of the parthenotes (Mellisho et al., Reference Mellisho, Velásquez, Nuñez, Cabezas, Cueto, Fader, Castro and Rodríguez-Álvarez2017). Figure 8 and Table 3 show the contents and effects of the EVs released by embryos on their environment and also other embryos.

Figure 8.

EVs released by embryos. The figure shows the environment of the EVs released by embryos and possible subvesicular localization of their components (enlarged images in digital format show the molecules in detail). Created with BioRender.com

4.4 New perspectives

miR-21 is considered anti-apoptotic, and is present in conditions of high cell proliferation such as carcinogenesis (Shen et al., Reference Shen, Xu, Lv, Dai, Chen and Chen2020) and embryogenesis (Lv et al., Reference Lv, Yu, Wang, Yi, Zeng and Xiao2018). Supplementing the embryo culture medium with EVs rich in this miRNA appears to improve embryonic development, although an excess could be harmful as the overexpression of miR-21 has a pro-apoptotic effect (Yan et al., Reference Yan, Rao, Yuan, Gao, Huang, Zhao and Ren2017). Conversely, and as discussed previously, oviductosomes, uterosomes and endometriosomes can play an important role during the different embryonic stages, as they interact with the embryo sequentially during its journey to the uterus. Therefore, mimicking the maternal physiological environment in the best possible way makes the sequential culture necessary with each of these types of EVs. This could significantly improve embryonic quality and development in vitro. Another way to support the development of embryos is through their co-culture with other embryos generated through simpler techniques, to increase the amount in the same well, such as embryos generated by SCNT co-cultured with parthenotes (Saadeldin et al., Reference Saadeldin, Kim, Choi and Lee2014), where the EVs secreted by the latter improve the quality and development of the former. However, to take full advantage of this methodology, it is imperative to outline the content and determine the effects of EVs of embryos generated by different techniques on early embryogenesis. Being very likely that the results obtained will be different between the species, due to the efficiency rates achieved with each in vitro production technique. For example, in bovine, IVF-derived embryos could secrete EVs with greater beneficial effects on embryo quality and development than ICSI-derived embryos, as they have higher quality (Salamone et al., Reference Salamone, Canel and Rodríguez2017). Such EVs could also support the remodelling of the maternal environment during embryo transfer (Bidarimath et al., Reference Bidarimath, Khalaj, Kridli, Kan, Koti and Tayade2017). So co-transfer with EVs secreted by good quality embryos would be another interesting perspective. To have a more accessible stock of EVs without having to generate embryos, an interesting alternative would be the derivation of embryonic stem cells from embryos generated through ARTs that work better for each species. Although EVs released from embryonic stem cells had been demonstrated to have similar effects to those released from embryos (Hur et al., Reference Hur, Feng, Wilson, Cerione and Antonyak2021), it was not reported whether there were differences in the profile of EVs secreted by stem cells derived from embryos of different quality, so further studies are needed.