Oviduct fluid affects sperm function

The fluid in the oviduct is highly viscous, unlike the culture medium in which studies of mammalian fertilization are usually performed. Fluid viscosity is often overlooked in studies of sperm function within the oviduct. More viscous fluid has more internal friction so the wake from a sperm swimming in viscous medium is relatively small compared with less viscous medium (Kirkman-Brown and Smith, Reference Kirkman-Brown and Smith2011). Studies of human sperm demonstrate that resistance of the fluid to be moved results in a sperm tail with multiple bends while beating (Kirkman-Brown and Smith, Reference Kirkman-Brown and Smith2011; Hyakutake et al., Reference Hyakutake, Suzuki and Yamamoto2015). In contrast, in less viscous medium, the tail has fewer bends and, instead, remains mostly straight while simply swinging or flapping back and forth (Kirkman-Brown and Smith, Reference Kirkman-Brown and Smith2011; Hyakutake et al., Reference Hyakutake, Suzuki and Yamamoto2015). Consequently, in viscous fluid, a motile sperm will have less side-to-side movement (yaw) than in a standard viscosity medium (Kirkman-Brown and Smith, Reference Kirkman-Brown and Smith2011). Sperm also tend to swim near and against solid surfaces, for example, epithelial walls or the corners of microchannels (Denissenko et al., Reference Denissenko, Kantsler, Smith and Kirkman-Brown2012). Sperm that are close to the channel wall swim faster than those moving in the center of the channel (El-Sherry et al., Reference El-Sherry, Elsayed, Abdelhafez and Abdelgawad2014). Viscoelastic medium induces bovine sperm to swim in coordinated groups that may facilitate sperm migration (Tung et al., Reference Tung, Lin, Harvey, Fiore, Ardon, Wu and Suarez2017). The majority of sperm orient their swimming so that they swim against the flow of medium when the flow rate is intermediate (33-134 μm/s) (Miki and Clapham, Reference Miki and Clapham2013; El-Sherry et al., Reference El-Sherry, Elsayed, Abdelhafez and Abdelgawad2014; Tung et al., Reference Tung, Ardon, Roy, Koch, Suarez and Wu2015a). This appears to guide sperm upstream in oviduct fluid (Miki and Clapham, Reference Miki and Clapham2013). There is controversy about whether a signaling process in sperm aids in orienting sperm in the upstream direction or if sperm rheotaxis is a passive process (Miki and Clapham, Reference Miki and Clapham2013; Hyakutake et al., Reference Hyakutake, Suzuki and Yamamoto2015).

Interestingly, the viscosity of oviduct fluid varies during the estrous cycle; tenacious mucus is found in the rabbit oviduct lumen at estrus and disappears after ovulation (Jansen, Reference Jansen1978). Most studies of sperm–oviduct interaction or fertilization have used standard culture medium and ignored its low viscosity, compared with oviduct fluid. A few have tried to recapitulate the viscosity of oviduct fluid by adding components like methylcellulose or polyvinylpyrrolidone to medium (Suarez and Dai, Reference Suarez and Dai1992; Alasmari et al., Reference Alasmari, Costello, Correia, Oxenham, Morris, Fernandes, Ramalho-Santos, Kirkman-Brown, Michelangeli, Publicover and Barratt2013; Gonzalez-Abreu et al., Reference Gonzalez-Abreu, Garcia-Martinez, Fernandez-Espin, Romar and Gadea2017). In addition to effects on normal motility, discussed above, physiological viscosity converts the wild thrashing motion and high yaw of hyperactivated sperm to motility with less yaw and a more forward movement (Suarez and Dai, Reference Suarez and Dai1992).

In addition to the rheological properties of oviduct fluid, specific components of oviduct fluid such as secreted proteins, proteoglycans and lipids may influence fertilization by affecting sperm function (Coy et al., Reference Coy, Lloyd, Romar, Satake, Matas, Gadea and Holt2010; Killian, Reference Killian2011). This complex fluid can affect sperm before encountering the oocyte and during fertilization (Rodriguez-Martinez, Reference Rodriguez-Martinez2007; Killian, Reference Killian2011). For example, bovine sperm take up phospholipids that are abundant in oviduct fluid (Evans and Setchell, Reference Evans and Setchell1978; Killian et al., Reference Killian, Chapman, Kavanaugh, Deaver and Wiggin1989). Oviduct fluid glutathione peroxidase, superoxide dismutase and catalase can protect bovine sperm from damage by reactive oxygen species that may otherwise reduce sperm viability and motility (Lapointe and Bilodeau, Reference Lapointe and Bilodeau2003). Proteoglycans found in oviduct fluid promote capacitation of bovine sperm through their glycosaminoglycan side chains (Parrish et al., Reference Parrish, Susko-Parrish, Handrow, Sims and First1989; Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006).

Oviduct fluid components, for example, glycosaminoglycans, can also cause proteolysis or loss of sperm membrane proteins, including those that are implicated in sperm binding to the oviduct epithelium. The best studied of these proteins originate from accessory gland secretions and bind to sperm at ejaculation. Some bovine Binder of Sperm (BSPs) and porcine sperm adhesins are lost as sperm are capacitated (Topfer-Petersen et al., Reference Topfer-Petersen, Ekhlasi-Hundrieser and Tsolova2008; Hung and Suarez, Reference Hung and Suarez2010). Although the significance of protein loss or proteolysis is uncertain, in sperm bound to the oviduct epithelium, it might contribute to their release before fertilization (Topfer-Petersen et al., Reference Topfer-Petersen, Ekhlasi-Hundrieser and Tsolova2008; Hung and Suarez, Reference Hung and Suarez2010).

In addition to losing proteins, sperm also gain proteins while they reside in the oviduct. The first of two examples is oviduct-specific glycoprotein (OGP) or oviductin, also known as OVGP1, found in oviducts of many mammals. Although it has homology to the chitinase family of proteins, OGP does not have enzymatic activity (Jaffe et al., Reference Jaffe, Arias, O’Day-Bowman, Donnelly, Mavrogianis and Verhage1996; Araki et al., Reference Araki, Nohara, Yoshida-Komiya, Kuramochi, Ito, Hoshi, Shinkai and Sendai2003). Bovine sperm incubated in OGP have improved motility and viability (Abe et al., Reference Abe, Sendai, Satoh and Hoshi1995). Hamster sperm treated with recombinant OGP have increased phosphorylation of tyrosine residues on proteins, an indication that capacitation was enhanced (Yang et al., Reference Yang, Zhao, Yang and Kan2015). There is also evidence in mice and swine that OGP binds to the zona pellucida to increase fertilization success by rendering the zona matrix more permissive to penetration by sperm (Lyng and Shur, Reference Lyng and Shur2009; Algarra et al., Reference Algarra, Han, Soriano-Ubeda, Aviles, Coy, Jovine and Jimenez-Movilla2016).

A second example of an oviduct protein that affects sperm is osteopontin. Although it is already bound to bovine sperm before semen is deposited in females (Erikson et al., Reference Erikson, Way, Chapman and Killian2007), addition of osteopontin during in vitro fertilization reduces polyspermy (Goncalves et al., Reference Goncalves, Chapman, Bertolla, Eder and Killian2008). Neither osteopontin nor OGP is necessary for fertility in mice because animals deficient in each are fertile (Rittling et al., Reference Rittling, Matsumoto, McKee, Nanci, An, Novick, Kowalski, Noda and Denhardt1998; Araki et al., Reference Araki, Nohara, Yoshida-Komiya, Kuramochi, Ito, Hoshi, Shinkai and Sendai2003).

In addition to oviduct fluid proteins being added as peripheral membrane proteins, integral membrane proteins could be added by fusion with sperm of oviductosomes secreted by the oviduct. For example, a portion of the major Ca²⁺ efflux pump is added to mouse sperm by oviduct exosomes (Al-Dossary et al., Reference Al-Dossary, Bathala, Caplan and Martin-DeLeon2015). The proteins secreted by bovine oviduct cells and found in oviduct fluid have recently been profiled and include growth factors, metabolic regulators, immune modulators, enzymes and extracellular matrix components (Lamy et al., Reference Lamy, Labas, Harichaux, Tsikis, Mermillod and Saint-Dizier2016; Pillai et al., Reference Pillai, Weber, Phinney and Selvaraj2017). They function in immune homeostasis, gamete maturation, fertilization and early development (Pillai et al., Reference Pillai, Weber, Phinney and Selvaraj2017). The abundance of some depend on the stage of the estrous cycle and whether they were found in oviducts ipsilateral or contralateral to the ovary that ovulated (Lamy et al., Reference Lamy, Labas, Harichaux, Tsikis, Mermillod and Saint-Dizier2016).

The oviduct as a functional sperm reservoir

The oviduct, along with the UTJ in some species, appears to be the major location in which sperm are stored before fertilization. In contrast, although sperm are retained in the cervix or uterus, it is not clear that they are eventually released to move to the oviduct. So the UTJ and oviduct appear to be the major sperm storage sites in many mammals. To be a true ‘functional sperm reservoir’, as coined by Hunter et al. (Reference Hunter, Nichol and Crabtree1980), in addition to retaining sperm, the oviduct must affect sperm function and lengthen sperm lifespan beyond the inherent longevity of sperm (Orr and Zuk, Reference Orr and Zuk2014). More than simple adhesion occurs because binding to the oviduct epithelium prolongs the lifespan of sperm and suppresses capacitation and motility (Pollard et al., Reference Pollard, Plante, King, Hansen, Betteridge and Suarez1991; Rodriguez-Martinez et al., Reference Rodriguez-Martinez, Saravia, Wallgren, Tienthai, Johannisson, Vazquez, Martinez, Roca, Sanz and Calvete2005; Rodriguez-Martinez, Reference Rodriguez-Martinez2007; Hung and Suarez, Reference Hung and Suarez2010). Thus, the oviduct isthmus meets these requirements. However, the ability of sperm reservoirs described in a variety of species to prolong the lifespan of a highly differentiated and transcriptionally inactive cell is enigmatic.

The reservoir also releases a finite number of stored sperm, acting as a buffer for sperm number to prevent polyspermy but still provide an appropriate number of fertile sperm to the upper oviduct (Hunter and Leglise, Reference Hunter and Leglise1971). The isthmic epithelium binds and retains preferentially sperm that have intact acrosomes and normal morphology (Teijeiro et al., Reference Teijeiro, Dapino and Marini2011; Teijeiro and Marini, Reference Teijeiro and Marini2012). All together, the isthmus functions to increase the probability that a suitable number of fertile sperm are present at the site of fertilization.

The oviduct epithelium retains sperm and modulates sperm function

In mammals, the oviduct epithelium binds and retains sperm so they accumulate to form the reservoir. Adhesion is very specific. The sperm head binds to oviduct epithelial cells but not all cells (Pacey et al., Reference Pacey, Hill, Scudamore, Warren, Barratt and Cooke1995; Kervancioglu et al., Reference Kervancioglu, Saridogan, Aitken and Djahanbakhch2000). Moreover, the ability of sperm binding to maintain viability is not a common property of all cells (Boilard et al., Reference Boilard, Bailey, Collin, Dufour and Sirard2002). The ability to maintain viability requires direct contact between sperm and oviduct epithelial cells (Dobrinski et al., Reference Dobrinski, Smith, Suarez and Ball1997; Murray and Smith, Reference Murray and Smith1997; Smith and Nothnick, Reference Smith and Nothnick1997). Adhesion to the oviduct regulates sperm capacitation (Dobrinski et al., Reference Dobrinski, Smith, Suarez and Ball1997; Boilard et al., Reference Boilard, Bailey, Collin, Dufour and Sirard2002; Fazeli et al., Reference Fazeli, Elliott, Duncan, Moore, Watson and Holt2003) and suppresses the normal increase in sperm intracellular free calcium that occurs during capacitation (Dobrinski et al., Reference Dobrinski, Ignotz, Thomas and Ball1996; Dobrinski et al., Reference Dobrinski, Smith, Suarez and Ball1997).

Studies performed in several mammals have concluded that glycans are the components in oviduct epithelial cells that bind sperm (Lefebvre et al., Reference Lefebvre, Lo and Suarez1997; Green et al., Reference Green, Bredl, Holt, Watson and Fazeli2001; Suarez, Reference Suarez2001; Cortes et al., Reference Cortes, Orihuela, Zuniga, Velasquez and Croxatto2004; Topfer-Petersen et al., Reference Topfer-Petersen, Ekhlasi-Hundrieser and Tsolova2008). The evidence in most studies underpinning a role for oviduct glycans is a competition assay in which different glycans are added to sperm before challenging these sperm by allowing them to bind oviduct epithelial cells in vitro. If few sperm bind to oviduct cells, this result is interpreted as an indication that the specific glycan is related to the authentic oviduct glycan that binds sperm. A frequent problem with these studies is that most test high concentrations of a small number of monosaccharides or small oligosaccharides.

Identification of glycans that bind porcine sperm using a glycan array

The development of glycans immobilized on an array provided an opportunity to test hundreds of glycans for their ability to bind sperm. Using such an array, nearly 400 glycans were tested for their ability to bind porcine sperm (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012). All the glycans that bound sperm contained one of two glycan motifs, either a Lewis X trisaccharide (Le^X) or a structure with core mannose and two antennae terminating in the sialylated lactosamine trisaccharide bi-SiaLN or in simply lactosamine (Figure 1). There were several examples demonstrating that sperm bound these two motifs with high specificity. In all sialic acid-containing structures that bound sperm, sialic acid was linked to the 6 position of galactose; structures that were identical except that sialic acid was attached to galactose at the 3 position did not bind sperm. Furthermore, the branched structure on a mannose core was required because single sialylated lactosamine trisaccharides (Neu5Acα2-6Galβ1-4GlcNAc) did not bind sperm (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012).

Figure 1

Structures of glycans that bind bovine (Le^A) and porcine sperm (bi-SiaLN, bi-LN, and Le^X), and the related glycan that does not (LN). Le^A is found on the bovine oviduct epithelium. bi-SiaLN is abundant on the epithelium of the porcine ampulla and isthmus including ciliated and non-ciliated cells. Le^X is found in the porcine isthmus but not the ampulla.

The Le^X trisaccharide was found as a monomer, dimer or trimer in the remaining glycans that bound sperm (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012). This trisaccharide is composed of Gal and Fuc linked to GlcNAc (Figure 1). The Le^X trisaccharide also bound sperm with high specificity; the closely related Lewis A trisaccharide (Le^A, a positional isomer; the carbons in GlcNAc to which Gal and Fuc are linked are exchanged) did not bind porcine sperm. Contrarily, bovine sperm bind Le^A but not Le^X (Suarez et al., Reference Suarez, Revah, Lo and Kolle1998). Binding specificity was further supported because porcine sperm did not bind to Galβ1-4GlcNAc; fucose substitution on Le^X was necessary (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012).

To confirm that the glycans on the array that bound sperm were present in the oviduct isthmus and to determine the complete structures of the oviduct glycans that bound sperm, oviduct glycans and glycolipid structures were identified by tandem MS (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012). The Le^X and branched sialylated motifs (bi-SiaLN) that bound sperm were found on larger structures that were the most abundant of the complex-type glycans on epithelial cells (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012). Nearly all of the complex-type oligosaccharides linked to proteins through asparagine residues were branched with two antennae and several had a sialyl residue on at least one terminus. Some biantennary glycans had both motifs, a sialyl residue on one terminus and a Lewis structure on the second. This kind of hybrid glycan was not present on the array but it is possible that, because it includes both motifs, it might bind sperm with even higher affinity than glycans with a single motif.

As tandem MS did not distinguish between Le^A and Le^X and between glycans with sialyl residues attached to the 6-carbon and the 3-carbon of Gal, an additional strategy was used. An antibody and specific lectin, Sambucus nigra agglutinin were used that recognize sialic acid attached to galactose in an α-2,6 linkage preferentially and not sialic acid attached to galactose in an α-2,3 linkage (Naito et al., Reference Naito, Takematsu, Koyama, Miyake, Yamamoto, Fujinawa, Sugai, Okuno, Tsujimoto, Yamaji, Hashimoto, Itohara, Kawasaki, Suzuki and Kozutsumi2007; Song et al., Reference Song, Yu, Chen, Lasanajak, Tappert, Air, Tiwari, Cao, Chokhawala, Zheng, Cummings and Smith2011). Both reagents detected 6-sialylated structures that were abundant on the epithelium throughout the oviduct including on ciliated and non-ciliated cells (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012).

Similarly, an antibody to Le^X was also used to confirm the identity of the oviduct Lewis trisaccharide structures identified by MS (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012). Interestingly, Le^X was found in a punctate pattern at the luminal surface of porcine isthmic epithelial cells (Machado et al., Reference Machado, Kadirvel, Daigneault, Korneli, Miller, Bovin and Miller2014) but was not found in the ampulla.

bi-SiaLN and Le^X glycan motifs bind to the porcine sperm head

The head is the portion of sperm that binds to the oviduct epithelium and is where (Suarez et al., Reference Suarez, Redfern, Raynor, Martin and Phillips1991) authentic receptors for glycans with bi-SiaLN and/or Le^X motifs should be localized. Fluorescein-labeled Le^X and bi-SiaLN bound preferentially to the apical edge of the head in 60%-70% sperm before capacitation (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012; Machado et al., Reference Machado, Kadirvel, Daigneault, Korneli, Miller, Bovin and Miller2014). Binding of fluoresceinated glycans could be displaced by an excess of the same glycan that did not have a fluorescent tag. The binding specificity was confirmed by testing sperm binding to oviduct glycans attached to Sepharose beads (Figure 2). Tethering a motile cell to a solid phase glycan rather than a soluble glycan more closely mimics sperm binding to the oviduct and requires a higher affinity.

Figure 2

Sperm bind to oviduct cell aggregates isolated from the isthmus (a; porcine sperm) and beads to which Lewis A (Le^A) trisaccharide has been attached (b; bovine sperm).

Porcine sperm binding to oviduct cells requires glycans with bi-SiaLN and Le^X

Experiments using immobilized glycans (i.e. the glycan array and glycans linked to Sepharose) showed that bi-SiaLN and Le^X were each sufficient to tether a motile sperm. Necessity experiments were performed in which either the glycans or putative receptors were blocked. The result of blocking was assessed by sperm binding to aggregates of epithelial cells stripped from the isthmus (Figure 2). Results of these experiments indicated that each glycan or glycan receptor was necessary for sperm to bind oviduct cells.

Receptors on sperm for oviduct glycans

The identity of the receptors that bind oviduct glycans are controversial. Sperm from different species bind different glycans and the receptors they use may also be unique. Using bovine cells, one group provided evidence that two oviduct proteins, the chaperones GRP78 and HSP60, bound to sperm, although the glycans each bound were not determined (Boilard et al., Reference Boilard, Reyes-Moreno, Lachance, Massicotte, Bailey, Sirard and Leclerc2004). A second group completed more detailed studies by and proposed that oviduct plasma membrane annexins containing fucose bind sperm proteins originating from accessory gland secretions added to sperm at ejaculation (Ignotz et al., Reference Ignotz, Cho and Suarez2007). This result was a bit surprising because annexins are usually considered cytosolic proteins and they lack signal peptides that would direct them through the secretory pathway to become fucosylated. A proteomic study found that annexin A1 is the most abundant protein in oviduct fluid (Lamy et al., Reference Lamy, Labas, Harichaux, Tsikis, Mermillod and Saint-Dizier2016). Perhaps it is released into fluid without passing through the secretory pathway. However, in the fluid, it would be expected to compete with annexin A1 located on oviduct epithelial cells and decrease sperm binding to the oviduct.

Studies of porcine sperm also implicated accessory gland secretions added to sperm (Ekhlasi-Hundrieser et al., Reference Ekhlasi-Hundrieser, Gohr, Wagner, Tsolova, Petrunkina and Topfer-Petersen2005; Topfer-Petersen et al., Reference Topfer-Petersen, Ekhlasi-Hundrieser and Tsolova2008). The spermadhesin AQN1 originating from accessory gland secretions is a glycan-binding protein (Ekhlasi-Hundrieser et al., Reference Ekhlasi-Hundrieser, Gohr, Wagner, Tsolova, Petrunkina and Topfer-Petersen2005; Topfer-Petersen et al., Reference Topfer-Petersen, Ekhlasi-Hundrieser and Tsolova2008). Spermadhesins represent 90% of the total boar seminal plasma protein and they become peripherally associated with the sperm plasma membrane after ejaculation (Sanz et al., Reference Sanz, Calvete, Mann, Gabius and Topfer-Petersen1993). Sperm AQN1 is reported to bind mannose and galactose residues on oviduct cells, but not Le^X or bi-SiaLN structures (Ekhlasi-Hundrieser et al., Reference Ekhlasi-Hundrieser, Gohr, Wagner, Tsolova, Petrunkina and Topfer-Petersen2005).

The observation that the accessory gland proteins do not bind Le^X and bi-SiaLN motifs (Ekhlasi-Hundrieser et al., Reference Ekhlasi-Hundrieser, Gohr, Wagner, Tsolova, Petrunkina and Topfer-Petersen2005) and sperm obtained from the cauda epididymis are still able to bind oviduct cells, although in reduced number (Petrunkina et al., Reference Petrunkina, Gehlhaar, Drommer, Waberski and Topfer-Petersen2001), suggested that other glycan receptors were important. Indeed, in cattle there is no evidence that the fertility of epididymal sperm, not exposed to accessory gland proteins, is lower that normal ejaculated semen that includes accessory gland secretions (Amann and Griel, Reference Amann and Griel1974). The fertility of cauda epididymal sperm motivated the investigation of glycan receptors on porcine sperm from the epididymis, which also avoided interference from the very abundant accessory gland proteins (Silva et al., Reference Silva, Kadirvel, Jiang, Bovin and Miller2014).

Membrane lysates from porcine cauda epididymal sperm were separated chromatographically and each fraction was subjected to SDS-PAGE, transferred to nitrocellulose and incubated with biotinylated Le^X and bi-SiaLN. This ‘glycan blot’ was used to identify proteins with appropriate glycan affinity. Several proteins were identified including the peripheral membrane protein MFG-E8, also known as lactadherin, P47 or SED1 (Silva et al., Reference Silva, Frost, Li, Bovin and Miller2017). Competition experiments showed that lactadherin bound to oviduct cells and that inhibition reduced sperm binding (Silva et al., Reference Silva, Frost, Li, Bovin and Miller2017).

Although there is compelling evidence that oviduct glycans are at least partially responsible for sperm binding, there is also evidence that sperm binding to oviduct epithelial cells is mediated to some degree by other interactions. Perturbation of glycans or their candidate receptors decreases sperm binding to oviduct cell aggregates by a maximum of 60% (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012; Machado et al., Reference Machado, Kadirvel, Daigneault, Korneli, Miller, Bovin and Miller2014). Protein-based interactions may account for the residual binding. For example, fibronectin from oviduct cells can bind α5β1 integrin on bovine sperm (Osycka-Salut et al., Reference Osycka-Salut, Castellano, Fornes, Beltrame, Alonso, Jawerbaum, Franchi, Diaz and Perez Martinez2017) and the adhesion protein E-cadherin is found in both sperm and oviduct epithelial cells (Pollard et al., Reference Pollard, Plante, King, Hansen, Betteridge and Suarez1991; Lefebvre et al., Reference Lefebvre, Chenoweth, Drost, LeClear, MacCubbin, Dutton and Suarez1995; Caballero et al., Reference Caballero, Gervasi, Veiga, Dalvit, Perez-Martinez, Cetica and Vazquez-Levin2014).

Oviduct epithelial cells respond to sperm binding

In addition to the effect of adhesion on sperm, sperm adhesion to the oviduct modifies the transcriptional profile of oviduct epithelial cells (Fazeli et al., Reference Fazeli, Affara, Hubank and Holt2004; Georgiou et al., Reference Georgiou, Sostaric, Wong, Snijders, Wright, Moore and Fazeli2005; Georgiou et al., Reference Georgiou, Snijders, Sostaric, Aflatoonian, Vazquez, Vazquez, Roca, Martinez, Wright and Fazeli2007; Lopez-Ubeda et al., Reference Lopez-Ubeda, Garcia-Vazquez, Romar, Gadea, Munoz, Hunter and Coy2015). Genes related to the inflammatory response, molecular transport, protein trafficking and cell-to-cell signaling are among those most affected by sperm (Lopez-Ubeda et al., Reference Lopez-Ubeda, Garcia-Vazquez, Romar, Gadea, Munoz, Hunter and Coy2015). In the sow, there is evidence that the ovary has a local effect on the transcriptome of the oviduct. Unilateral ovariectomy reduces expression of genes believed to be involved in sperm survival and early embryonic development (Lopez-Ubeda et al., Reference Lopez-Ubeda, Munoz, Vieira, Hunter, Coy and Canovas2016). The effect of sperm on the sperm reservoir appears conserved between birds and mammals. Infiltration of porcine sperm into the UTJ and rooster sperm into the chicken utero-vaginal junction alters the expression of genes involved in pH regulation and immune-modulation (Atikuzzaman et al., Reference Atikuzzaman, Alvarez-Rodriguez, Vicente-Carrillo, Johnsson, Wright and Rodriguez-Martinez2017). Even more surprisingly, the transcriptional response of oviduct cells is different in response to insemination of either X chromosome- or Y chromosome-bearing sperm (Alminana et al., Reference Alminana, Caballero, Heath, Maleki-Dizaji, Parrilla, Cuello, Gil, Vazquez, Vazquez, Roca, Martinez, Holt and Fazeli2014). Thus, the presence of sperm changes the behavior of oviduct cells in addition to its consequences for sperm. The result of altered production of specific proteins by oviduct cells is not clear.

Sperm release from oviduct epithelial cells

For successful fertilization, sperm must be released from the reservoir in the isthmus to meet the oocyte in the ampulla. There are several hypotheses to explain how sperm are released. The first is that a signal, perhaps from follicular fluid or the ovulated cumulus–oocyte complex stimulates the release of sperm. This would assure that some sperm are released at the appropriate time. An alternate hypothesis is that a small fraction of sperm is released almost continuously so that there is always a small number of sperm prepared to fertilize an oocyte. It is possible that both mechanisms exist; that is, release in response to a signal is superimposed on top of the more spontaneous release of fractions of sperm. In any case, sperm release is due to a change in sperm behavior, in oviduct cell function or in the oviduct fluid surrounding the cells.

An important maturation that sperm complete in the oviduct is capacitation. After capacitation, sperm have a reduced ability to bind oviduct glycans (Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012; Machado et al., Reference Machado, Kadirvel, Daigneault, Korneli, Miller, Bovin and Miller2014), supporting the hypothesis that during capacitation, glycan receptors are modified. How capacitation might affect glycan receptors is unclear, but there is some preliminary evidence in cattle and swine that, during capacitation, they may be targeted for proteolysis. The molecular mass of one of the BSPs is altered during capacitation (Hung and Suarez, Reference Hung and Suarez2012). Furthermore, a candidate glycan receptor on porcine sperm, MFG-E8, co-precipitates in sperm lysates with a proteasomal subunit suggesting it may also be degraded (Miles et al., Reference Miles, O’Gorman, Zhao, Samuel, Walters, Yi, Sutovsky, Prather, Wells and Sutovsky2013).

Another alternative is that the development of hyperactivated motility may be sufficient to detach a sperm from the oviduct epithelium (Curtis et al., Reference Curtis, Kirkman-Brown, Connolly and Gaffney2012). In support of this, mouse sperm deficient in CatSper calcium channels that cannot hyperactivate do not detach from the oviduct (Ho et al., Reference Ho, Wolff and Suarez2009).

There is evidence that the cumulus cells of the ovulated cumulus–oocyte complex can release chemical signals, such as progesterone (Schoenfelder et al., Reference Schoenfelder, Schams and Einspanier2003; Tosca et al., Reference Tosca, Uzbekova, Chabrolle and Dupont2007), which might activate localized sperm release by promoting Ca²⁺ influx through CatSper channels (Lishko et al., Reference Lishko, Kirichok, Ren, Navarro, Chung and Clapham2012). Release may also be controlled by components from the oviduct itself, such as disulfide reducants (Talevi et al., Reference Talevi, Zagami, Castaldo and Gualtieri2007; Brussow et al., Reference Brussow, Ratky and Rodriguez-Martinez2008), glycosidases that cleave oviduct glycans from the epithelium (Carrasco et al., Reference Carrasco, Coy, Aviles, Gadea and Romar2008a and Reference Carrasco, Romar, Aviles, Gadea and Coy2008b), and oviduct smooth muscle contractions (Chang and Suarez, Reference Chang and Suarez2012). There is evidence that locally produced anandamide activates cannabinoid receptors and TRPV1 to induce a Ca²⁺ influx and sperm release (Gervasi et al., Reference Gervasi, Osycka-Salut, Sanchez, Alonso, Llados, Castellano, Franchi, Villalon and Perez-Martinez2016). Anandamide may also activate nitric oxide production by sperm to promote their release (Osycka-Salut et al., Reference Osycka-Salut, Gervasi, Pereyra, Cella, Ribeiro, Franchi and Perez-Martinez2012). Finally, the production of unknown sulfated glyconjugates may release sperm by competing for binding sites on the oviduct epithelium (Talevi and Gualtieri, Reference Talevi and Gualtieri2010). The dynamic nature of sperm interaction with the oviduct suggests that a variety of factors may regulate sperm release that may aid in providing a constant supply of competent fertilizing sperm.

Immunological tolerance of sperm in the oviduct

The oviduct lumen must maintain an aseptic state for successful fertilization and early embryonic development while regulating maternal responses to allogenic sperm and semi-allogenic embryos (Marey et al., Reference Marey, Yousef, Kowsar, Hambruch, Shimizu, Pfarrer and Miyamoto2016). Under pathologic conditions, the mucosal immune system produces a proinflammatory response. However, bovine sperm binding to oviduct epithelial cells induces an upregulation of IL-10, TGFβ and increased production of prostaglandin E₂, inducing an anti-inflammatory response (Marey et al., Reference Marey, Yousef, Kowsar, Hambruch, Shimizu, Pfarrer and Miyamoto2016; Yousef et al., Reference Yousef, Marey, Hambruch, Hayakawa, Shimizu, Hussien, Abdel-Razek, Pfarrer and Miyamoto2016). This produces an environment that suppresses sperm phagocytosis by PMNs and allows sperm greater opportunity to survive in the oviduct and fertilize oocytes. In essence, sperm induce their own protection from an immune response in the oviduct.