Implications
Artificial insemination is used for almost 20% of breeding cows worldwide. There is a need for semen extenders that enhance and prolong semen fertility beyond that which is possible using existing commercial formulations for cryopreservation or liquid semen storage. This could allow the use of reduced numbers of sperm per insemination, enhance the use of bulls of high genetic merit, and facilitate the greater application of fresh and sex-sorted semen. Here we review current information on the microenvironments of sperm as they mature and migrate. We also discuss how this knowledge can be used in the design of next-generation semen diluents.
Introduction
Artificial insemination (AI) has facilitated improvements to the genetic merit of dairy herds worldwide through provision of greater access to sires with favourable genetic traits, such as those relating to milk production and health. AI also allows easier herd management, reduces the risk of venereal disease, and improves farm safety. The semen extenders employed to successfully maintain the fertility of sperm are central to the widespread and commercial use of AI and these have been extensively reviewed (Vishwanath and Shannon, Reference Vishwanath and Shannon2000; Vishwanath, Reference Vishwanath2003). However, current drawbacks to storage of sperm in a cryopreserved or fresh (liquid) state highlight the need for improvements to semen diluents.
Cryopreservation of sperm facilitates its storage for indefinite periods. It is an indispensable technology to the AI industry internationally with 95% of AI worldwide carried out using frozen-thawed sperm (Thibier and Wagner, Reference Thibier and Wagner2002). However, higher concentrations of sperm must be used per insemination dose to achieve non-return rates (NRRs) comparable to AI with fresh sperm (Vishwanath and Shannon, Reference Vishwanath and Shannon2000). Sperm damage occurs during sperm cryopreservation as a result of mechanical stress to the plasma membrane and the generation of excess reactive oxygen species (ROS) (Bailey et al., Reference Bailey, Bilodeau and Cormier2000). This cryodamage is likely to account for the reduced viability of frozen-thawed compared with fresh sperm. However, cryocapacitation, a phenomenon of sperm cryopreservation (Bailey et al., Reference Bailey, Bilodeau and Cormier2000), could account in part for reduced fertility of frozen-thawed compared with fresh sperm (Thundathil et al., Reference Thundathil, Gil, Januskauskas, Larsson, Soderquist, Mapletoft and Rodriguez-Martinez1999). The ability to counteract or minimise damage associated with cryopreservation of sperm could allow lower concentrations of frozen-thawed sperm to be used per insemination dose without compromising fertility. Consequently, there would be increased availability of frozen-thawed sperm of popular bulls.
Currently, the fertility of fresh sperm can be maintained for 3 to 5 days in commercial diluents such as Caprogen® (Vishwanath and Shannon, Reference Vishwanath and Shannon1997 and Reference Vishwanath and Shannon2000). However, a major impediment to the widespread use of fresh sperm is its limited shelf-life (Vishwanath and Shannon, Reference Vishwanath and Shannon2000; Vishwanath, Reference Vishwanath2003). Although the use of fresh sperm for AI is confined primarily to countries that operate on a grass-based production system and have a narrow breeding season, prolonged storage of fresh sperm would permit more efficient and intensive use of ejaculates of elite sires in these countries during the peak breeding season. Factors contributing to a decline in the fertility of fresh sperm stored at ambient temperature are likely to include the production of ROS and the interaction of sperm with seminal plasma components (Vishwanath and Shannon, Reference Vishwanath and Shannon1997).
Further to the limitations of diluents used for frozen-thawed and fresh sperm, the advancement of reproductive technologies has placed extra demands on diluents. Such an advance in reproductive technology is the emergence of sex-sorted sperm. Conception rates are lower using sex-sorted sperm for AI compared with unsorted sperm (Borchersen and Peacock, Reference Borchersen and Peacock2009; DeJarnette et al., Reference DeJarnette, Nebel and Marshall2009; Frijters et al., Reference Frijters, Mullaart, Roelofs, van Hoorne, Moreno, Moreno and Merton2009). A recent analysis of conception rates following insemination of 4456 Holstein heifers in Australia with conventional (unsorted) or sex-sorted, frozen-thawed sperm of 56 dairy sires revealed conception rates of 32.1% and 21.3%, respectively (Healy et al., Reference Healy, House and Thomson2013). In the United States, conception rates following AI of Holstein heifers were 56% and 39% for conventional and sex-sorted sperm, respectively, while for cows conception rates were 30% and 25% (Norman et al., Reference Norman, Hutchison and Miller2010). A combination of both lower sperm doses and reduced sperm lifespan as a result of the sorting process contribute to the lower fertility rates observed with sex-sorted sperm when used for AI (Vazquez et al., Reference Vazquez, Parrilla, Gil, Cuello, Caballero, Vazquez, Roca and Martinez2008; Frijters et al., Reference Frijters, Mullaart, Roelofs, van Hoorne, Moreno, Moreno and Merton2009). Fresh, sex-sorted sperm could help overcome the negative effects associated with frozen-thawed, sex-sorted sperm such as the reduced conception rates resulting in reduced farm profitability (Hutchinson et al., Reference Hutchinson, Shalloo and Butler2013). However, better semen extenders that improve the survival of sex-sorted sperm would also be helpful.
Perhaps a limitation on the development and design of improved semen extenders is the expense and time-consuming nature of fertility trials. A range of parameters are used in preliminary, in vitro tests to predict the fertility of sperm. However, the determination of extender efficacy is ultimately assessed by its effect on fertility as in vitro parameters of sperm quality such as motility do not always accurately predict fertility (Bailey et al., Reference Bailey, Robertson and Buhr1994; Watson Reference Watson1995; Vishwanath and Shannon, Reference Vishwanath and Shannon1997).
The formulation of existing semen extenders has been based on a series of somewhat haphazard and empirical discoveries, combined with fundamental insights into sperm energy metabolism. However, research into the mechanisms of natural sperm storage and fertility enhancement in the male and female reproductive tracts are now revealing exciting mechanisms that could be exploited to improve our ability to support the fertility of frozen or fresh, stored sperm. The natural microenvironments experienced by sperm from spermatogenesis, storage in the epididymis, through admixture with seminal fluid, followed by capacitation within the female and onward transit to fertilisation in the ampulla, all contribute to sperm maturation and survival. These events, which ultimately influence the capacity of sperm to bring about a viable conception, are providing us with essential leads that could inform the development of better semen diluents through rational design.
Influence of the male reproductive tract
The role of the male reproductive tract cannot be overlooked if an in depth understanding of the mechanisms of mammalian sperm storage and maturation are sought. The male reproductive tract influences the fertility of sperm through maturation, subsequent storage and even following ejaculation through various factors secreted into the seminal plasma.
Cauda epididymis
Apart from creating a suitable microenvironment for sperm maturation, the epididymis also maintains sperm in a viable state until ejaculation (Jones, Reference Jones2004). The duration of epididymal storage can range from hours as in birds to months as is the case for bats and reptiles (Setchell et al., Reference Setchell, Sanchez-Partida and Chairussyuhur1993). Potentially fertile and motile bull sperm can be stored in aviable state in the bovine cauda epididymis for 4 to 6 weeks before ejaculation (Kirillov and Mozorov, Reference Kirillov and Mozorov1936). Understanding the mechanisms by which sperm are stored and sustained by the epididymis could potentially provide important insights for improving the storage of sperm in vitro.
It has been demonstrated for species within various mammalian clades that co-incubation with epididymis epithelial cells (EECs) supports sperm survival (Moore and Hartman, Reference Moore and Hartman1986; Moore et al., Reference Moore, Curry, Penfold and Pryor1992; Lin et al., Reference Lin, Zhang, Murdoch and Aitken2000). This was also shown for bovines (Gagnon et al., Reference Gagnon, Sullivan and Sirard2000). Molecules secreted by bovine primary epididymal cells, into epididymal fluid, have been shown to interact with bovine sperm, and certain soluble factors in EEC conditioned medium support sperm motility (Reyes-Moreno et al., Reference Reyes-Moreno, Boilard, Sullivan and Sirard2002a and Reference Reyes-Moreno, Boilard, Sullivan and Sirard2002b). Reyes-Moreno et al. (Reference Reyes-Moreno, Boilard, Sullivan and Sirard2002a) demonstrated that protein extracts, >10 kDa in size, from bovine cauda epididymal fluid prolong the duration for which bovine sperm are progressively motile. Sperm incubated in media to which these proteins were added had a significantly greater percentage of motile sperm after 6 h than media with or without control proteins. Furthermore, the percentage of sperm exhibiting progressive motility was significantly greater in samples containing epididymal proteins. Specific proteins in this epididymal fluid that interacted with bovine sperm, and which could potentially be responsible for eliciting these effects, were identified as α and β clusterin subunits, β-adrenergic receptor kinase 2, antithrombin-III (AT-III) and fibrinogen γ-B chains. Identification of factors such as these that influence sperm motility or survival could be applicable to better diluent formulation if they are found to prolong or improve the fertility of fresh or frozen-thawed sperm when used for AI or if they reveal novel mechanisms through which stored sperm can be positively manipulated.
Various environmental conditions of the cauda epididymis such as low oxygen tension (Free et al., Reference Free, Schluntz and Jaffe1976), hyperosmolarity of epididymal fluid (Liu and Foote, Reference Liu and Foote1998) and low pH (Acott and Carr, Reference Acott and Carr1984; Carr et al., Reference Carr, Usselman and Acott1985) have been shown to favour sperm survival. Verberckmoes et al. (Reference Verberckmoes, Van Soom, Dewulf, De Pauw and de Kruif2004) successfully preserved fresh bovine semen in CEP-2, a diluent designed around the ionic composition of cauda epididymal fluid. Analysis of the ionic composition of cauda epididymal fluid was carried out using fluid extracted in vivo from Holstein bulls using cannulation and in vitro through micropuncture of epididymides obtained from a slaughterhouse (Verberckmoes, Reference Verberckmoes2004). The osmolarity was adjusted to 338 mOsm (the average of the in vitro and in vivo collected cauda epididymal plasma) using sorbitol as it proved to be the best polyol additive for maintenance of progressive motility across 8 days (Verberckmoes et al., Reference Verberckmoes, Van Soom, Dewulf, De Pauw and de Kruif2004). An egg-yolk concentration of 10% was included in CEP-2 as this was found to be optimal for this diluent when progressive motility was assessed (Verberckmoes et al., Reference Verberckmoes, Van Soom, Dewulf, De Pauw and de Kruif2004).
The ability of the newly developed CEP-2 diluent to maintain progressive motility over 6 days was compared with that of a Tris diluent (Verberckmoes et al., Reference Verberckmoes, Van Soom, Dewulf, De Pauw and de Kruif2004). Samples incubated in CEP-2 exhibited a slower decrease in the percentage of progressively motile sperm compared with sperm in the Tris diluent. Similarly, when compared with storage in commercial diluents (Triladyl® and Caprogen® at 5°C) it was found that sperm stored in CEP-2 swam significantly faster and straighter after 6 days (Verberckmoes et al., Reference Verberckmoes, Van Soom, Dewulf and de Kruif2005). In vitro fertilisation (IVF) rates of sperm stored in CEP-2 for 6 days at 5°C were at least 50%, which is comparable to Caprogen® but significantly higher than Triladyl® (Verberckmoes et al., Reference Verberckmoes, Van Soom, Dewulf and de Kruif2005).
The development of the CEP-2 diluent is a prime example of how identifying the characteristics of the unique microenvironments of sperm that favour their survival can be applied to the design of new diluents. However, there is currently no data available on the in vivo fertility of fresh sperm stored in CEP-2.
Seminal plasma
Seminal plasma comprises secretions from the testes, epididymis, and accessory glands and has been a productive area of investigation into factors that mediate interactions between sperm and the female reproductive tract (FRT). These factors include various heparin binding proteins (HBPs) such as binder of sperm (BSP) proteins, fertility associated antigen (FAA), and type 2 tissue Inhibitor of metalloproteinase (TIMP-2). Their in vivo functions, for which we have an increased understanding, are providing mechanisms that could be exploited to regulate oviduct binding and improve sperm transit or viability.
BSP proteins are the most abundant proteins in seminal plasma, accounting for 40% to 57% of the total protein present within seminal plasma (Nauc and Manjunath, Reference Nauc and Manjunath2000). Several members of the BSP family of proteins have been described (Nauc and Manjunath, Reference Nauc and Manjunath2000). Interaction between bovine sperm and BSP1, BSP3 and BSP5 seems to be important for sperm binding to the oviduct as they confer on epididymal sperm increased binding affinity for oviduct epithelial cells (OECs) (Gwathmey et al., Reference Gwathmey, Ignotz, Mueller, Manjunath and Suarez2006). Interestingly, each of these BSP proteins, in the presence of oviduct apical plasma membrane vesicles, have been shown to maintain a significantly greater percentage of frozen-thawed and fresh, epididymal sperm in a progressively motile state than controls over a 5 and 8 h incubation period, respectively (Gwathmey et al., Reference Gwathmey, Ignotz, Mueller, Manjunath and Suarez2006). However, all three of these proteins are capable of blocking the interaction of ejaculated sperm with OECs (Gwathmey et al., Reference Gwathmey, Ignotz, Mueller, Manjunath and Suarez2006) and would, therefore, probably not be suitable for addition to extenders intended for ejaculated sperm. Furthermore, high levels of BSP proteins in semen have been negatively correlated with fertility (Moura et al., Reference Moura, Koc, Chapman and Killian2006; Roncoletta et al., Reference Roncoletta, Morani Eda, Esper, Barnabe and Franceschini2006; D’Amours et al., Reference D’Amours, Frenette, Fortier, Leclerc and Sullivan2010; Lessard et al., Reference Lessard, Siqueira, D'Amours, Sullivan, Leclerc and Palmer2011).
HBPs have been shown to have deleterious effects on various in vitro post-thaw parameters of frozen-thawed buffalo sperm quality when added before freezing (Harshan et al., Reference Harshan, Singh, Arangasamy, Ansari and Kumar2006). Free seminal BSP proteins may leave sperm susceptible to damage during cryopreservation by stimulating efflux of cholesterol and phospholipid from the sperm plasma membrane (Thérien et al., Reference Thérien, Souberyrand and Manjunath1997). Srivastava et al. (Reference Srivastava, Srivastava, Ghosh, Singh, Prasad, Kumar, Perumal, Jerome and Thamizharasan2012) employed an innovative method to sequester BSP1 from the semen during collection. Tubes for semen collection were coated with an antibody raised against BSP1. Semen from six dairy crossbred bulls (Bos taurus×Bos indicus) was collected into these tubes before dilution of the semen in a Tris-egg yolk-glycerol cryopreservation diluent. Control samples of semen were collected in tubes that were not coated with the antibody. Viability, motility and acrosome integrity were assessed before freezing and following thaw. Before freezing, sperm that were collected into tubes lined with anti-BSP1 antibody had significantly greater viability and acrosome integrity than control samples. Motility, as well as viability and acrosome integrity was significantly greater in the anti-BSP1 exposed sperm, post-thaw.
Perhaps collection methods developed to selectively sequester excess BSP proteins in the semen could improve the in vivo fertility of fresh or frozen-thawed sperm and lead to improved outcome when used for AI. However, a more detailed understanding of the subtle differences in the biological roles of BSP proteins may be required to implement this approach. Release of sperm from the oviductal reservoir is thought to be facilitated by modifications of the BSP protein profile on bovine sperm (Ardon and Suarez, Reference Ardon and Suarez2013). Changes that occur include the loss of most BSP5, alterations to the size of BSP3 while BSP1 is retained (Hung and Suarez, Reference Hung and Suarez2012). Disrupting the normal interaction of BSP proteins with sperm or the oviduct could potentially be detrimental to fertility. Furthermore, a low level of BSP5 in semen seems to be associated with higher in vivo fertility (Moura et al., Reference Moura, Koc, Chapman and Killian2006). The levels of BSP proteins present in the semen may be more important in determining fertility than their mere presence or absence. Perhaps this will be considered in the development of next-generation extenders.
FAA and TIMP-2 bind to epididymal sperm as part of a complex and increase their ability to respond to heparin and zona pellucida (ZP) proteins (Miller et al., Reference Miller, Winer and Ax1990; Nass et al., Reference Nass, Miller and Winer1990; Killian et al., Reference Killian, Chapman and Rogowski1993). Interestingly, bulls containing FAA in their semen were found to have greater in vivo fertility than those without FAA (Sprott et al., Reference Sprott, Harris, Forrest, Young, Zhang, Oyarzo, Bellin and Ax2000). FAA and TIMP-2 are likely to influence fertility by increasing sperm binding to glycosaminoglycans (GAGs) in the FRT, therefore, facilitating capacitation. However, FAA may also improve sperm fertility through degradation of neutrophil extracellular traps (NETs) in the FRT that would otherwise entangle sperm (Alghamdi and Foster, Reference Alghamdi and Foster2005).
Recombinant versions of FAA (rFAA) and TIMP-2 (rTIMP-2) were recently shown to significantly improve fertility of frozen-thawed sperm in an AI field trial when added to a Tris-egg yolk-citrate extender (Alvarez-Gallardo et al., Reference Alvarez-Gallardo, Kjelland, Moreno, Welsh, Randel, Lammoglia, Perez-Martinez, Lara-Sagahon, Esperon-Sumano and Romo2013). rFAA and rTIMP-2 were added to a semen sample, each at a concentration of 25 µg/ml, before dilution and cryopreservation in the extender. Interestingly, FAA seems to reduce post-thaw acrosome damage when added before freezing (Alvarez-Gallardo et al., Reference Alvarez-Gallardo, Kjelland, Moreno, Welsh, Randel, Lammoglia, Perez-Martinez, Lara-Sagahon, Esperon-Sumano and Romo2013). A second aliquot of semen which was diluted and frozen without these proteins served as the control. AI was carried out on 100 crossbred heifers (B. indicus×B. taurus) (50 each for control and treatment groups). Pregnancy rates, as diagnosed 45 days after insemination were 40% and 16% for heifers inseminated with the treated and untreated semen, respectively.
Although the number of inseminations and semen samples used in this experiment were low, it demonstrates the potential for factors that naturally influence sperm fertility in vivo to improve semen extenders.
Influence of the FRT
Sperm storage in the FRT occurs in a range of taxa (Holt and Lloyd, Reference Holt and Lloyd2010; Holt, Reference Holt2011). The storage periods can reach months and even years for various species of birds, reptiles, amphibians, sharks and rays. However, among eutherian mammals, with the exception of bats, storage durations are significantly shorter (Holt and Lloyd, Reference Holt and Lloyd2010; Holt, Reference Holt2011). For example, in the reproductive tract of cows, sows and ewes, ejaculated sperm normally survive for 24 to 48 h (Hunter and Nichol, Reference Hunter and Nichol1983; Hunter, Reference Hunter1984; Hunter and Wilmut, Reference Hunter and Wilmut1984) while in the FRT of the dog viable sperm have been recovered 11 days after copulation (Doak et al., Reference Doak, Hall and Dale1967). Mimicking the sperm storage mechanism employed by the FRT could facilitate significant advances in sperm diluent design for fresh and frozen-thawed sperm AI.
After ejaculation in cattle, sperm migrate along the FRT and upon reaching the oviduct they adhere to the epithelium of the oviductal isthmus (Hunter and Wilmut, Reference Hunter and Wilmut1984; Lefebvre et al., Reference Lefebvre, Chenoweth, Drost, LeClear, MacCubbin, Dutton and Suarez1995). Fucose residues on the surface of epithelial cells of the bovine oviduct bind sperm at this site (Lefebvre et al., Reference Lefebvre, Lo and Suarez1997) via the Ca2+-dependent lectin PDC-109 (Suarez et al., Reference Suarez, Revah, Lo and Kolle1998; Ignotz et al., Reference Ignotz, Lo, Perez, Gwathmey and Suarez2001) (since reclassified as BSP1 (Manjunath et al., Reference Manjunath, Lefebvre, Jois, Fan and Wright2009)). Binding of sperm to the oviductal isthmus in vivo serves to form a sperm reservoir and a lengthening of the fertile lifespan of sperm is achieved (Hunter and Wilmut, Reference Hunter and Wilmut1984; Suarez, Reference Suarez2001 and Reference Suarez2002). Maintenance of sperm viability at this oviductal reservoir is thought to be facilitated by delayed capacitation (Murray and Smith, Reference Murray and Smith1997; Petrunkina et al., Reference Petrunkina, Simon, Gunzel-Apel and Topfer-Petersen2003 and Reference Petrunkina, Simon, Gunzel-Apel and Topfer-Petersen2004).
Regulation of capacitation
Capacitation is a process that ultimately leads to the acquisition of ZP binding ability and subsequent fertilisation of an oocyte. It involves various changes such as increases in intracellular Ca2+ levels (Parrish et al., Reference Parrish, Susko-Parrish and Graham1999), protein tyrosine phosphorylation (Galantino-Homer et al., Reference Galantino-Homer, Visconti and Kopf1997), and alterations to membrane and cell surface components (Suzuki and Yanagimachi, Reference Suzuki and Yanagimachi1989; Taitzoglou et al., Reference Taitzoglou, Kokoli and Killian2007; Hung and Suarez, Reference Hung and Suarez2012). Delaying these processes could lengthen the fertile lifespan of sperm as premature capacitation and acrosomal exocytosis would lead to death in the absence of fertilisation.
Dobrinski et al. (Reference Dobrinski, Suarez and Ball1996) compared the intracellular Ca2+ concentration ([Ca2+]i) of stallion sperm incubated with OECs to that of sperm that were not exposed to OECs. They found the [Ca2+]i of sperm incubated with OECs was maintained at a lower level over the 6 h incubation than that of control sperm that had no access to OECs (Dobrinski et al., Reference Dobrinski, Suarez and Ball1996). Capacitation and acrosomal exocytosis involve an increase in [Ca2+]i. Thus, maintenance of low [Ca2+]i in sperm bound to the oviduct could function to delay capacitation.
The ability to regulate or delay capacitation in vitro could facilitate improvements to the effectiveness of extenders for cryopreservation of bovine sperm. Capacitation that results from freezing (cryocapacitation) is thought to impact on the fertility of frozen-thawed sperm as the proportion of viable, uncapacitated sperm in frozen-thawed samples is positively correlated with in vivo fertility as measured by the day 56 NRRs (Thundathil et al., Reference Thundathil, Gil, Januskauskas, Larsson, Soderquist, Mapletoft and Rodriguez-Martinez1999). Cryocapacitation could increase the susceptibility of sperm to premature or spontaneous acrosome reaction, thereby, compromising their potential for fertilisation.
A typical capacitation-associated change observable in cryopreserved sperm is an increase in [Ca2+]i. Although this resembles a normal feature of capacitation, frozen-thawed bovine sperm suffer a reduced ability to regulate [Ca2+]i (Bailey and Buhr, Reference Bailey and Buhr1994). This seems to adversely affect their fertility when used for AI. It was found that in vivo fertility rates (60 to 90 days NRRs) of frozen-thawed bovine sperm are correlated with the capacity of the sperm to regulate [Ca2+]i (Bailey et al., Reference Bailey, Robertson and Buhr1994). Low intracellular and high extracellular Ca2+ concentrations favour the in vivo fertility of frozen-thawed sperm (Bailey et al., Reference Bailey, Robertson and Buhr1994; Collin et al., Reference Collin, Sirard, Dufour and Bailey2000).
Recent results from Okazaki et al. (Reference Okazaki, Yoshida, Teshima and Shimada2011) provide evidence that regulation of [Ca2+]i in frozen-thawed sperm can lead to improvements in fertility. The Ca2+ chelators ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA) were added in combination, each at a concentration of 6 mM, to the thawing solution of frozen-thawed boar sperm. Significant suppression of increases in [Ca2+]i was observable in frozen-thawed sperm treated with the combination of EDTA and EGTA. Furthermore, protein tyrosine phosphorylation events were suppressed, acrosomal integrity improved and motility was significantly better in samples treated with EDTA and EGTA. However, in the context of improving semen extenders, the most interesting result comes from the in vivo fertility experiments. The application of this combinational treatment of EGTA and EDTA to the thawing solution of frozen-thawed boar sperm used for AI in 33 sows resulted in significant improvements in the fertilisation rate as assessed by the number of fertilised oocytes per total number of corpora lutea 36 h post-insemination. AI using control frozen-thawed boar sperm generated a fertilisation rate of 33% compared with 82% for treated frozen-thawed sperm.
Further investigations are required to understand the details of the interactions between sperm and OECs that ultimately lead to regulation of [Ca2+]i and capacitation in sperm. However, knowledge of such details could permit the development of semen extenders that regulate the capacitation-associated increases in [Ca2+]i that occur during freeze-thawing of sperm. Thus, the fertility of frozen-thawed sperm could be improved for AI as evidenced by the results of Okazaki et al. (Reference Okazaki, Yoshida, Teshima and Shimada2011). Sex-sorting of sperm also leads to capacitation-like changes (Maxwell and Johnson, Reference Maxwell and Johnson1999) indicating that advances in diluent formulation that mitigate premature capacitation could be particularly useful in this context. Furthermore, given that the fertile lifespan of freshly ejaculated sperm can be extended in the oviduct through regulation of capacitation, it is likely that these mechanisms could also be employed to prolong the storage of fresh sperm in vitro for AI.
Bovine, luteal and follicular phase oviductal fluid from the isthmus and ampulla has been shown to be capable of inducing capacitation (Parrish et al., Reference Parrish, Susko-Parrish, Handrow, Sims and First1989; McNutt and Killian, Reference McNutt and Killian1991; Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006). Clearly oviductal fluid could provide an insight into factors that regulate capacitation.
A number of such factors have been identified which could be influencing sperm capacitation in vivo including GAGs (Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006), calcium (Visconti et al., Reference Visconti, Westbrook, Chertihin, Demarco, Sleight and Diekman2002), bicarbonate (Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006) and anandamide (Gervasi et al., Reference Gervasi, Osycka-Salut, Caballero, Vazquez-Levin, Pereyra, Billi, Franchi and Perez-Martinez2011).
The sulphated GAG (S-GAG), heparin, is commonly employed for in vitro capacitation of bovine sperm and GAGs have been proposed as in vivo capacitators of sperm (Bergqvist and Rodriguez-Martinez, Reference Bergqvist and Rodriguez-Martinez2006). Another S-GAG, heparan sulphate, has been proposed as a potential inducer of capacitation in oviductal fluid since heparin is not present in oviductal fluid in vivo (Kjellen and Lindahl, Reference Kjellen and Lindahl1991; Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006). Bergqvist et al. (Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006) compared the capacity of various GAGs to induce bovine sperm capacitation to that of oviductal fluid and bicarbonate. They found that hyaluronic acid (HA) significantly increases the percentage of capacitated sperm without killing them. However, it seems to be the least potent GAG at inducing the acrosome reaction. As HA is present in the oviductal fluid (Bergqvist et al., Reference Bergqvist, Yokoo, Heldin, Frendin, Sato and Rodriguez-Martinez2005), this could have some influence on sperm in vivo. Chondroitin sulphate B has been implicated in the capacitation of bovine sperm within the oviduct in vivo and its ability to induce the capacitation of bovine sperm in vitro has been confirmed (Thérien et al., Reference Thérien, Bergeron, Bousquet and Manjunath2005; Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006). Despite the effects of GAGs on capacitation, natural oviductal fluid was more effective, suggesting that other factors within it were also bioactive (Bergqvist et al., Reference Bergqvist, Ballester, Johannisson, Hernandez, Lundeheim and Rodriguez-Martinez2006). Previous studies have indicated that bicarbonate, which stimulates protein kinase A-dependent phosphorylation in boar sperm (Harrison, Reference Harrison2004), may be more potent at capacitating bovine sperm than GAGs alone (Sostaric et al., Reference Sostaric, van de Lest, Colenbrander and Gadella2005).
Gervasi et al. (Reference Gervasi, Rapanelli, Ribeiro, Farina, Billi, Franchi and Perez Martinez2009) found that cannabinoid receptors CB1 and CB2, and fatty acid amide hydrolase, an enzyme that regulates endogenous levels of anandamide (AEA), are expressed by bovine sperm and OECs. They demonstrated that AEA, an endogenous cannabinoid receptor agonist, at physiological levels, and an analogue of AEA (Met-AEA) were capable of reducing the numbers of sperm bound to oviductal cells in vitro. These effects were shown to be due to an interaction with CB1 rather than CB2 (Gervasi et al., Reference Gervasi, Rapanelli, Ribeiro, Farina, Billi, Franchi and Perez Martinez2009). It was later found that AEA and Met-AEA are capable of inducing capacitation through CB1 and the transient receptor potential vanilloid type 1 (TRPV1) (Gervasi et al., Reference Gervasi, Osycka-Salut, Caballero, Vazquez-Levin, Pereyra, Billi, Franchi and Perez-Martinez2011). Furthermore, CB1 and TRPV1 antagonists blocked heparin-induced capacitation suggesting that heparin and anadamides act through the same molecular mechanisms to capacitate sperm (Gervasi et al., Reference Gervasi, Osycka-Salut, Caballero, Vazquez-Levin, Pereyra, Billi, Franchi and Perez-Martinez2011). Interestingly AEA levels in bovine oviductal fluid were recently found to be greatest around ovulation suggesting that the endocannabinoid system could play an important role in regulating sperm function in vivo (Gervasi et al., Reference Gervasi, Marczylo, Lam, Rana, Franchi, Konje and Perez-Martinez2013).
Identification of factors such as GAGs and anandamide that elicit capacitation, together with their receptors, could be useful for designing semen extenders that delay capacitation through agents capable of specific receptor blockade.
Other mechanisms of regulating sperm viability in the oviduct
Apical plasma membrane proteins
It has been shown that purified apical plasma membranes (APMs) from fresh and cultured OECs are capable of significantly improving viability and longevity of bovine (Boilard et al., Reference Boilard, Bailey, Collin, Dufour and Sirard2002) and porcine sperm (Fazeli et al., Reference Fazeli, Elliott, Duncan, Moore, Watson and Holt2003; Holt et al., Reference Holt, Elliott, Fazeli, Satake and Watson2005). Boilard et al. (Reference Boilard, Bailey, Collin, Dufour and Sirard2002) showed that frozen-thawed bovine sperm incubated over a 6 h period with these APMs exhibited significantly greater motility than controls. It was found that a subset of soluble apical plasma membrane (sAPM) proteins derived from APM vesicles could bind to sperm (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). Among these proteins, heat shock 70 kDa protein 8 (HSPA8) was specifically identified (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). Anti-HSPA8 antibodies blocked the effect of sAPM proteins on the longevity of boar sperm. Furthermore, recombinant bovine HSPA8 generated the same sperm survival effects in boar and bull sperm as sAPM proteins (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). The concentration of HSPA8 (0.25 µg/ml) required to elicit these effects was considerably lower than that of sAPM proteins (200 µg/ml).
Significant improvements in the viability of bull and boar sperm, as measured by membrane integrity over 48 h, have been consistently demonstrated using HSPA8 at low concentrations (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). HSPA8 was found to positively influence fertility parameters other than membrane integrity. For example, brief exposure of boar sperm to HSPA8 before IVF of oocytes resulted in an improved rate of embryo development (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). Similar observations were recorded for sperm treated with sAPM before fertilisation (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). Monospermy was also increased using HSPA8 (Elliott et al., Reference Elliott, Lloyd, Fazeli, Sostaric, Georgiou, Satake, Watson and Holt2009). The addition of HSPA8 to the commercial ram sperm extender INRA96 at a concentration of 4 µg/ml gave significantly greater survival over ram sperm stored in INRA96 alone when maintained at 17°C over a 48 h period (Lloyd et al., Reference Lloyd, Fazeli, Watson and Holt2012). These are encouraging results but it remains to be seen whether HSPA8 can be used routinely to improve the formulation of currently available bovine semen extenders.
Antioxidants
High concentrations of ROS form during sperm cryopreservation (Bailey et al., Reference Bailey, Bilodeau and Cormier2000) and storage of sperm at ambient temperatures (Vishwanath and Shannon, Reference Vishwanath and Shannon2000). These can damage sperm through protein and lipid peroxidation, and loss of sulphydryls, thus, affecting fertility. Sperm stored for 24 to 48 h at ambient temperature in Caprogen® diluent to which catalase was added were significantly more fertile than controls as measured by the day 49 NRRs (Vishwanath and Shannon, Reference Vishwanath and Shannon2000). However, only a limited number of diluents contain catalase and these are used for ambient or refrigerated storage of sperm (Vishwanath and Shannon, Reference Vishwanath and Shannon2000). Furthermore, Klinc and Rath (Reference Klinc and Rath2007) demonstrated that catalase provides protection to sperm during sex-sorting indicating that decreased survival of sex-sorted sperm could be largely attributable to the production of ROS.
Oviductal fluid contains many antioxidant proteins such as catalase (Lapointe et al., Reference Lapointe, Sullivan and Sirard1998), superoxide dismutase (Roy et al., Reference Roy, Gauvreau and Bilodeau2008) and glutathione peroxidase (Lapointe and Bilodeau, Reference Lapointe and Bilodeau2003). These antioxidants are likely to influence sperm in the FRT by offering protection from oxidative and proteolytic damage, consequently, maintaining sperm membrane integrity and prolonging the fertile lifespan of sperm. Incorporating these oviductal proteins or reflecting this natural mechanism of protecting sperm in the design of novel diluents intended for sperm cryopreservation or, indeed, ambient temperature storage, could bring about improvements in sperm fertility.
The antioxidant cytochrome C was recently shown to give improvements in the in vivo fertility of frozen-thawed sperm when added to the cryopreservation diluent after thawing (Zahariev et al., Reference Zahariev, Stefanov, Sabev, Miteva and Nikolov2007). Zahariev et al. (Reference Zahariev, Stefanov, Sabev, Miteva and Nikolov2007) reported on more than 2000 inseminations of Holstein–Friesian cows inseminated with the frozen-thawed sperm of two Holstein–Friesian bulls to which 1.5 mg/ml cytochrome C was either added post-thaw or not at all in the case of controls. Addition of cytochrome C to the extender post-thaw resulted in significant improvements in day 60 NRRs (Zahariev et al., Reference Zahariev, Stefanov, Sabev, Miteva and Nikolov2007). The effect of antioxidants on sperm cryopreservation is currently a rich area of investigation. However there are a limited number of field fertility trials available.
Oviductal fluid
Oviductal fluid has been shown to prolong bovine sperm viability and motility (Killian, Reference Killian2011). Boquest et al. (Reference Boquest, Smith, Briggs, Duganzich and Summers1999) separated out proteins >8 kDa in size from the luteal phase oviductal fluid of B. indicus cows and assessed their effect on pooled samples of frozen-thawed sperm from four Friesian–Holstein bulls. Sperm were incubated with 1 mg/ml of these oviductal proteins in a ram semen diluent, RSD-1. Control samples had extra bovine serum albumin (BSA) added to match the protein concentration in treated samples. After 5 h of incubation the percentage of live sperm in samples treated with oviductal fluid proteins was significantly greater than the control group. The percentage of acrosome reacted sperm in samples treated with oviductal proteins was significantly lower than control samples after 24 h. This could indicate that oviductal proteins play a role in delaying capacitation.
Imam et al. (Reference Imam, Ansari, Ahmed and Kumaresan2008) separated the heparin-binding and non-HBPs from non-luteal oviductal fluid and sperm samples were treated with either fraction at a concentration of 0.652 and 2 mg/ml, respectively, in a Tris-egg yolk-citrate extender. Oviductal fluid proteins were omitted from the extender in control samples. All three treatment groups were cryopreserved. A variety of in vitro parameters were assessed in each sample before freezing and after thawing. The percentage motility, viability and acrosomal integrity of sperm treated with heparin-binding proteins before and after freezing were all significantly greater than the control, untreated group. Sperm treated with the heparin-unbound fraction had significantly greater percentages of viability and acrosomal integrity than controls pre-freeze and post-thaw. However, the percentage motility was not greater than controls.
These reports of the effect of oviductal fluid proteins on sperm in vitro exemplify how factors that favour sperm survival during its natural transit through the FRT can be applied to bovine semen extenders to improve sperm quality throughout its storage and potentially improve sperm fertility when used for AI.
Perhaps the most promising result of this study was that the positive effects of the oviductal fluid proteins in this study were detectable both before freezing and after thawing. This indicates that the properties of these proteins could be valuable for improvements to both fresh semen diluents and cryopreservation diluents. An investigation of the in vivo fertility of sperm treated with these proteins is warranted as in vitro parameters of sperm quality do not always give an accurate representation of fertility.
Specific oviductal fluid proteins that influence sperm
Specific proteins such as osteopontin (OPN) (Gabler et al., Reference Gabler, Chapman and Killian2003) and oviduct specific glycoprotein (OGP) (Boice et al., Reference Boice, Geisert, Blair and Verhage1990; Sendai et al., Reference Sendai, Abe, Kikuchi, Satoh and Hoshi1994; Buhi, Reference Buhi2002) have been identified as secretory products in the bovine oviduct and were found to interact with gametes while also improving the efficiency of IVF (Martus et al., Reference Martus, Verhage, Mavrogianis and Thibodeaux1998; Killian, Reference Killian2011). OPN is an extracellular matrix protein (McKee and Nanci, Reference McKee and Nanci1996) that, as well as being present in bovine seminal plasma (Cancel et al., Reference Cancel, Chapman and Killian1997), is secreted by the oviduct mucosa. OPN can improve sperm viability, facilitate bovine sperm capacitation, ZP binding, fertilisation and embryo development and reduce polyspermy (Killian, Reference Killian2011). It has also been shown to be more prevalent in the seminal plasma of higher fertility bulls (Cancel et al., Reference Cancel, Chapman and Killian1997).
OGPs (Boice et al., Reference Boice, Geisert, Blair and Verhage1990; Sendai et al., Reference Sendai, Abe, Kikuchi, Satoh and Hoshi1994; Buhi, Reference Buhi2002) are present in the oviduct of every mammal studied to date. They are virtually exclusive to oviduct tissue and their production is greatest during oestrus and ovulation (Buhi, Reference Buhi2002). OGPs have been found to influence a wide range of reproductive phenomena. Satoh et al. (Reference Satoh, Abe, Sendai, Iwata and Hoshi1995) purified one such OGP, a 72 kDa sialylated glycoprotein which was secreted by the bovine oviductal epithelium during the follicular phase. This was characterised as bovine oviduct specific glycoprotein (BOGP). BOGP at both 100 and 500 µg/ml in Tyrode’s medium containing lactate and pyruvate (TLP) was capable of sustaining significantly greater bovine sperm viability in vitro than control TLP containing oviduct extract over 3 h (Satoh et al., Reference Satoh, Abe, Sendai, Iwata and Hoshi1995). The effects of BOGP on sperm in Tyrode’s medium containing albumin, lactate, and pyruvate (TALP) were found to be dose dependent (Abe et al., Reference Abe, Sendai, Satoh and Hoshi1995). Furthermore, maintenance of bovine viability and motility across 12 h in a TALP-BOGP medium was more effective than in TALP medium supplemented with egg albumin, lactalbumin, BSA or gastric mucin (Abe et al., Reference Abe, Sendai, Satoh and Hoshi1995). The ability of bovine sperm to undergo capacitation and fertilise oocytes was increased following treatment with BOGP (King et al., Reference King, Anderson and Killian1994; Martus et al., Reference Martus, Verhage, Mavrogianis and Thibodeaux1998).
New frontiers: sperm-mucin interactions and their relevance to diluent development
During natural copulation in cattle, ejaculation occurs in the cranial part of the vagina. After insemination, sperm are in continual contact with mucus and its principal polymeric component, hydrated mucin. Mucins are a family of large molecular weight, filamentous glycoproteins (Lai et al., Reference Lai, Wang, Wirtz and Hanes2009; Hickey et al., Reference Hickey, Patel, Fahey and Wira2011). Over 60% of the molecular weight of mucins is O-linked oligosaccharides commonly presenting terminal sialic acid, resulting in a large degree of negative charge (Yudin et al., Reference Yudin, Hanson and Katz1989; Gipson, Reference Gipson2001; Cone, Reference Cone2009; Lai et al., Reference Lai, Wang, Wirtz and Hanes2009). Some mucins may also be sulphated; this too confers negative charge (Robbe et al., Reference Robbe, Michalski and Capon2006). The rheological properties of mucus such as viscosity and elasticity are thought to be central to the resistance faced by sperm at the cervical mucus barrier (Suarez and Pacey, Reference Suarez and Pacey2006) while porosity, hydration and ionic content of mucus are also implicated in sperm transit through the reproductive tract (Katz et al., Reference Katz, Slade and Nakajima1997; Hansson, Reference Hansson2010).
Literature on the interaction of bovine sperm with mucin is sparse when compared with other aspects of sperm’s natural migration, though they present a rich diversity of carbohydrate structures with the potential for specific and non-specific interaction. The cervix is the principal source of secreted mucus in the FRT and the fact that bovine AI bypasses the cervix, thereby, largely evading cervical mucus, may explain the paucity of research in this area. However, this does not imply that sperm’s interaction with mucus is irrelevant to fertility or that it cannot offer insights into sperm fertility that might ultimately be applicable to better diluent design. Mucus is also present in the uterus and uterine tube where it arises from cervical secretions (Suarez and Pacey, Reference Suarez and Pacey2006) and shedding of endometrial and oviductal membrane bound mucins (Gipson, Reference Gipson2001). Furthermore, a growing body of evidence is pointing towards the possibility that mucin-sperm interactions at a biochemical level are relevant to sperm function.
Understanding these mechanisms could provide opportunities to advance the design of next-generation semen extenders capable of enhancing sperm transit through the FRT and improving fertility. This proposal is based on (i) the mounting evidence that appropriate sperm surface characteristics rather than motility are paramount to facilitating mucus penetration and therefore fertility and (ii) increasing evidence for the importance of carbohydrate-based interactions between sperm and their different microenvironments.
Evidence for electronegative repulsion between sperm and mucins
Research into the association of β-defensin 126 (DEFB126), a protein of epididymal origin, with macaque sperm has provided valuable insights into the potential significance of cervical mucins during sperm transit. Tollner et al. (Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b) demonstrated that loss of DEFB126 reduces cervical mucus penetration ability. Surprisingly, the surface characteristic of the sperm; more specifically the presence or absence of normal DEFB126; was found to be more important in determining mucus penetration ability than the type of motility (hyperactivated v. non-hyperactivated) (Tollner et al., Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b).
DEFB126 coating confers a high negative charge upon macaque sperm due to its presentation of sialylated oligosaccharides (Yudin et al., Reference Yudin, Generao, Tollner, Treece, Overstreet and Cherr2005). Interestingly, sialidase treatment of macaque sperm was found to reduce mucus penetrating ability (Tollner et al., Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b). Furthermore, this reduction was comparable to that achieved with removal of DEFB126 (Tollner et al., Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b) indicating that sialic acid may account in full for the role of DEFB126 in mucus penetration. These observations are compatible with a potential importance of electronegative repulsion between defensin-coated sperm and sialylated mucin as a factor that promotes mucus penetration.
DEFB126 is lost from the macaque sperm surface during in vitro capacitation (Tollner et al., Reference Tollner, Yudin, Treece, Overstreet and Cherr2004). The loss of DEFB126 from the sperm surface during capacitation is logical as DEFB126 mediates sperm oviduct binding (Tollner et al., Reference Tollner, Yudin, Tarantal, Treece, Overstreet and Cherr2008a). This loss would facilitate the final release of sperm from the tubal reservoir before fertilisation. However, its presence on the sperm surface before capacitation is also of importance because of a probable protective and disadhesive role during transit of sperm through cervical mucus.
Mechanisms for mucus penetration by sperm analogous to that in macaques may also exist in other species. The sperm of various species have been shown to acquire a high negative charge throughout maturation in the epididymis (Tollner et al., Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b). Furthermore, increased surface sialylation is suspected to account for this as sialidase treatment eliminates this negative charge on ram and bull sperm (Holt, Reference Holt1980). Moreover, loss of surface negative charge seems to occur during capacitation. Bull sperm, for example have reduced electrophoretic migration towards the anode following capacitation (Iqbal and Hunter, Reference Iqbal and Hunter1995). Capacitation has also been shown to reduce the levels of wheat germ agglutinin (WGA) binding to bovine sperm which could be indicative of reduced surface sialylation (Taitzoglou et al., Reference Taitzoglou, Kokoli and Killian2007). Human sperm lose approximately half of their surface negative charge during capacitation (Rosado et al., Reference Rosado, Velazquez and Lara-Ricalde1973) and it has recently been reported that capacitation of human sperm is associated with increased sialidase activity on the sperm, and loss of surface sialic acid (Ma et al., Reference Ma, Wu, Deng, Secrest, Zhao, Varki, Lindheim and Gagneux2012).
The acquisition of high negative charge of bovine sperm and, indeed, the sperm of other mammals, during epididymal storage and its subsequent loss during capacitation closely resembles the pattern of acquisition and loss of surface negative charge by macaque sperm. Perhaps a mechanism for mucus penetration by sperm similar to that demonstrated in macaques, may also exist in cattle. HA gels (artificial mucus) are used experimentally as a proxy for cervical mucus as they have similar charge characteristics and create a viscous medium (Tollner et al., Reference Tollner, Dong and VandeVoort2011). Recently, Al Naib et al. (Reference Al Naib, Ward, Kelly, Wade, Marti and Lonergan2011) reported that the ability of fresh bovine sperm to penetrate artificial mucus is almost absent after 2 days of storage at ambient temperature in Caprogen® diluent. Studies of AI carried out in New Zealand demonstrated that storage of sperm at ambient temperature in Caprogen® diluent maintains sperm fertility (day 24 NRRs) at ~70% for 4 days while even after 6 days of storage NRRs are ~65% (Vishwanath and Shannon, Reference Vishwanath and Shannon1997). However, fresh sperm stored at ambient temperature in Caprogen® diluent is only used for 2 to 3 days for AI in Ireland as it is generally considered that fertility rates drop subsequently (Al Naib et al., Reference Al Naib, Ward, Kelly, Wade, Marti and Lonergan2011; Murphy et al., Reference Murphy, Fahey, Shafat and Fair2013). The loss of artificial mucus penetration ability observed by Al Naib et al. (Reference Al Naib, Ward, Kelly, Wade, Marti and Lonergan2011) coincides with the loss of fertility of fresh sperm stored in Caprogen® diluent.
Although loss of mucus penetration may not fully explain the decline in fertility of fresh sperm when used for AI, it could partially contribute to it. Ruminant uterine and oviductal epithelia secrete a variety of mucin/mucin-like glycoproteins through which the sperm must pass (Lagow et al., Reference Lagow, DeSouza and Carson1999) and cervical mucus may also ascend into the uterus around oestrus (Suarez and Pacey, Reference Suarez and Pacey2006). Furthermore, the results of Tollner et al. (Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b) revealed how a simple change in sperm surface characteristics can influence a fundamental aspect of sperm function.
This could also be of relevance to AI using frozen-thawed sperm as it was demonstrated that frozen-thawed macaque sperm have a significantly reduced ability to penetrate macaque cervical mucus and HA gel compared with fresh sperm (Tollner et al., Reference Tollner, Dong and VandeVoort2011). When equal numbers of progressively motile fresh and frozen-thawed macaque sperm were compared, it was found that despite retaining >80% of the progressive motility of fresh sperm, frozen-thawed sperm exhibited only 18.6% of the cervical mucus penetration ability and 16.2% of the artificial mucus penetration ability of fresh sperm. In cattle, it is known that when equal numbers of motile fresh and frozen-thawed sperm are inseminated, frozen-thawed sperm are less fertile (Watson, Reference Watson1995). However, the degree to which this is attributable to loss of mucus penetration ability in frozen-thawed sperm is not yet known.
This mechanistic insight could be exploited to improve mucus penetration by sperm in vivo if loss of surface charge is proven to have a direct influence on in vivo fertility in response to AI.
Evidence for potential carbohydrate-based sperm-mucin interactions
Beyond the electronegative repulsion effect suggested by Tollner et al. (Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b), current understanding of the relationship between sperm and sialylated glycoproteins is of relevance to the improved design of semen diluents. Mucins and other sialylated glycoproteins of the FRT appear to significantly influence sperm function, most likely through direct sperm-carbohydrate interactions.
Human cervical mucins have been shown to positively influence human sperm motility (Eriksen et al., Reference Eriksen, Carlstedt, Uldbjerg and Ernst1998). Mucins bear a wide diversity of carbohydrate structures that could have the potential for interaction with sperm surface proteins (Pluta et al., Reference Pluta, Irwin, Dolphin, Richardson, Fitzpatrick, Gallagher, Reid, Crowe, Roche, Lonergan, Carrington and Evans2011). To the best of our knowledge, a similar influence of cervical mucin on bovine sperm has not yet been demonstrated. However, it is plausible that bovine sperm could interact specifically with mucin glycans as bovine sperm have been shown to have affinity for such sugars (Lefebvre et al., Reference Lefebvre, Lo and Suarez1997; Velasquez et al., Reference Velasquez, Canovas, Barajas, Marcos, Jimenez-Movilla, Gallego, Ballesta, Aviles and Coy2007).
Satoh et al. (Reference Satoh, Abe, Sendai, Iwata and Hoshi1995) deduced that mucin-type oligosaccharides with terminally exposed sialic acid residues could exist on BOGP, as affinity for WGA was high. The effect of BOGP on sperm viability and motility was abrogated by pretreatment with sialidase indicating that its sialylation was required for its bioactivity (Satoh et al., Reference Satoh, Abe, Sendai, Iwata and Hoshi1995). BOGP binding to sperm was later localised to the tail and the posterior region of the head (Abe et al., Reference Abe, Sendai, Satoh and Hoshi1995). BOGP may interact with specific receptors on the sperm in these regions. However, to the best of our knowledge, these have not been investigated.
The prospect of a functional relationship between bovine sperm and a sialoglycoprotein in the FRT is intriguing in the context of cervical mucus penetration. Potential interactions between these putative BOGP binding sites and bovine cervical mucin may be functionally relevant. As suggested by Tollner et al. (Reference Tollner, Yudin, Treece, Overstreet and Cherr2008b), negatively charged sialoglycoproteins on the surface of sperm may be required for cervical mucus penetration. However, release of this glycoprotein occurs during capacitation and this could expose binding sites required for other functionally relevant interactions at the appropriate time and location. This is a plausible scenario for bovines given that sialic acid recognition seems to be important for BOGP binding, and is also involved in ZP binding (Velasquez et al., Reference Velasquez, Canovas, Barajas, Marcos, Jimenez-Movilla, Gallego, Ballesta, Aviles and Coy2007). Sperm must still negotiate a mucin-rich environment in the oviduct before reaching these sites. Given the structural diversity of different forms of sialic acid (Cohen and Varki, Reference Cohen and Varki2010), and the number of sperm-sialic acid interactions characterised for a range of species (DeMott et al., Reference DeMott, Lefebvre and Suarez1995; Cortes et al., Reference Cortes, Orihuela, Zuniga, Velasquez and Croxatto2004; Pang et al., Reference Pang, Chiu, Lee, Chang, Panico, Morris, Haslam, Khoo, Clark, Yeung and Dell2011; Kadirvel et al., Reference Kadirvel, Machado, Korneli, Collins, Miller, Bess, Aoki, Tiemeyer, Bovin and Miller2012), such interactions are likely to be functionally sequenced through topographical segregation along the natural migratory path of sperm, and strongly regulated. Indeed, the high degree of sialylation and surface negative charge of sperm before capacitation may function not only to facilitate mucus penetration through electronegative repulsion but may also prevent premature, specific interactions between sperm and mucins in the FRT.
This complex link between sperm and mucins or other sialoglycoproteins in the FRT is fascinating and we are only beginning to fully understand its influence over sperm’s potential to successfully fertilise an oocyte. Unveiling the intricacies of this relationship could lead to insights that drive improvements in sperm diluent design.
Conclusion
Many factors have been identified that contribute to the maintenance of sperm viability and enhancement of sperm fertility in the bovine male and FRT. These have the potential to be exploited in bovine semen extenders to overcome current issues in bovine AI relating to sperm quality and lifespan. Increased understanding of functionally relevant changes that sperm undergo at different developmental and migratory stages has arisen from research into the interaction between sperm and their different microenvironments. This knowledge has great potential to permit the design of extenders that prevent the premature capacitation of sperm and enhance the effectiveness of AI. Research into the interaction of bovine sperm with cervical mucus could lead to innovative diluents through insights gained into the regulation of sperm transit and sperm physiology. The environments experienced by sperm throughout their journey all influence the likelihood of successful fertilisation and greater understanding of the interactions between sperm and these microenvironments is helping to create a bright picture for the future of bovine semen diluents.
Acknowledgements
J.A. McGetrick is funded by the Irish Research Council (IRC) under an EMBARK scholarship. The contribution of S.D. Carrington to this paper was supported by Science Foundation Ireland under 07/SRC/B1156: The Reproductive Biology Research Cluster.
