Emerging Roles of Seminal Plasma in Sperm Function

Seminal plasma is comprised of the extracellular sperm milieu from spermatogenesis in the testes, through to maturation in the epididymis, as well as secretions from the accessory sex glands. Consisting primarily of proteins, this substance has a multitude of functions in mammalian reproduction. Seminal plasma influences sperm physiology, success of semen freezing, migration of spermatozoa through the cervix (in sheep) and induction of ovulation (in camelids). Global proteomic studies have begun to reveal the complexity of the seminal plasma proteome and the variation that exists between species and individuals. More targeted studies have focused on the relationship between protein composition and functional phenotypes. Such research is highlighting proteins of interest for further investigation of their physiological effect on sperm. It is hoped that such information will not only improve our basic understanding of sperm function but underlie artificial breeding program advancements. 1.0 Introduction During ejaculation, spermatozoa from the tail of the epididymis mix with secretions from the accessory sex glands (prostate, vesicular, ampulla and bulbourethral glands). Known collectively as seminal plasma, these secretions were initially thought to act merely as a transport medium for spermatozoa, but have since been recognised as a substance with profound influence over sperm physiology and other reproductive processes [1]. Despite these advances, debate continues over the precise role of seminal plasma in sperm function and male fertility as its effects have often been contradictory and can vary between species, season of collection and individuals [2,3]. This has been particularly evident in the numerous studies which have investigated the use of seminal plasma supplementation to offset the deleterious effects of semen processing for cryopreservation or sex-sorting. This short review will briefly summarise the current knowledge on seminal plasma composition, function and variation and discuss the most recent proteomic investigations of this biological fluid. Particular emphasis will be placed on ruminant and camelid seminal plasma. 2.0 Biophysical characteristics of ruminant and camelid semen The ejaculates of the bull and ram are somewhat similar due to comparable accessory sex gland structure (large vesicular glands and small or disseminated prostate and bulbourethral glands). Ejaculates are of relatively low volume, but exhibit very high sperm concentration, most likely as an evolutionary adaptation to rapid vaginal semen deposition. Camelids also have low ejaculate volumes due to the small size of their Published in IVIS with the permission of the AAAA Close this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 94 accessory sex glands (small prostate and bulbourethral glands with vesicular glands completely absent), but have a much lower sperm concentration than ruminants as they also possess smaller testes [4]. As such, seminal plasma comprises approximately 85% of a camelid ejaculate [5] as opposed to roughly 70% in ruminants [6]. Camelid seminal plasma is also highly viscous, which complicates basic semen processing, such as dilution, and has thus far prevented application of routine techniques such as semen cryopreservation. 3.0 Biochemical composition of seminal plasma Seminal plasma is a complex secretion composed of inorganic ions, sugars, organic salts, lipids, enzymes, prostaglandins, proteins and various other factors produced by the testes, epididymides and accessory sex glands of the male [1,6]. Of these components, the most significant by weight and by theorised function is protein. As such, it is the composition, variation and effect of seminal plasma protein(s) which are of central interest. Proteins are highly specific molecules whose physiological role is related both to their fold and assembly as well as the environment in which they exert their function. The seminal plasma proteins of most species are generally divided into three distinct families: spermadhesins, proteins with fibronectin Type-2 domains (the BSPs or Binder of Sperm Proteins) and Cysteine Rich Secretory Proteins (CRISPs). The majority of proteins in seminal plasma are derived from the accessory sex glands. In the ram, spermadhesins are secreted from the seminal vesicles and account for the vast majority of total seminal plasma protein present by weight [7]. They exhibit a diverse range of ligand binding abilities to substances such as carbohydrates, sulfated glycosaminoglycans, phospholipids and protease inhibitors [8] and are thought to be involved in sperm capacitation, more specifically the prevention of premature capacitation [7,8]. The BSP family of proteins in the ram (also known as RSPs), are characterised by a fibronectin Type-2 domains and are also produced by the seminal vesicles. Ram seminal plasma contains four BSP proteins termed, RSP-14, RSP-16, RSP-22 and RSP-24, which account for 20% of total ram seminal plasma proteins by weight [7]. Thus, ram seminal plasma shows a highly unbalanced composition due to the dominance of BSP proteins and spermadhesins. In fact, a recent investigation of seminal plasma by GeLC MS/MS found that 5% of proteins in ram seminal plasma (39 out of total 727 identified) accounted for 50 % of total spectra (Druart et al., 2014, unpublished observations). Until recently, virtually nothing was known about the protein composition of camelid seminal plasma aside from its SDS-PAGE protein profile but it was thought to be involved in the induction of ovulation [9]. The application of global proteomics [i.e. tandem mass spectrometry (LC-MS/MS)] to camelid species has begun to shed light on their seminal plasma proteomes. A preliminary list of 89 proteins has been identified in alpaca seminal plasma [4] and 19 proteins in camel seminal plasma [10] using various separation methods combined with LC-MS/MS. These two significant advances are evidence of a wider global push over the last decade to apply mass spectrometry to reproductive research. Indeed LC-MS/MS has been used to identify over 2000 proteins in human seminal plasma [13-15], as well as new proteins in bull [16,17] and ram [18] seminal plasma. We have Published in IVIS with the permission of the AAAA Close this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 95 recently applied this tool in the first major cross species comparison of seminal plasma proteins identifying 323 proteins in a total of 7 separate species (including the bull, ram, alpaca and camel) using geLC-MS/MS and 2DLC-MS/MS [19]. Such comparison between the species allows identification of proteins essential to reproduction in all species (see Table 1) as well as species specific phenomena. For example, beta nerve growth factor, protein is highly abundant in alpaca, llama and camel seminal plasma and is most likely responsible for induction of ovulation in all camelids. However, smaller amounts were also detected in the bull, ram, and horse. It remains to be seen whether this protein has similar effects on the hypothalamicpituitary-gonadal axis of other induced ovulators or even spontaneous ovulators such as sheep and cattle. No doubt there is added interest on the potential use of this protein in controlled breeding of species with long follicular or oestrus periods such as the sow and mare. Mucin5b was also identified as highly abundant in alpaca seminal plasma and is thought to be responsible for the high viscosity of alpaca semen [4]. 4.0 Function and effect of seminal plasma In simplest terms, seminal plasma acts as a transport medium for spermatozoa through the male and the initial parts of the female reproductive tract. Inorganic components regulate osmolarity of the ejaculate, simple sugars provide an energy source, buffers protect against pH changes and antioxidant systems protect against the deleterious effects of reactive oxygen species. Combined, these factors act to keep spermatozoa within the ejaculate viable and functional as they are deposited in the vagina or uterus and begin their passage to the site of fertilisation. The proteaceous component of seminal plasma has been shown to influence fertilisation associated events such as maturation [20,21], capacitation [22,23] and interaction with the oviduct [24,25] and oocyte [8]. Extensive work in the bull has shown BSP proteins bind to choline phospholipids of the sperm membrane in a rapid (half time less than one second) and specific manner [26,27]. During natural mating this association acts as a protective glycoprotein coat which stabilises the sperm membrane during transit through the reproductive tract. Upon reaching the oviduct, BSP proteins may also assist in the establishment of a bovine oviductal sperm reservoir as in vitro supplementation of BSP proteins to epididymal spermatozoa enhances sperm-oviductal epithelial cell binding [24,25]. Finally, secondary messengers in the oviductal fluid, such as high-density lipoprotein and heparin-like glycosaminoglycans, also bind BSP proteins and are thought to coordinate the onset and progression of capacitation [28]. When presented in this textbook manner, the function of the major proteins of seminal plasma appears resolved, but closer examination of the literature reveals that considerable ambiguity remains. A large confounder is the analysis of protein function in vitro which typically involves co-incubation of the protein of interest with sperm for extended periods of time. This is quite distinct from the situation in vivo in which proteins from the accessory sex glands are only in brief contact with spermatozoa. In fact, under the artificial conditions involved in semen processing, BSPs are quite detrimental to bull spermatozoa. Extended association of bull spermatozoa and BSP proteins causes phospholipid and cholesterol efflux from the sperm membrane in a dose and time-dependent manner [29,30]. This destabilises the sperm membrane and renders Published in IVIS with the permission of the AAAA Close this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 96 it more susceptible to stress [31-34]. Fortunately, the common use of egg yolk and skim milk in cryoprotective diluents has unintentionally afforded some protection as the low density lipoproteins and casein micelles present in these diluents preferentially bind BSP proteins, reducing the number available to bind to the sperm membrane [31]. Such detailed work has not been completed in the ram but extensive investigation suggests ram seminal plasma is not detrimental to ram spermatozoa function in vitro despite its high concentration of BSP proteins. In sheep, seminal plasma supplementation protects sperm from handling induced damage associated with sex-sorting and freezing and has been noted to improve motility, viability, capacitation status, ability to penetrate cervical mucus in vitro and to survive an oxidative challenge [35-43]. Seminal plasma proteins have also been shown to prevent and/or reverse cold shock damage, improving the viability and heterogeneity (assessed by centrifugal counter current distribution) of the sperm sample and reducing protein tyrosine phosphorylation [44-47]. Interestingly, it is the BSPs products of ram seminal plasma that has been attributed to the protective effect during cold shock. Size exclusion fractionation of ram seminal plasma has shown the main protective component to be a 14 kDa Fn-2 protein (likely RSP-15) and a unidentified 20 kDa protein [48] with co-incubation of washed diluted spermatozoa partly preventing [46] or reverting [44] coldshock damage. The reasons underlying this divergent behaviour are numerous, and are the subject of an earlier review [3,49], but it shows that despite the ram and bull sharing common ancestry, similar mating strategies and seminal plasma profiles the influence of shared proteins families can still be quite divergent. Unfortunately improved ram sperm function in vitro from seminal plasma supplementation is yet to translate into consistently improved pregnancy outcomes [37,50,51], making it difficult to recommend the use of seminal plasma in semen processing protocols. However, recent research has at least shed light on the biological purpose of seminal plasma with conclusive evidence from in vivo fertility trials, and in utero cell imaging, that seminal plasma contributes to the migration of epididymal spermatozoa through the cervix and into the uterus (Rickard et al., 2014, unpublished observations). The idea that there is inter-male variation in seminal plasma composition which can be used as a phenotypic marker is alluring. Correlations between protein profiles and the fertilising ability [52,53] or freezability [54,55] of individual males have been made, but correlations are not evidence of direct effects on sperm function. An alternative is studies which demonstrate that seminal plasma from one male can alter the function of spermatozoa from another male.. Such studies have been completed in the stallion [56] and boar [57], but not in those species of direct relevance to this review. We have recently shown that ram spermatozoa from males with high freezing resilience is able to change the cryotolerance of spermatozoa sourced from males with low freezing resilience (and vice versa; Rickard et al., 2014, unpublished results). Proteomic identification of the differences between these two seminal plasma phenotypes is ongoing in an effort to elucidate the proteins responsible for the aforementioned stimulatory and inhibitory effects of seminal plasma. Finally, while the main effects of seminal plasma are no doubt related to sperm function, this substance can also play a significant role in female reproductive physiology. For example, exposure to seminal plasma elicits Published in IVIS with the permission of the AAAA Close this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 97 increased uterine blood flow and activation of immune complement and oedema, particularly in uterine depositors such as the horse and pig [58]. It is also postulated that hormones such as prostaglandin found within seminal plasma may aid contractions of the uterus and thus sperm transport following mating [1]. 5.0 Conclusion Evidence continues to build that seminal plasma proteins are key to the function and fertility of spermatozoa and that variation in its composition can greatly influence its effect. The use of modern proteomic techniques to characterise species specific seminal plasma proteomes is developing an understanding of the complex formation of this biological fluid. Considerable work ahead is required to elucidate function of the multitude of proteins which have been, and continue to be identified. Comparative proteomic studies of seminal plasma types that alter the ability of spermatozoa to survive sex-sorting or cryopreservation will lead the discovery of proteins which prove beneficial in an artificial breeding context. In time, it is hoped this research will boost the fertility of spermatozoa in artificial insemination programs across a number of species. 6.0 Conflict of interest declarationThe authors declare no conflicts of interest which may influence the work presented in this manuscript. References[1] Maxwell WMC, de Graaf SP, El-Hajj Ghaoui R, Evans G. 2007. Seminal plasma effects on sperm handling andfemale fertility. 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Relations between the fertilizing ability, motility and maturation processin the boar. Annals NY Acad Sci 438: 526-529.[21] Dacheux JL, Druart X, Fouchecourt S, Syntin P, Gatti JL, Okamura N, Dacheux F. 1998. Role of epididymalsecretory proteins in sperm maturation with particular reference to the boar. J Reprod Fertil 99-107.[22] Chang MC. 1957. A detrimental effect of seminal plasma on the fertilizing capacity of sperm. Nature 179:258-259.[23] Manjunath P, Thérien I. 2002. Role of seminal plasma phospholipids-binding proteins in sperm membranelipid modification that occurs during capacitation. J Reprod Immun. 58 109-119.[24] Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS. 2006. Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biol Reprod. 75: 501-507.[25] Gwathmey TM, Ignotz GG, Suarez SS. 2003. PDC-109 (BSP-A1/A2) promotes bull sperm binding tooviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod. 69:809-815.[26] Desnoyers L, Manjunath P. 1992. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J Biol Chem. 267: 10149-10155.Published in IVIS with the permission of the AAAAClose this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 99[27] Muller P, Erlemann KR, Muller K, Calvete JJ, Topfer-Petersen E, Marienfeld K, Herrmann A. 1998. Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipidvesicles. Europ Biophysics J Biophysics Leters 27: 33-41.[28] Lane ME, Therien I, Moreau R, Manjunath P. 1999. Heparin and high density lipoprotein mediate bovinesperm capacitation by different mechanisms. Biol Reprod. 60: 169-175.[29] Therien I, Moreau R, Manjunath P. 1999. Bovine seminal plasma phospholipid-binding proteins stimulatephospholipid efflux from epididymal sperm. Biol Reprod. 61: 590-598.[30] Therien I, Moreau R, Manjunath P. 1998. Major proteins of bovine seminal plasma and high-densitylipoprotein induce cholesterol efflux from epididymal sperm. Biol Reprod. 59: 768-776.[31] Manjunath P, Nauc V, Bergeron A, Menard M. 2002. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk. Biol Reprod. 67: 1250-1258.[32] Bergeron A, Manjunath P. 2006. New insights towards understanding the mechanisms of spermprotection by egg yolk and milk. Molec Reprod Devel. 73: 1338-1344.[33] Therien I, Soubeyrand S, Manjunath P. 1997. Major proteins of bovine seminal plasma modulate spermcapacitation by high-density lipoprotein. Biol Reprod. 57: 1080-1088.[34] Manjunath P, Therien I. 2002. Role of seminal plasma phospholipid-binding proteins in sperm membranelipid modification that occurs during capacitation. J Reprod Immun. 53: 109-119.[35] Graham JK. 1994. Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa ofthe ram and bull during the cryopreservation process. Theriogenology 41: 1151-1162.[36] El-Hajj Ghaoui R, Thomson PC, Leahy T, Evans G, Maxwell WMC. 2007. Autologous whole ram seminalplasma and its vesicle-free fraction improve motility characteristics and membrane status but not in vivofertility of frozen-thawed ram spermatozoa. Reprod Domest Anim 42: 541-549.[37] Maxwell WMC, Evans G, Mortimer ST, Gillan L, Gellatly ES, McPhie CA. 1999. Normal fertility in ewes aftercervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reprod FertilDevel 11: 123-126.[38] El-Hajj Ghaoui R, Gillan L, Thomson PC, Evans G, Maxwell WMC. 2007. Effect of seminal plasma fractionsfrom entire and vasectomized rams on the motility characteristics, membrane status, and in vitro fertility ofram spermatozoa. J Androl. 28: 109-122.[39] Leahy T, Marti JI, Evans G, Maxwell WMC. 2010. Seasonal variation in the protective effect of seminalplasma on frozen-thawed ram spermatozoa. Anim Reprod Sci. 119: 147-153.[40] Leahy T, Marti JI, Evans G, Maxwell WMC. 2009. Seminal plasma proteins protect flow-sorted ramspermatozoa from freeze-thaw damage. Reprod Fertil Devel. 21: 571-578.[41] Leahy T, Celi P, Bathgate R, Evans G, Maxwell WMC, Marti JI. 2010. Flow-sorted ram spermatozoa arehighly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase. ReprodFerti Devel. 22: 1131-1140.[42] Catt SL, O'Brien JK, Maxwell WMC, Evans G. 1997. Assessment of ram and boar spermatozoa during cell-sorting by flow cytometry. Reprod Domest Anim. 32: 251-258.Published in IVIS with the permission of the AAAAClose this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 100[43] Maxwell WMC, Welch GR, Johnson LA. 1996. Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Devel. 8:1165-1178.[44] Barrios B, Perez-Pe R, Gallego M, Tato A, Osada J, Muino-Blanco T, Cebrian-Perez JA. 2000. Seminal plasmaproteins revert the cold-shock damage on ram sperm membrane. Biol Reprod. 63: 1531-1537.[45] Garcia-Lopez N, Ollero M, Cebrian-Perez JA, Muino-Blanco T. 1996. Reversion of thermic-shock effect onram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phasesystems. J Chroma B Biomed Appl. 680: 137-143.[46] Perez-Pe R, Cebrian-Perez JA, Muino-Blanco T. 2001. Semen plasma proteins prevent cold-shockmembrane damage to ram spermatozoa. Theriogenology 56: 425-434. [47] Perez-Pe R, Grasa P, Fernandez-Juan M, Peleato M, Cebrian-Perez J, Muino-Blanco T. 2002. Seminalplasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ramspermatozoa. Mol Reprod Devel. 61: 226-233.[48] Muino-Blanco T, Perez-Pe R, Cebrian-Perez JA. 2008. Seminal plasma proteins and sperm resistance tostress. Reprod Domest Anim. 43: 18-31.[49] Leahy T, de Graaf SP. 2012. Seminal plasma and its effect on ruminant spermatozoa during processing.Reprod Domest Anim. 47: 207-213.[50] O'Meara CM, Donovan A, Hanrahan JP, Duffy P, Fair S, Evans ACO, Lonergan P. 2007. Resuspending ramspermatozoa in seminal plasma after cryopreservation does not improve pregnancy rate in cervicallyinseminated ewes. Theriogenology 67: 1262-1268.[51] Leahy T, Evans G, Maxwell WMC, Marti JI. 2010. Seminal plasma proteins do not consistently improvefertility after cervical insemination of ewes with non-sorted or sex-sorted frozen-thawed ram spermatozoa.Reprod Fertil Devel. 22: 606-612.[52] Killian GJ, Chapman DA, Rogowski LA. 1993. Fertility associated proteins in Holstein bull seminal plasma .Biol Reprod. 49: 1202-1207.[53] Ollero M, Cebrian-Perez JA, Muino-Blanco T. 1997. Improvement of cryopreserved ram spermheterogeneity and viability by addition of seminal plasma. J Androl. 18: 732-739.[54] Jobim MIM, Oberst ER, Salbego CG, Souza DO, Wald VB, Tramontina F, Mattos RC. 2004. Two-dimensionalpolyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semenfreezability. Theriogenology 61: 255-266.[55] Corcini CD, Varela AS, Pigozzo R, Rambo G, Goularte KL, Calderam K, Leon PMM, Bongalhardo DC, Lucia T.2012. Pre-freezing and post-thawing quality of boar sperm for distinct portions of the ejaculate and as afunction of protein bands present in seminal plasma. Livestock Sci 145: 28-33.[56] Aurich JE, Kuhne A, Hoppe H, Aurich C. 1996. Seminal plasma affects membrane integrity and motility ofequine spermatozoa after cryopreservation. Theriogenology 46: 791-797.[57] Hernandez M, Roca J, Calvete JJ, Sanz L, Muino-Blanco T, Cebrian-Perez JA, Vazquez JM, Martinez EA.2007. Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars. J Androl. 28: 689-697.Published in IVIS with the permission of the AAAAClose this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 101[58] Troedsson MHT, Desvousges A, Alghamdi AS, Dahms B, Dow CA, Hayna J, Valesco R, Collahan PT, Macpherson ML, Pozor M, Buhi WC. 2005. Components in seminal plasma regulating sperm transport andelimination. Anim Reprod Sci. 89: 171-186.Published in IVIS with the permission of the AAAAClose this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia Proceedings, Association for Applied Animal Andrology, 9 Biennial Conference, August 2014 102Table 1. Seminal plasma proteins identified in at least 4 different species. The detection of a specific protein inthe seminal plasma of each species is denoted by a black box [modified from [19]]Published in IVIS with the permission of the AAAAClose this window to return to IVIS 9th Biennial Conference of the Association for Applied Animal Andrology, 2014 Newcastle, Australia