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Characterisation of the stallion sperm proteome
Journal of Equine Veterinary Science 34 (2014) 35
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Characterisation of the stallion sperm proteome A. Swegen*, R.J. Aitken University of Newcastle, Priority Research Centre for Reproductive Biology, Callaghan, NSW, Australia
1. Introduction The quest for improved understanding of stallion fertility and its biomarkers has resulted in the identification of a handful of functional sperm proteins in recent years [15]; however, the greater part of the equine sperm proteome has not been previously characterised. This represents a valuable yet untapped resource in light of the recent characterisation of the mouse, rat, and human sperm proteomes, which documented w1000 non-redundant gene products present in the sperm of each species [5] and provided new insights into the specialised processes that regulate spermatozoa. Such information bears particular pertinence to spermatozoa when one considers that these cells possess no capacity for transcription or translation of genes, making genomic approaches far less useful in the investigation of the significant structural and functional changes experienced by sperm in their lifetime. In this context, mass spectrometry and bioinformatics were employed to document the proteome of equine spermatozoa, revealing a large number of gene products and providing a solid foundation for further in-depth investigation of the molecular mechanisms that regulate sperm function.
2. Materials and methods Spermatozoa were isolated from undiluted, gel-free semen, collected from fertile stallions (n¼2), by discontinuous Percoll gradient and washing in protein-free media. Soluble protein fraction was obtained by addition of lysis buffer, then reduced and alkylated before methanol/chloroform precipitation. Samples were fractionated by 1D SDS gel electrophoresis and sectioning of gel lanes. After trypsin digestion, peptides were extracted, and analysed by liquid chromatography and tandem mass spectrometry. * Presenting author
Peptide sequence data obtained were used to search protein and gene sequence databases to acquire protein IDs and respective encoding genes. Interrogation of DAVID Bioinformatics database allowed investigation of gene ontology of the detected peptides with respect to inferred cellular localisation, molecular function and biological processes pertaining to each identified protein.
3. Results Mass spectrometry and extensive bioinformatics analysis allowed detection of 4262 peptides leading to identification of 942 proteins present in stallion spermatozoa. These were mapped to 810 gene symbols that could then be used to investigate the potential functional and location characteristics attributable to each gene product. Gene ontology analysis for inferred cellular localisation indicated that 90.7% of annotatable proteins (555 of 612) are intracellular, with 26.4% of the proteins localised to mitochondria. This is consistent with analyses of rat, mouse and human sperm proteomes, where mitochondria are the most highly represented intracellular organelle. Molecular function analysis indicated that the vast majority of proteins (538 of 623 annotated) participate in various binding roles. Catalytic activity was attributed to 56.5% of annotated proteins (352 of 623). Antioxidant activity and roles in electron transport were attributed to 1% and 4.2% of proteins, respectively. Categorization according to biological process indicated metabolism as the dominant process, with 64.8% of annotated proteins (333 of 514) involved in primary metabolic processes. A category of significant interest is that of proteins involved in response to chemical stimuli, highlighting 67 proteins as potential players in the interactions of sperm with the extracellular environment. Among these are a number of receptor/signalling proteins, chaperonins, phosphorylation candidates and proposed decapacitation factor proteins; selected proteins of interest are listed in Table 1.
36
Abstracts / Journal of Equine Veterinary Science 34 (2014) 36
Table 1 A selection of proteins and corresponding genes identified in stallion spermatozoa chaperonins
chaperonincontaining complex/TRiC
heat shock protein
Receptor proteins
receptor
associated proteins
Candidate decapacitation factor proteins
t-complex 1
TCP1
chaperonin containing TCP1, subunit 2 (beta) chaperonin containing TCP1, subunit 3 (gamma) chaperonin containing TCP1, subunit 4 (delta) chaperonin containing TCP1, subunit 5 (epsilon) chaperonin containing TCP1, subunit 6A (zeta 1) chaperonin containing TCP1, subunit 6B (zeta 2) chaperonin containing TCP1, subunit 7 (eta) chaperonin containing TCP1, subunit 8 (theta) heat shock 10kDa protein 1 (chaperonin 10) heat shock 60kDa protein 1 (chaperonin) heat shock protein, alpha-crystallin-related, B9 stress-70 protein, mitochondrial heat shock-related 70 kDa protein 2 heat shock cognate 71 kDa protein heat shock 70kDa protein 5 heat shock 70kDa protein 4-like heat shock 70kDa protein 1-like Heat shock protein HSP 90-alpha G protein-coupled receptor, family C, group 5, member A
CCT2 CCT3 CCT4 CCT5 CCT6A CCT6B CCT7 CCT8 HSPE1 HSPD1 HSPB9 HSPA9 HSPA2 HSPA8 HSPA5 HSPA4L HSPA1L HSP90AA1 GPRC5A
hyaluronan-mediated motility receptor (RHAMM) parathyroid hormone 2 receptor peroxisome proliferator-activated receptor g protein tyrosine phosphatase, receptor type, G protein tyrosine phosphatase, receptor type, T olfactory receptor, family 5, subfamily W, member 2 adrenergic B receptor kinase 2 low density lipoprotein receptor-related protein associated protein 1 regulator of G-protein signaling 22 transforming growth factor B associated protein 1 phospholipase C, zeta 1
HMMR PTH2R PPARG PTPRG PTPRT OR5W2 ADRBK2 LRPAP1 RGS22 TRAP1 PLCZ1
aspartate aminotransferase phosphatidiethanolamine binding protein 1 phosphatidyiethanolamine-binding protein 4 cysteine-rich secretory protein-2
GOT2 PEBP1 PEBP4 CRISP-2
4. Discussion A large number of the identified proteins are involved in housekeeping and cell metabolism functions; interspersed among these, however, are several groups of proteins that stand out with regard to their potential roles in the specialised functions of spermatozoa. Receptor proteins are frequently responsible for the interaction of spermatozoa with the extracellular environment and play key roles in maturation, transport and capacitation. Sperm surface remodelling is influenced by secretions encountered in the male and female reproductive tract [7] and further clarification of specific receptor roles in such interactions will shed light on the acquisition of functional competence by equine spermatozoa. While presence of the identified G-protein-coupled receptor GPRC5A is regulated by retinoic acid, its exact biological function is uncertain. The receptor has been shown to be involved in cAMP signaling and possibly regulation of
apoptosis [8] and thus may have important implications for survival and capacitation of equine sperm. Olfactory receptors have long been known to be present on spermatozoa of various species [9], with roles in chemotaxis and hypermotility [10, 11]; however the olfactory receptor identified in this analysis (OR2W5) has not been previously associated with spermatozoa. Functional characterisation of sperm OR2W5 may reveal novel liganddriven mechanisms for equine sperm transport and hyperactivation. The effects of peroxisome proliferator-activated receptor gamma (PPARg) ligands on sperm function have been investigated recently in boar and human, providing evidence of the receptor’s involvement in regulation of sperm metabolism, cholesterol efflux and tyrosine phosphorylation [12, 13]. The PPARg mechanism is also likely to be responsible for prostaglandin-induced enhancement of sperm motility and acrosome reaction, given that prostaglandins function as natural PPARg ligands and successfully
Abstracts / Journal of Equine Veterinary Science 34 (2014) 37
produced such effects in vitro [12]. Furthermore, the PPARg receptor-ligand system may provide a link between capacitation signalling and regulation of energy metabolism to explain the elevated metabolic state seen in capacitated spermatozoa. Other proteins of interest identified include the full set of subunits that constitute the chaperonin containing complex, which has demonstrated roles in capacitationassociated sperm surface remodelling and zona pellucida adhesion in the mouse [14], suggesting that these mechanisms may be conserved in horses. Analysis of the stallion sperm proteome has allowed identification of several hundred gene products, with bioinformatics yielding inferences about their functions and subcellular location. While these 900+ proteins are unlikely to constitute the entire proteome contained within equine spermatozoa, they will serve as a solid foundation for new insights into processes such as maturation, transport, capacitation and fertilization, and allow greater understanding of the species-specific features of these complex cells. References [1] Gamboa S, Ramalho-Santos J. SNA-FREE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility. Theriogenology 2005;64:275–91. [2] Gonzalez-Fernandez L, Macias-Garcia B, Loux SC, Varner DD, Hinrichs K. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm. Biology of Reproduction 2013;88:138. [3] Meyers SA, Rosenberger AE. A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
37
sperm and is associated with spermatozoal function. Biology of Reproduction 1999;61:444–51. Novak S, Smith TA, Paradis F, Burwash L, Dyck MK, Foxcroft GR, et al. Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. Theriogenology 2010;74:956–67. Ramalho-Santos J, Gamboa S. Snare proteins are present on the acrosome of equine sperm: Implications for stallion fertility. Biology of Reproduction 2003;68:175. Baker MA, Aitken RJ. Proteomic insights into spermatozoa: critiques, comments and concerns. Expert Review of Proteomics 2009;6:691– 705. Brewis IA, Gadella BM. Sperm surface proteomics: from protein lists to biological function. Molecular Human Reproduction 2010;16:68– 79. Hirano M, Zang LQ, Oka T, Ito Y, Shimada Y, Nishimura Y, et al. Novel reciprocal regulation of cAMP signaling and apoptosis by orphan Gprotein-coupled receptor GPRC5A gene expression. Biochemical and Biophysical Research Communications 2006;351:185–91. Conti M, Livera G, Horner K, Xie F, Jaiswal B, Storm D. Olfactory receptor signaling pathways in sperm. Biology of Reproduction 2003;68:89. Fukuda N, Yomogida K, Okabe M, Touhara K. Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility. Journal of Cell Science 2004;117:5835–45. Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Nauhaus EM, et al. Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis. Journal of Biological Chemistry 2004;279:40194–203. Aquila S, Bonofiglio D, Gentile M, Middea E, Gabriele S, Belmonte M, et al. Peroxisome proliferator-activated receptor (PPAR)gamma is expressed by human spermatozoa: Its potential role on the sperm physiology. Journal of Cellular Physiology 2006;209:977–86. Santoro M, Guido C, De Amicis F, Sisci D, Vizza D, Gervasi S, et al. Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPAR gamma). Journal of Experimental Biology 2013;216:1085–92. Dun MD, Smith ND, Baker MA, Lin M, Aitken RJ, Nixon B. The Chaperonin Containing TCP1 Complex (CCT/TRiC) Is Involved in Mediating Sperm-Oocyte Interaction. Journal of Biological Chemistry 2011;286:36875–87.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Characterisation of the stallion sperm proteome A. Swegen*, R.J. Aitken University of Newcastle, Priority Research Centre for Reproductive Biology, Callaghan, NSW, Australia
1. Introduction The quest for improved understanding of stallion fertility and its biomarkers has resulted in the identification of a handful of functional sperm proteins in recent years [15]; however, the greater part of the equine sperm proteome has not been previously characterised. This represents a valuable yet untapped resource in light of the recent characterisation of the mouse, rat, and human sperm proteomes, which documented w1000 non-redundant gene products present in the sperm of each species [5] and provided new insights into the specialised processes that regulate spermatozoa. Such information bears particular pertinence to spermatozoa when one considers that these cells possess no capacity for transcription or translation of genes, making genomic approaches far less useful in the investigation of the significant structural and functional changes experienced by sperm in their lifetime. In this context, mass spectrometry and bioinformatics were employed to document the proteome of equine spermatozoa, revealing a large number of gene products and providing a solid foundation for further in-depth investigation of the molecular mechanisms that regulate sperm function.
2. Materials and methods Spermatozoa were isolated from undiluted, gel-free semen, collected from fertile stallions (n¼2), by discontinuous Percoll gradient and washing in protein-free media. Soluble protein fraction was obtained by addition of lysis buffer, then reduced and alkylated before methanol/chloroform precipitation. Samples were fractionated by 1D SDS gel electrophoresis and sectioning of gel lanes. After trypsin digestion, peptides were extracted, and analysed by liquid chromatography and tandem mass spectrometry. * Presenting author
Peptide sequence data obtained were used to search protein and gene sequence databases to acquire protein IDs and respective encoding genes. Interrogation of DAVID Bioinformatics database allowed investigation of gene ontology of the detected peptides with respect to inferred cellular localisation, molecular function and biological processes pertaining to each identified protein.
3. Results Mass spectrometry and extensive bioinformatics analysis allowed detection of 4262 peptides leading to identification of 942 proteins present in stallion spermatozoa. These were mapped to 810 gene symbols that could then be used to investigate the potential functional and location characteristics attributable to each gene product. Gene ontology analysis for inferred cellular localisation indicated that 90.7% of annotatable proteins (555 of 612) are intracellular, with 26.4% of the proteins localised to mitochondria. This is consistent with analyses of rat, mouse and human sperm proteomes, where mitochondria are the most highly represented intracellular organelle. Molecular function analysis indicated that the vast majority of proteins (538 of 623 annotated) participate in various binding roles. Catalytic activity was attributed to 56.5% of annotated proteins (352 of 623). Antioxidant activity and roles in electron transport were attributed to 1% and 4.2% of proteins, respectively. Categorization according to biological process indicated metabolism as the dominant process, with 64.8% of annotated proteins (333 of 514) involved in primary metabolic processes. A category of significant interest is that of proteins involved in response to chemical stimuli, highlighting 67 proteins as potential players in the interactions of sperm with the extracellular environment. Among these are a number of receptor/signalling proteins, chaperonins, phosphorylation candidates and proposed decapacitation factor proteins; selected proteins of interest are listed in Table 1.
36
Abstracts / Journal of Equine Veterinary Science 34 (2014) 36
Table 1 A selection of proteins and corresponding genes identified in stallion spermatozoa chaperonins
chaperonincontaining complex/TRiC
heat shock protein
Receptor proteins
receptor
associated proteins
Candidate decapacitation factor proteins
t-complex 1
TCP1
chaperonin containing TCP1, subunit 2 (beta) chaperonin containing TCP1, subunit 3 (gamma) chaperonin containing TCP1, subunit 4 (delta) chaperonin containing TCP1, subunit 5 (epsilon) chaperonin containing TCP1, subunit 6A (zeta 1) chaperonin containing TCP1, subunit 6B (zeta 2) chaperonin containing TCP1, subunit 7 (eta) chaperonin containing TCP1, subunit 8 (theta) heat shock 10kDa protein 1 (chaperonin 10) heat shock 60kDa protein 1 (chaperonin) heat shock protein, alpha-crystallin-related, B9 stress-70 protein, mitochondrial heat shock-related 70 kDa protein 2 heat shock cognate 71 kDa protein heat shock 70kDa protein 5 heat shock 70kDa protein 4-like heat shock 70kDa protein 1-like Heat shock protein HSP 90-alpha G protein-coupled receptor, family C, group 5, member A
CCT2 CCT3 CCT4 CCT5 CCT6A CCT6B CCT7 CCT8 HSPE1 HSPD1 HSPB9 HSPA9 HSPA2 HSPA8 HSPA5 HSPA4L HSPA1L HSP90AA1 GPRC5A
hyaluronan-mediated motility receptor (RHAMM) parathyroid hormone 2 receptor peroxisome proliferator-activated receptor g protein tyrosine phosphatase, receptor type, G protein tyrosine phosphatase, receptor type, T olfactory receptor, family 5, subfamily W, member 2 adrenergic B receptor kinase 2 low density lipoprotein receptor-related protein associated protein 1 regulator of G-protein signaling 22 transforming growth factor B associated protein 1 phospholipase C, zeta 1
HMMR PTH2R PPARG PTPRG PTPRT OR5W2 ADRBK2 LRPAP1 RGS22 TRAP1 PLCZ1
aspartate aminotransferase phosphatidiethanolamine binding protein 1 phosphatidyiethanolamine-binding protein 4 cysteine-rich secretory protein-2
GOT2 PEBP1 PEBP4 CRISP-2
4. Discussion A large number of the identified proteins are involved in housekeeping and cell metabolism functions; interspersed among these, however, are several groups of proteins that stand out with regard to their potential roles in the specialised functions of spermatozoa. Receptor proteins are frequently responsible for the interaction of spermatozoa with the extracellular environment and play key roles in maturation, transport and capacitation. Sperm surface remodelling is influenced by secretions encountered in the male and female reproductive tract [7] and further clarification of specific receptor roles in such interactions will shed light on the acquisition of functional competence by equine spermatozoa. While presence of the identified G-protein-coupled receptor GPRC5A is regulated by retinoic acid, its exact biological function is uncertain. The receptor has been shown to be involved in cAMP signaling and possibly regulation of
apoptosis [8] and thus may have important implications for survival and capacitation of equine sperm. Olfactory receptors have long been known to be present on spermatozoa of various species [9], with roles in chemotaxis and hypermotility [10, 11]; however the olfactory receptor identified in this analysis (OR2W5) has not been previously associated with spermatozoa. Functional characterisation of sperm OR2W5 may reveal novel liganddriven mechanisms for equine sperm transport and hyperactivation. The effects of peroxisome proliferator-activated receptor gamma (PPARg) ligands on sperm function have been investigated recently in boar and human, providing evidence of the receptor’s involvement in regulation of sperm metabolism, cholesterol efflux and tyrosine phosphorylation [12, 13]. The PPARg mechanism is also likely to be responsible for prostaglandin-induced enhancement of sperm motility and acrosome reaction, given that prostaglandins function as natural PPARg ligands and successfully
Abstracts / Journal of Equine Veterinary Science 34 (2014) 37
produced such effects in vitro [12]. Furthermore, the PPARg receptor-ligand system may provide a link between capacitation signalling and regulation of energy metabolism to explain the elevated metabolic state seen in capacitated spermatozoa. Other proteins of interest identified include the full set of subunits that constitute the chaperonin containing complex, which has demonstrated roles in capacitationassociated sperm surface remodelling and zona pellucida adhesion in the mouse [14], suggesting that these mechanisms may be conserved in horses. Analysis of the stallion sperm proteome has allowed identification of several hundred gene products, with bioinformatics yielding inferences about their functions and subcellular location. While these 900+ proteins are unlikely to constitute the entire proteome contained within equine spermatozoa, they will serve as a solid foundation for new insights into processes such as maturation, transport, capacitation and fertilization, and allow greater understanding of the species-specific features of these complex cells. References [1] Gamboa S, Ramalho-Santos J. SNA-FREE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility. Theriogenology 2005;64:275–91. [2] Gonzalez-Fernandez L, Macias-Garcia B, Loux SC, Varner DD, Hinrichs K. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm. Biology of Reproduction 2013;88:138. [3] Meyers SA, Rosenberger AE. A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
37
sperm and is associated with spermatozoal function. Biology of Reproduction 1999;61:444–51. Novak S, Smith TA, Paradis F, Burwash L, Dyck MK, Foxcroft GR, et al. Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions. Theriogenology 2010;74:956–67. Ramalho-Santos J, Gamboa S. Snare proteins are present on the acrosome of equine sperm: Implications for stallion fertility. Biology of Reproduction 2003;68:175. Baker MA, Aitken RJ. Proteomic insights into spermatozoa: critiques, comments and concerns. Expert Review of Proteomics 2009;6:691– 705. Brewis IA, Gadella BM. Sperm surface proteomics: from protein lists to biological function. Molecular Human Reproduction 2010;16:68– 79. Hirano M, Zang LQ, Oka T, Ito Y, Shimada Y, Nishimura Y, et al. Novel reciprocal regulation of cAMP signaling and apoptosis by orphan Gprotein-coupled receptor GPRC5A gene expression. Biochemical and Biophysical Research Communications 2006;351:185–91. Conti M, Livera G, Horner K, Xie F, Jaiswal B, Storm D. Olfactory receptor signaling pathways in sperm. Biology of Reproduction 2003;68:89. Fukuda N, Yomogida K, Okabe M, Touhara K. Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility. Journal of Cell Science 2004;117:5835–45. Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Nauhaus EM, et al. Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis. Journal of Biological Chemistry 2004;279:40194–203. Aquila S, Bonofiglio D, Gentile M, Middea E, Gabriele S, Belmonte M, et al. Peroxisome proliferator-activated receptor (PPAR)gamma is expressed by human spermatozoa: Its potential role on the sperm physiology. Journal of Cellular Physiology 2006;209:977–86. Santoro M, Guido C, De Amicis F, Sisci D, Vizza D, Gervasi S, et al. Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPAR gamma). Journal of Experimental Biology 2013;216:1085–92. Dun MD, Smith ND, Baker MA, Lin M, Aitken RJ, Nixon B. The Chaperonin Containing TCP1 Complex (CCT/TRiC) Is Involved in Mediating Sperm-Oocyte Interaction. Journal of Biological Chemistry 2011;286:36875–87.