BACKGROUND
male infertility is a major problem for mammalian reproduction.
the nature of sub-fertility due to the male is as complex as that of the female  <cit> .
infertility due to male factor contributes approximately 40% of the infertility cases in humans.
for this reason it is very important to investigate the factors that affect male fertility.
here we used bovine spermatozoa to model human male fertility because cattle provide several advantages as a model for male factor infertility.
these include good breeding records fertility data records and progeny records.
in cattle breeding, artificial insemination , a common breeding technique, utilizes semen from genetically superior sires to inseminate cows.
in the united states more than ~70% of cows are bred by ai but only ~50% of these matings result in successful full term pregnancy  <cit> .
the underlying molecular events/mechanisms that determine the fertilizing potential of a semen sample are not well defined.
a thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder.
fertility traits of semen can be categorized as compensable or uncompensable  <cit> .
defects in compensable traits  can be overcome by increasing the number of spermatozoa per insemination  <cit> .
defects in uncompensable traits affect the function of spermatozoa during the later stages of fertilization and in embryonic development  <cit>  and as such cannot be compensated.
uncompensable traits include nuclear vacuoles  <cit> , morphological deficiencies that do not suppress movement  <cit> , defective chromatin structure  <cit> .
low fertility in bulls has an uncompensable component that includes reduced cleavage rate and delayed pronuclear formation following in vitro fertilization  <cit> .
currently available fertility assays assess the defects that affect functional competence of spermatozoa   <cit> , however these cannot definitively predict fertility.
at present, the molecular nature of sperm fertility defects or biomarkers for accurate fertility prediction is not known  <cit> .
spermatozoa are transcriptionally inactive so the only comprehensive method to understand the molecular functions in spermatozoa is via proteomics  <cit> .
published proteomic studies with bull spermatozoa described the sub-proteome of the sperm and functions of proteins from its surrounding cells.
accessory gland  proteins were shown to modulate important sperm functions after ejaculation and in the female reproductive tract such as capacitation, acrosome reaction, sperm-oocyte interaction, and sperm protection  <cit> .
it is known that fertile associated antigen , a heparin binding protein from seminal vesicles and prostate glands, binds to spermatozoa membrane and modulates heparin-sperm interactions that are indicative of fertility  <cit> .
two seminal plasma proteins such as, prostaglandin-d-synthetase and osteoponin were more abundant in the semen of high fertility bulls when compared to low fertility bulls  <cit> .
here we describe a comprehensive proteomic analysis of bull sperm using differential detergent fractionation  two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry .
we compared protein expression profiles of sperm from high and low fertility bulls to characterize the differences in fertility at the protein level.
our results show that expression of  <dig> and  <dig> proteins was specific to high and low fertility bull spermatozoa, respectively and  <dig> proteins were common to both.
differential expression of  <dig> proteins was significant between high and low fertility bull spermatozoa and these proteins are potential biomarkers for bovine male fertility.
biological systems utilize highly complex, interrelated metabolic and signaling pathways to function.
therefore, to identify signaling pathways involved in fertility, we carried out systems modeling of our proteomic datasets using gene ontology  and ingenuity pathway analysis .
we identified differences in the signaling pathways between high and low fertility bull spermatozoa and found that egf and pdgf signaling pathways were specific to high fertility.
RESULTS
proteome profiles of spermatozoa from high and low fertility bulls
we identified  <dig> and  <dig> proteins in high and low fertility group spermatozoa respectively .
among these  <dig>  were common to both groups and  <dig> and  <dig> proteins were unique to high and low fertility groups respectively .
only those proteins identified by at least three peptides were included in the analysis for differential expression and we identified  <dig> proteins as differentially-expressed between the high and low fertility spermatozoa.
compared to low fertility bull spermatozoa, expression of  <dig> proteins increased and there was a decrease in the expression of  <dig> proteins in high fertility spermatozoa .
only a small proportion of proteins identified in this study have been previously described ).
the majority of the identified proteins are 'predicted' .
we contributed to the annotation of the newly sequenced bovine genome by experimentally confirming the in vivo expression of  <dig>  electronically predicted proteins .
we also identified  <dig> % and  <dig> % 'hypothetical'  proteins specific to high fertility and low fertility spermatozoa respectively.
list of differentially expressed proteins in high fertility  group spermatozoa when compared to low fertility group.
spermatozoa in this table we provided the information about number of peptides, sequest cross correlation  score and p value for each protein in high and low fertility group spermatozoa respectively.
predicted and hypothetical proteins do not have any functional annotation associated with them and they represent ~80% of differentially expressed proteins between high and low fertility spermatozoa .
this poses a problem for meaningful biological modeling of our data without carrying out some functional annotation first.
therefore, we annotated all differentially expressed proteins in our data sets using agbase go resources.
membrane and nuclear proteins
membrane and nuclear proteins are fundamental for inter and intra cellular signaling and are thus fundamental for modeling cell-cell interactions.
sperm oocyte fusion is a key element for fertilization.
this process is facilitated by sperm surface proteins and leads to specific binding of the sperm surface-active component with the egg zona pellucida and, ultimately, sperm-egg fusion  <cit> .
to identify proteins from the sperm membrane and the nucleus which function in cell fusion, we focused on membrane and nuclear proteins identified in our datasets.
based on the go associations of known proteins,  <dig> %  are membrane proteins.
we also identified  <dig> nuclear proteins based on go associations.
biological process annotation of membrane proteins revealed that majority of membrane proteins involved in transport , cell communication  and metabolism .
we go annotated all differentially expressed proteins and applied the generic go slim  <cit>  to identify  <dig> functional super-categories represented in differentially expressed proteins in high fertility spermatozoa.
most go slim categories, including processes such as metabolism, cell communication and cell motility showed overall up regulation of protein expression in the high fertility group while transport proteins showed an overall down regulation in the high fertility group .
high fertility and low fertility sperm proteomes: molecular network and pathway analysis
protein identification from biological samples on a global scale is important.
however, there is a need to move beyond this level of analysis; instead of simply enumerating a list of proteins, the analysis needs to include their interactions as parts of complexes, pathways and biological networks.
to achieve this level of analysis with our high fertility and low fertility spermatozoa proteomic datasets we used ingenuity pathway analysis .
at ipa thresholds for significance,  <dig>  and  <dig> networks and  <dig>  and  <dig> functions/diseases were significantly represented in the proteomes of high fertility and low fertility spermatozoa respectively.
the top  <dig> functions/diseases , and the associated signaling pathways are shown in table  <dig> and table  <dig> for proteomes of high and low fertility groups respectively.
analysis of the top  <dig> functions revealed that functions like cellular movement, cell to cell signaling and interaction were identified only in the high fertility sperm proteome .
whereas, functions like cell death and reproductive system disease were identified only in the low fertility sperm proteome .
egf: epidermal growth factor; pdgf: platelet derived growth factor; igf1: insulin growth factor- <dig>
mapk: mitogen activated protein kinase; vegf: vascular endothelial growth factor; pten: phosphatase and tensin homolog deleted on chromosome ten.
compared to low fertility sperm proteome , the high fertility sperm proteome  had a 2-fold enrichment in signaling pathways.
however, the number of significant metabolic pathways represented was comparable between the low  and high  fertility spermatozoa.
epidermal growth factor  signaling was the most prominent signaling pathway specific to high fertility sperm .
egf signaling is known to promote proliferation, survival, and differentiation of a wide variety of mammalian cells  <cit> .
in addition to the egf signaling pathway, platelet derived growth factor  signaling, peroxisome proliferated activator receptor  signaling, interleukin - <dig> signaling, nf-kβ signaling, chemokine signaling, and insulin growth factor - <dig> signaling were identified only in high fertility spermatozoa.
in low the fertility group, cell cycle: g2/m dna damage check point regulation was the most significant pathway followed by integrin signaling.
proteins with significantly altered expression: molecular network and pathway analysis
systems analysis of global proteomes revealed that some signaling pathways are differentially represented between the high and low fertility group spermatozoa.
to further analyze these differentially expressed pathways, we carried out ipa analysis with just the  <dig> differentially expressed proteins.
in high fertility spermatozoa, expression of  <dig> proteins was increased when compared to low fertility spermatozoa.
ipa analysis identified three significant networks with scores of  <dig>   <dig>  and  <dig> respectively.
proteins identified in the top three networks are participants in egf signaling, pdgf signaling, oxidative phosophorylation, and pyruvate metabolism pathways.
expression of two proteins, atp synthase, h+ transporting, mitochondrial f <dig> complex , and cytochrome c oxidase subunit iii  involved in oxidative phosphorylation and casein kinase ii involved in egf signaling and pdgf signaling were higher in the high fertility spermatozoa compared to low fertility spermatozoa .
ipa also identified pyruvate metabolism as the most significant pathway in up regulated proteins of high fertility spermatozoa.
in the low fertility sperm proteome, expression of  <dig> proteins increased when compared to high fertility spermatozoa.
ipa analysis identified two significant networks in highly expressed proteins of low fertility sperm.
proteins identified in the top two significant networks are participants in integrin signaling and estrogen receptor signaling.
discussion
male fertility can be described as the success by spermatozoa to fertilize oocytes and of the resulting zygotes continue on through embryonic and fetal development until birth  <cit> .
in this study we used bovine spermatozoa to study fertility as it can serve as a model for understanding human male infertility and reproductive diseases.
studying bovine male fertility on its own merit has implications in agro-economics involving cattle industry worldwide.
a spermatozoon must reach the site of fertilization and be capacitated for successful fertilization to occur.
a subsequent step is the acrosome reaction characterized by fusion of a spermatozoon outer acrosomal membrane with overlying plasma membrane  <cit> .
the molecular mechanisms and signal transduction pathways mediating the processes of capacitation and acrosome reaction have been partially defined  <cit> .
bull sperm cytosolic fraction proteomic analysis showed enrichment for tyrosine kinases which are essential for phosphorylation of specific sperm proteins during capacitation  <cit> .
the abundance of a variety of proteins from cells surrounding the sperm has been proposed to indicate male fertility  <cit> .
most of the studies used 2-dimensional electrophoresis  for isolation and identification of sperm proteins  <cit> .
to our knowledge this is the first comprehensive non-electrophoretic proteomic study of bull sperm proteome.
the aim of our study was to identify proteins that were differentially expressed between high and low fertility bull spermatozoa and interrelated metabolic and signaling pathways that have a role in fertility.
we identified  <dig> proteins as differentially expressed in between the high and low fertility sperm even though  <dig> proteins were common to both groups and about  <dig> were unique to each.
the reasons for this apparent discrepancy are that we took a conservative approach to the statistical analysis: only proteins identified by at least three peptides were included in the analysis for differential expression and the statistical method used in protquant is very conservative.
protquant specifically address the issue of "missing" mass spectra that occurs in all 2-d lc ms <dig> -based expression proteomics methods.
no other published method  addresses this issue.
missing mass spectra are due to the inherent limitations of the mass spectrometers, the probabilistic nature of sampling and the cutoffs used to determine "true" assignments of peptides to mass spectra  <cit> .
protquant is highly conservative method which is based on sum of xcorr method itself increases the specificity of spectral counting and reduce the type i errors of differential expression.
regardless, proteins were analyzed from each of three of the areas represented in figure  <dig> and differentially-expressed proteins occurred in all three .
from proteome profiles of specific cells or tissues, one acquires large datasets that are inherently complex.
as a result we consider it beneficial to model our bovine sperm proteome data sets using go and ipa.
from go associations of differentially expressed proteins we found that there was a comparative up regulation of three biological processes in high fertility spermatozoa: metabolism, cell communication and cell motility .
up regulation of metabolism is consistent with the fact that capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration  <cit> .
pyruvate metabolism and glycolysis were the top most significant metabolic pathways represented in high fertility sperm proteome by ipa.
in glycolysis, expression of pyruvate kinase  was higher in high fertility spermatozoa.
pkm <dig> catalyzes the production of pyruvate and atp from phosphoenol pyruvate.
pyruvate formed in this process serves as an energy source for cells  <cit> .
impaired or lower pyruvate metabolism could limit the cell's ability to produce energy and this could be one of the reasons for reduced fertility in the low fertility group.
expression of cox  <dig> and atp5b involved in oxidative respiration was higher in high fertility spermatozoa compared to low fertility spermatozoa.
cox <dig> is a member of the large transmembrane protein complex found in the mitochondrion and is the last protein in the electron transport chain.
coupling of electron transport to oxidative respiration maintains the high mitochondrial transmembrane potential required for mitochondrial atp production  <cit> .
atp5b catalyzes the production of atp from adp in the presence of a proton gradient across the mitochondrial membrane and this atp is utilized for the motility of sperm and capacitation  <cit> .
communication between sperm and oocyte is critical for successful fertilization.
we found that there was up regulation of cell communication in the high fertility sperm proteome when compared to low fertility sperm proteome .
to bring about cell to cell communication several signaling pathways are necessary.
egf signaling and pdgf signaling were the top two significant signaling pathways identified in high fertiliy spermatozoa.
egf and pdgf signaling pathways stimulate tyrosine phosphorylation of various map kinases and their upstream activators mek <dig>  mek <dig> and mekk  <cit> .
egf signaling has an important role in sperm capacitation as it stimulates tyrosine phosphorylation of many proteins  <cit> .
in addition, egf signaling also activates phospholipase c   <cit>  .
plc is important for the acrosome reaction , fertilization and embryo development.
plc catalyzes the production of inositol  <dig>   <dig>  5-triphosphate  from phosphatidylinositol  <dig>  5-biphosphate.
ip <dig> generated by plc activates the extra cellular calcium influx required for the ar via binding to the ip <dig> receptor  gated calcium channel located on the acrosome membrane  <cit> .
mutations in mouse plcb <dig> reduced the ar rate, fertilization rate and embryo development  <cit> .
egf signaling was specific to high fertility bull sperm.
defects in egf signaling in low fertility spermatozoa may prevent capacitation.
expression of casein kinase  <dig>  prime poly peptide in egf signaling was higher in high fertility spermatozoa compared to low fertility spermatozoa .
ckii is preferentially expressed in late stages of spermatogeneis and is involved in sperm chromatin decondensation after sperm oocyte fusion  <cit> .
ckii deficient mice are infertile with oligospermia and globozoospermia <cit> .
egf signaling also induces actin polymerization in bovine sperm capcitation  <cit> .
actin polymerization is essential for incorporation of sperm into egg cytoplasm  <cit>  and for sperm nuclei decondensation  <cit> .
comparing the proteome profiles of bull sperm of high and low fertility showed some molecular features associated with low fertility.
cell cycle: g2/m dna damage check point regulation was the topmost significant signaling pathway followed by integrin signaling in low fertility bull sperm .
the g2/m dna damage checkpoint could help in maintaining the integrity of the genome during different stages of development.
progression through different phases of the cell cycle requires the sequential activation of various cyclin dependent kinases and these kinases in turn are regulated by integrin signaling.
integrin signals are necessary for cells to traverse the cell division cycle  <cit> .
these two pathways may be a compensatory response for reproductive system disease function which was identified only in low fertility sperm .
in addition to differences in signaling and metabolic pathways between high and low fertility spermatozoa, we identified differences in protein expression that had implications in sperm motility.
expression of a-kinase anchor protein- <dig>  was significantly higher in high fertility spermatozoa .
akap <dig> is a major fibrous sheath protein of the principal piece of the sperm flagellum.
akap <dig> recruits protein kinase a to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function in humans  <cit> .
it also serves as a scaffolding protein for signaling proteins and proteins involved in metabolism.
higher expression of akap <dig> in the high fertility group sperm could result in higher motility.
CONCLUSIONS
in summary, this is the first comprehensive description of the spermatozoa proteome of bovine.
comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.
we observed marked differences in signaling and metabolic pathways between high fertility and low fertility spermatozoa that have implications in sperm capacitation, acrosomal reaction and sperm-oocyte communication.

 male infertility is a major problem for mammalian reproduction. the nature of sub-fertility due to the male is as complex as that of the female. infertility due to male factor contributes approximately 40% of the infertility cases in humans. for this reason it is very important to investigate the factors that affect male fertility. here we used bovine spermatozoa to model human male fertility because cattle provide several advantages as a model for male factor infertility. these include good breeding records fertility data records and progeny records. in cattle breeding, artificial insemination, a common breeding technique, utilizes semen from genetically superior sires to inseminate cows. in the united states more than ~70% of cows are bred by ai but only ~50% of these matings result in successful full term pregnancy. the underlying molecular events/mechanisms that determine the fertilizing potential of a semen sample are not well defined. a thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. fertility traits of semen can be categorized as compensable or uncompensable. defects in compensable traits can be overcome by increasing the number of spermatozoa per insemination. defects in uncompensable traits affect the function of spermatozoa during the later stages of fertilization and in embryonic development and as such cannot be compensated. uncompensable traits include nuclear vacuoles, morphological deficiencies that do not suppress movement, defective chromatin structure. low fertility in bulls has an uncompensable component that includes reduced cleavage rate and delayed pronuclear formation following in vitro fertilization. currently available fertility assays assess the defects that affect functional competence of spermatozoa, however these cannot definitively predict fertility. at present, the molecular nature of sperm fertility defects or biomarkers for accurate fertility prediction is not known. spermatozoa are transcriptionally inactive so the only comprehensive method to understand the molecular functions in spermatozoa is via proteomics. published proteomic studies with bull spermatozoa described the sub-proteome of the sperm and functions of proteins from its surrounding cells. accessory gland proteins were shown to modulate important sperm functions after ejaculation and in the female reproductive tract such as capacitation, acrosome reaction, sperm-oocyte interaction, and sperm protection. it is known that fertile associated antigen, a heparin binding protein from seminal vesicles and prostate glands, binds to spermatozoa membrane and modulates heparin-sperm interactions that are indicative of fertility. two seminal plasma proteins such as, prostaglandin-d-synthetase and osteoponin were more abundant in the semen of high fertility bulls when compared to low fertility bulls. here we describe a comprehensive proteomic analysis of bull sperm using differential detergent fractionation two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry. we compared protein expression profiles of sperm from high and low fertility bulls to characterize the differences in fertility at the protein level. our results show that expression of and proteins was specific to high and low fertility bull spermatozoa, respectively and proteins were common to both. differential expression of proteins was significant between high and low fertility bull spermatozoa and these proteins are potential biomarkers for bovine male fertility. biological systems utilize highly complex, interrelated metabolic and signaling pathways to function. therefore, to identify signaling pathways involved in fertility, we carried out systems modeling of our proteomic datasets using gene ontology and ingenuity pathway analysis. we identified differences in the signaling pathways between high and low fertility bull spermatozoa and found that egf and pdgf signaling pathways were specific to high fertility.  proteome profiles of spermatozoa from high and low fertility bulls we identified and proteins in high and low fertility group spermatozoa respectively. among these were common to both groups and and proteins were unique to high and low fertility groups respectively. only those proteins identified by at least three peptides were included in the analysis for differential expression and we identified proteins as differentially-expressed between the high and low fertility spermatozoa. compared to low fertility bull spermatozoa, expression of proteins increased and there was a decrease in the expression of proteins in high fertility spermatozoa. only a small proportion of proteins identified in this study have been previously described ). the majority of the identified proteins are'predicted'. we contributed to the annotation of the newly sequenced bovine genome by experimentally confirming the in vivo expression of electronically predicted proteins. we also identified % and %'hypothetical' proteins specific to high fertility and low fertility spermatozoa respectively. list of differentially expressed proteins in high fertility group spermatozoa when compared to low fertility group. spermatozoa in this table we provided the information about number of peptides, sequest cross correlation score and p value for each protein in high and low fertility group spermatozoa respectively. predicted and hypothetical proteins do not have any functional annotation associated with them and they represent ~80% of differentially expressed proteins between high and low fertility spermatozoa. this poses a problem for meaningful biological modeling of our data without carrying out some functional annotation first. therefore, we annotated all differentially expressed proteins in our data sets using agbase go resources. membrane and nuclear proteins membrane and nuclear proteins are fundamental for inter and intra cellular signaling and are thus fundamental for modeling cell-cell interactions. sperm oocyte fusion is a key element for fertilization. this process is facilitated by sperm surface proteins and leads to specific binding of the sperm surface-active component with the egg zona pellucida and, ultimately, sperm-egg fusion. to identify proteins from the sperm membrane and the nucleus which function in cell fusion, we focused on membrane and nuclear proteins identified in our datasets. based on the go associations of known proteins, % are membrane proteins. we also identified nuclear proteins based on go associations. biological process annotation of membrane proteins revealed that majority of membrane proteins involved in transport, cell communication and metabolism. we go annotated all differentially expressed proteins and applied the generic go slim to identify functional super-categories represented in differentially expressed proteins in high fertility spermatozoa. most go slim categories, including processes such as metabolism, cell communication and cell motility showed overall up regulation of protein expression in the high fertility group while transport proteins showed an overall down regulation in the high fertility group. high fertility and low fertility sperm proteomes: molecular network and pathway analysis protein identification from biological samples on a global scale is important. however, there is a need to move beyond this level of analysis; instead of simply enumerating a list of proteins, the analysis needs to include their interactions as parts of complexes, pathways and biological networks. to achieve this level of analysis with our high fertility and low fertility spermatozoa proteomic datasets we used ingenuity pathway analysis. at ipa thresholds for significance, and networks and and functions/diseases were significantly represented in the proteomes of high fertility and low fertility spermatozoa respectively. the top functions/diseases, and the associated signaling pathways are shown in table and table for proteomes of high and low fertility groups respectively. analysis of the top functions revealed that functions like cellular movement, cell to cell signaling and interaction were identified only in the high fertility sperm proteome. whereas, functions like cell death and reproductive system disease were identified only in the low fertility sperm proteome. egf: epidermal growth factor; pdgf: platelet derived growth factor; igf1: insulin growth factor- mapk: mitogen activated protein kinase; vegf: vascular endothelial growth factor; pten: phosphatase and tensin homolog deleted on chromosome ten. compared to low fertility sperm proteome, the high fertility sperm proteome had a 2-fold enrichment in signaling pathways. however, the number of significant metabolic pathways represented was comparable between the low and high fertility spermatozoa. epidermal growth factor signaling was the most prominent signaling pathway specific to high fertility sperm. egf signaling is known to promote proliferation, survival, and differentiation of a wide variety of mammalian cells. in addition to the egf signaling pathway, platelet derived growth factor signaling, peroxisome proliferated activator receptor signaling, interleukin - signaling, nf-kβ signaling, chemokine signaling, and insulin growth factor - signaling were identified only in high fertility spermatozoa. in low the fertility group, cell cycle: g2/m dna damage check point regulation was the most significant pathway followed by integrin signaling. proteins with significantly altered expression: molecular network and pathway analysis systems analysis of global proteomes revealed that some signaling pathways are differentially represented between the high and low fertility group spermatozoa. to further analyze these differentially expressed pathways, we carried out ipa analysis with just the differentially expressed proteins. in high fertility spermatozoa, expression of proteins was increased when compared to low fertility spermatozoa. ipa analysis identified three significant networks with scores of and respectively. proteins identified in the top three networks are participants in egf signaling, pdgf signaling, oxidative phosophorylation, and pyruvate metabolism pathways. expression of two proteins, atp synthase, h+ transporting, mitochondrial f complex, and cytochrome c oxidase subunit iii involved in oxidative phosphorylation and casein kinase ii involved in egf signaling and pdgf signaling were higher in the high fertility spermatozoa compared to low fertility spermatozoa. ipa also identified pyruvate metabolism as the most significant pathway in up regulated proteins of high fertility spermatozoa. in the low fertility sperm proteome, expression of proteins increased when compared to high fertility spermatozoa. ipa analysis identified two significant networks in highly expressed proteins of low fertility sperm. proteins identified in the top two significant networks are participants in integrin signaling and estrogen receptor signaling. discussion male fertility can be described as the success by spermatozoa to fertilize oocytes and of the resulting zygotes continue on through embryonic and fetal development until birth. in this study we used bovine spermatozoa to study fertility as it can serve as a model for understanding human male infertility and reproductive diseases. studying bovine male fertility on its own merit has implications in agro-economics involving cattle industry worldwide. a spermatozoon must reach the site of fertilization and be capacitated for successful fertilization to occur. a subsequent step is the acrosome reaction characterized by fusion of a spermatozoon outer acrosomal membrane with overlying plasma membrane. the molecular mechanisms and signal transduction pathways mediating the processes of capacitation and acrosome reaction have been partially defined. bull sperm cytosolic fraction proteomic analysis showed enrichment for tyrosine kinases which are essential for phosphorylation of specific sperm proteins during capacitation. the abundance of a variety of proteins from cells surrounding the sperm has been proposed to indicate male fertility. most of the studies used 2-dimensional electrophoresis for isolation and identification of sperm proteins. to our knowledge this is the first comprehensive non-electrophoretic proteomic study of bull sperm proteome. the aim of our study was to identify proteins that were differentially expressed between high and low fertility bull spermatozoa and interrelated metabolic and signaling pathways that have a role in fertility. we identified proteins as differentially expressed in between the high and low fertility sperm even though proteins were common to both groups and about were unique to each. the reasons for this apparent discrepancy are that we took a conservative approach to the statistical analysis: only proteins identified by at least three peptides were included in the analysis for differential expression and the statistical method used in protquant is very conservative. protquant specifically address the issue of "missing" mass spectra that occurs in all 2-d lc ms -based expression proteomics methods. no other published method addresses this issue. missing mass spectra are due to the inherent limitations of the mass spectrometers, the probabilistic nature of sampling and the cutoffs used to determine "true" assignments of peptides to mass spectra. protquant is highly conservative method which is based on sum of xcorr method itself increases the specificity of spectral counting and reduce the type i errors of differential expression. regardless, proteins were analyzed from each of three of the areas represented in figure and differentially-expressed proteins occurred in all three. from proteome profiles of specific cells or tissues, one acquires large datasets that are inherently complex. as a result we consider it beneficial to model our bovine sperm proteome data sets using go and ipa. from go associations of differentially expressed proteins we found that there was a comparative up regulation of three biological processes in high fertility spermatozoa: metabolism, cell communication and cell motility. up regulation of metabolism is consistent with the fact that capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration. pyruvate metabolism and glycolysis were the top most significant metabolic pathways represented in high fertility sperm proteome by ipa. in glycolysis, expression of pyruvate kinase was higher in high fertility spermatozoa. pkm catalyzes the production of pyruvate and atp from phosphoenol pyruvate. pyruvate formed in this process serves as an energy source for cells. impaired or lower pyruvate metabolism could limit the cell's ability to produce energy and this could be one of the reasons for reduced fertility in the low fertility group. expression of cox and atp5b involved in oxidative respiration was higher in high fertility spermatozoa compared to low fertility spermatozoa. cox is a member of the large transmembrane protein complex found in the mitochondrion and is the last protein in the electron transport chain. coupling of electron transport to oxidative respiration maintains the high mitochondrial transmembrane potential required for mitochondrial atp production. atp5b catalyzes the production of atp from adp in the presence of a proton gradient across the mitochondrial membrane and this atp is utilized for the motility of sperm and capacitation. communication between sperm and oocyte is critical for successful fertilization. we found that there was up regulation of cell communication in the high fertility sperm proteome when compared to low fertility sperm proteome. to bring about cell to cell communication several signaling pathways are necessary. egf signaling and pdgf signaling were the top two significant signaling pathways identified in high fertiliy spermatozoa. egf and pdgf signaling pathways stimulate tyrosine phosphorylation of various map kinases and their upstream activators mek mek and mekk. egf signaling has an important role in sperm capacitation as it stimulates tyrosine phosphorylation of many proteins. in addition, egf signaling also activates phospholipase c. plc is important for the acrosome reaction, fertilization and embryo development. plc catalyzes the production of inositol 5-triphosphate from phosphatidylinositol 5-biphosphate. ip generated by plc activates the extra cellular calcium influx required for the ar via binding to the ip receptor gated calcium channel located on the acrosome membrane. mutations in mouse plcb reduced the ar rate, fertilization rate and embryo development. egf signaling was specific to high fertility bull sperm. defects in egf signaling in low fertility spermatozoa may prevent capacitation. expression of casein kinase prime poly peptide in egf signaling was higher in high fertility spermatozoa compared to low fertility spermatozoa. ckii is preferentially expressed in late stages of spermatogeneis and is involved in sperm chromatin decondensation after sperm oocyte fusion. ckii deficient mice are infertile with oligospermia and globozoospermia. egf signaling also induces actin polymerization in bovine sperm capcitation. actin polymerization is essential for incorporation of sperm into egg cytoplasm and for sperm nuclei decondensation. comparing the proteome profiles of bull sperm of high and low fertility showed some molecular features associated with low fertility. cell cycle: g2/m dna damage check point regulation was the topmost significant signaling pathway followed by integrin signaling in low fertility bull sperm. the g2/m dna damage checkpoint could help in maintaining the integrity of the genome during different stages of development. progression through different phases of the cell cycle requires the sequential activation of various cyclin dependent kinases and these kinases in turn are regulated by integrin signaling. integrin signals are necessary for cells to traverse the cell division cycle. these two pathways may be a compensatory response for reproductive system disease function which was identified only in low fertility sperm. in addition to differences in signaling and metabolic pathways between high and low fertility spermatozoa, we identified differences in protein expression that had implications in sperm motility. expression of a-kinase anchor protein- was significantly higher in high fertility spermatozoa. akap is a major fibrous sheath protein of the principal piece of the sperm flagellum. akap recruits protein kinase a to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function in humans. it also serves as a scaffolding protein for signaling proteins and proteins involved in metabolism. higher expression of akap in the high fertility group sperm could result in higher motility.  in summary, this is the first comprehensive description of the spermatozoa proteome of bovine. comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype. we observed marked differences in signaling and metabolic pathways between high fertility and low fertility spermatozoa that have implications in sperm capacitation, acrosomal reaction and sperm-oocyte communication. 
this is the first comprehensive description of the bovine spermatozoa proteome. compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. male infertility is a major problem for mammalian reproduction. however, molecular details including the underlying mechanisms of male fertility are still not known. a thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype. we next used quantitative systems biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. our data also suggests a hypothesis that low fertility sperm dna integrity may be compromised because cell cycle: g2/m dna damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology and identified putative biomarkers of fertility.
 male infertility is a major problem for mammalian reproduction. however, molecular details including the underlying mechanisms of male fertility are still not known. a thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder.  using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology and identified putative biomarkers of fertility. we next used quantitative systems biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. our data also suggests a hypothesis that low fertility sperm dna integrity may be compromised because cell cycle: g2/m dna damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa.  this is the first comprehensive description of the bovine spermatozoa proteome. comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype. 