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In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation., (© 2019 Wiley Periodicals, Inc.)

Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen.

Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real-time RT-PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. Biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 μM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 μM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment., (© 2015 Wiley Periodicals, Inc.)

Glioma pathogenesis-related 1-like protein1 (GliPr1L1) was identified by liquid chromatography-tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine-rich secretory proteins, Antigen 5 and pathogenesis-related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N-glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N-glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti-GliPr1L1 showed that this protein is involved in sperm-zona pellucida interaction., (Copyright © 2012 Wiley Periodicals, Inc.)

Previously, we showed that epididymal sperm binding protein 1 (ELSPBP1) characterizes spermatozoa already dead before ejaculation in bovine. In this study, we investigated the presence of ELSPBP1 in bull genital tract as well as its acquisition by spermatozoa during epididymal transit. As assessed by real-time RT-PCR, ELSPBP1 was highly expressed in the caput and the corpus epididymis but was present in lower expression levels in the testis and the cauda epididymis. Immunohistochemistry revealed the same expression pattern. However, Western blot on tissue homogenates showed some discrepancies, as ELSPBP1 was found in a comparable concentration all along the epididymis. This difference was due to the presence of ELSPBP1 in the epididymal fluid. In both caput and cauda epididymal fluid, ELSPBP1 was associated with the epididymosomes, small membranous vesicles secreted by epithelial cells of the epididymis and implicated in the transfer of proteins to spermatozoa. As assessed by immunocytometry, ELSPBP1 was found on a subset of dead spermatozoa in caput epididymis but was found on all dead spermatozoa in cauda epididymis. To assess ELSPBP1 acquisition by spermatozoa, caput epididymal spermatozoa were incubated with cauda epididymosomes under various conditions. ELSPBP1 detection by immunocytometry assay revealed that only spermatozoa already dead before incubation were receptive to ELSPBP1 transfer by epididymosomes. This receptivity was enhanced by the presence of zinc in the incubation medium. This specificity for a sperm subpopulation suggests that an underlying mechanism is involved and that ELSPBP1 could be a tag for the recognition of dead spermatozoa during epididymal transit.

During their epididymal maturation, stabilizing factors such as cholesterol sulfate are associated with the sperm plasma membrane. Cholesterol is sulfated in epididymal spermatozoa by the enzyme estrogen sulfotransferase. Because of its role in the efflux of sulfate conjugates formed intracellularly by sulfotransferases, the ATP-binding cassette membrane transporter G2 (ABCG2) might have a role in the translocation of this compound across the plasma membrane. In the present study we showed that ABCG2 is present in the plasma membrane overlaying the acrosomal region of spermatozoa recovered from testis, epididymis, and after ejaculation. Although ABCG2 is also present in epididymosomes, the transporter is not transferred to spermatozoa via this mechanism. Furthermore, although epididymal sperm ABCG2 was shown to be functional, as determined by its ability to extrude Hoechst 33342 in the presence of the specific inhibitor Fumitremorgin C, ABCG2 present in ejaculated sperm was found to be nonfunctional. Additional experiments demonstrated that phosphorylation of ABCG2 tyrosyl residues, but not its localization in lipid rafts, is the mechanism responsible for its functionality. Dephosphorylation of ABCG2 in ejaculated spermatozoa is proposed to cause a partial protein relocalization to other intracellular compartments. Prostasomes are proposed to have a role in this process because incubation with this fraction of seminal plasma induces a decrease in the amount of ABCG2 in the associated sperm membrane fraction. These results demonstrate that ABCG2 plays a role in epididymal sperm maturation, but not after ejaculation. The loss of ABCG2 function after ejaculation is proposed to be regulated by prostasomes.

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.

During their transit along the epididymis, mammalian spermatozoa acquire new proteins that are necessary for their acquisition of forward motility and fertility. By using the bovine model, we previously showed that small membranous vesicles named epididymosomes are secreted in the epididymal intraluminal compartment. Epididymosomes from caput and cauda are different, and interact sequentially with the transiting spermatozoa. In fact, selected proteins of epididymosomes are transferred to different subcompartments of the maturing spermatozoa. In this study, we investigate the possibility that different subpopulations of epididymosomes are present in the caudal portion of the epididymis. Through the use of discontinuous sucrose gradient ultracentrifugation, we isolated two distinct populations that differ in their protein and lipid compositions. Although they have similar diameters, the ultrastructural appearance of these two populations was very different. The low-density (Ld) vesicles are enriched in cholesterol, sphingomyelin, and ganglioside M1, suggesting the existence of detergent-resistant membrane domains or rafts. The high-density (Hd) vesicles show a high protein concentration, including ACTB and VAMP8. When each subpopulation of biotinylated cauda epididymosomes was coincubated with caput spermatozoa, a subset of biotinylated proteins was transferred to the sperm; the Ld and Hd vesicles transferring the same pattern of proteins. In vitro competition assays of protein transferred from Ld or Hd epididymosomes to sperm confirm the similarity in the selected transferred proteins. Electrospray tandem mass spectrometry (ES-MS/MS) analysis of proteins associated with the two populations of vesicles confirm the epididymal origin of some of them, the possible involvement of others in transmembrane signaling systems, and the identification of proteins for which functions in sperm physiology remain to be determined. Mass spectrometry analysis also revealed that ELSPBP1 and GBB2 were transferred from epididymosomes to spermatozoa. Results are discussed with regard to the functions of these two cauda epididymosome populations in sperm physiology.

Intrinsic factors such as proteins modulate the fertilising ability of male gametes. We compared detergent-extracted sperm protein composition of bulls with different fertility indexes in order to highlight putative fertility markers of sperm. Frozen semen from 23 Holstein bulls with documented fertility was used. According to their 'fertility solution' (SOL), as calculated by the Canadian dairy network, bulls were divided into four groups: high fertility (HF) (SOL>3.0; n=6), medium-HF (2.9>SOL>2.0; n=5), medium-low fertility (-2.8>SOL>-4.9; n=8) and low fertility (LF; SOL<-5.0; n=4), with a SOL=0 being the average. Triton X-100 protein extracts from ejaculated spermatozoa were subjected to two-dimensional difference gel electrophoresis, and polypeptide maps were quantitatively analysed by ImageMaster software. Nine protein spots showed significant differences between the HF and LF groups, and eight of these proteins were identified by liquid chromatography-tandem mass spectrometry. T-complex protein 1 subunits epsilon and (CCT5 and CCT8), two isoforms of epididymal sperm-binding protein E12 (ELSPBP1), proteasome subunit alpha type-6 and binder of sperm 1 (BSP1) were more expressed in the LF group than in the HF group. On the other hand, adenylate kinase isoenzyme 1 (AK1) and phosphatidylethanolamine-binding protein 1 (PEBP1) were more expressed in the HF group than in the LF group. The presence and expression level of ELSPBP1, BSP1, AK1 and PEBP1 were confirmed by western blot. A linear regression model established that CCT5 and AK1 explained 64% (P<0.001) of the fertility scores. The reported functions of these proteins are in agreement with a putative involvement in defective sperm physiology, where lower or higher levels can jeopardise sperm ability to reach and fertilise the oocyte.

Estrogen is found in high concentrations in the excurrent duct, where it regulates the expression of genes involved in water reabsorption. Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific sulfonation with a high affinity for estrogens. Because sulfated estrogens do not bind to estrogen receptors, they are considered to be hormonally inactive. EST may thus determine where along the male tract estrogenic environment predominates. Sulfotransferase activity increases along the epididymis and may also play a role in sperm physiology during the epididymal transit. Using a bovine model, we investigated the distribution of EST along the excurrent duct and the possibility that sterols associated with spermatozoa can be substrates of this enzyme. Reverse transcription polymerase chain reactions showed that mRNA encoding EST was expressed in the testis and all along the epididymis. A highly specific antiserum was raised against the bovine recombinant EST and used in Western blots and immunohistologic studies. Western blots of tissue homogenates showed that EST was localized all along the excurrent duct with a higher signal in the caput and corpus epididymidis. EST was detectable in the intraluminal compartment only in the caput epididymidis, where it was associated with epididymosomes and spermatozoa. EST was undetectable in different fractions of fluids collected in the cauda segment. In immunohistologic studies, EST was restricted to the acrosomal region of the caput, but not the cauda epididymal spermatozoa, and detectable in the cytoplasm of the epithelium bordering the lumen all along the epididymis as well as in the rete testis and vas efferens. This enzyme was also associated with the nucleus in the caput and corpus as well as with the apical membrane of the corpus epididymal epithelium. When recombinant EST was incubated in vitro in the presence of caput and cauda spermatozoa, it was able to add sulfate to sperm membrane cholesterol. Our study shows that EST is present in both the intracellular and intraluminal compartments of the epididymis, suggesting that this enzyme plays different roles along the excurrent duct.