Sperm Surface Protein Research Articles

The proteomic landscape of sperm surface deciphers its maturational and functional aspects in buffalo.

Buffalo is a dominant dairy animal in many agriculture-based economies. However, the poor reproductive efficiency (low conception rate) of the buffalo bulls constrains the realization of its full production potential. This in turn leads to economic and welfare issues, especially for the marginal farmers in such economies. The mammalian sperm surface proteins have been implicated in the regulation of survival and function of the spermatozoa in the female reproductive tract (FRT). Nonetheless, the lack of specific studies on buffalo sperm surface makes it difficult for researchers to explore and investigate the role of these proteins in the regulation of mechanisms associated with sperm protection, survival, and function. This study aimed to generate a buffalo sperm surface-specific proteomic fingerprint (LC-MS/MS) and to predict the functional roles of the identified proteins. The three treatments used to remove sperm surface protein viz. Elevated salt, phosphoinositide phospholipase C (PI-PLC) and in vitro capacitation led to the identification of N = 1,695 proteins (≥1 high-quality peptide-spectrum matches (PSMs), p < 0.05, and FDR<0.01). Almost half of these proteins (N = 873) were found to be involved in crucial processes relevant in the context of male fertility, e.g., spermatogenesis, sperm maturation and protection in the FRT, and gamete interaction or fertilization, amongst others. The extensive sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth of identification of reproduction-specific cell-surface proteins and can provide a potential framework for further studies on the functional aspects of buffalo spermatozoa.

Mammalian fertilization: Does sperm IZUMO1 mediate fusion as well as adhesion?

The molecular mechanism of sperm-egg fusion is a long-standing mystery in reproduction. Brukman and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202207147) now provide evidence that the sperm surface protein IZUMO1, which is essential for mammalian fertilization, can induce membrane fusion in cultured cells.

Is there a relationship between infertility and fertilin β protein distribution?

IntroductionFertilin β is a sperm surface protein that can mediate sperm-egg membrane interaction. This study was conducted to determine whether the expression of fertilin β after intrauterine insemination (IUI) in donors with normal parameters after standard semen analysis is related to low success rate or failure of fertilization. MethodsWe examined the sperm of 30 male donors who have normal as controls, oligozoospermia, and unexplained infertility as the clinically indication for IUI. Fertilin β has been labeled with the ADAM2 antibody by indirect immunofluorescence (IF) assay. To evaluate the reproducibility of the test, we selected four sperm samples scale of 0 to +++ according to the distribution of fluorescence label. ResultsThe results were highly correlated with the corrected total cell fluorescence (CTCF) (Rp=0.9972, P<0.05). We suggest that the relationship between infertility and fertilin β may be due to the distribution of this protein on the sperm surface. Male partners of couples with unexplained infertility showed a low distribution of fertilin β by a decrease of the fluorescence signal in the IF labeling (scale of +++ by 7.4±10.32%, P<0.0001, ±SD). DiscussionAbnormal fertilin β function may be a potential mechanism that could lead to fertilization failure.

Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose.

Simple SummaryCryopreservation of sperm is an important process used in livestock breeding. It is used to facilitate artificial insemination and helps to maintain genetic diversity by utilizing storage in cryobanks. Boar sperm are extremely sensitive to the conditions present during cryopreservation. Glycerol is the most used permeable cryoprotectant, showing both protective activity and some level of sperm toxicity. To improve the insemination rates, it is necessary to develop new cryoprotective agents. The goal of this paper is to evaluate the effect of the highly biocompatible, non-toxic polysaccharides dextran and pentaisomaltose on the cryopreservation of boar sperm. Additionally, we used computational modeling for the prediction of their cryoprotective action through interaction with selected sperm surface proteins. Our results show a lower impact of cryopreservation on sperm qualitative parameters when the extender is modified by pentaisomaltose. This approach represents a promising option for the complete removal of toxic glycerol in cryopreservation.The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.

Selection and Characterization of DNA Aptamers Targeting hLCN6 Protein for Sperm Capture.

It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science. In this project, we developed a DNA aptamer-based system for sperm separation and purification from mixed stain samples by targeting sperm surface proteins. Human lipocalin 6 (hLCN6) is an epididymal secreted protein that binds to the head and tail of sperm cells and associated with sperm maturation. Using systematic evolution of ligands by exponential enrichment (SELEX) technology, aptamers that bind with high affinity and specificity to hLCN6 were screened from a random single-stranded DNA (ssDNA) library using magnetic bead-bound hLCN6 as target. The enriched library was obtained after 15 SELEX rounds. Of hLCN6-binding aptamer variants, 19 were further classified into one of the four groups based on their N60 random sequence regions, wherein one representative from each group was characterized. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Binding capability of selected aptamers was investigated by quantitative PCR, and aptamer H2 was found to be the most specific aptamer to sperm cells. The dissociation constant (Kd) of H2 aptamer was calculated as 3.21 ± 0.75nM. Furthermore, H2 aptamer-coupled magnetic beads can recognize and capture sperm cells, which establishes the foundation of an approach for rapidly isolating sperm cells from mixed stains based on nucleic acid-protein interaction.

Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated thatthe abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

BackgroundLow conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins.ResultsThe raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments.ConclusionsThe surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

Sperm-oocyte signaling: the role of IZUMO1R and CD9 in PTK2B activation and actin remodeling at the sperm binding site†.

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

Localization and expression of ADAM2 in the dromedary camel testis, epididymis and sperm during rutting season.

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.

Comparative transcriptome analysis of three gonadal development stages reveals potential genes involved in gametogenesis of the fluted giant clam (Tridacna squamosa)

BackgroundGonad development and differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. However, the mechanisms underlying gonad development and gametogenesis remain unclear in Tridacna squamosa, a large-size bivalve of great ecological value. They are protandrous simultaneous hermaphrodites, with the male gonad maturing first, eventually followed by the female gonads. In this study, nine gonad libraries representing resting, male and hermaphrodite stages in T. squamosa were performed to identify the molecular mechanisms.ResultsSixteen thousand four hundred ninety-one unigenes were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 5091 and 7328 unigenes were assigned to Gene Ontology categories and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database, respectively. A total of 4763 differentially expressed genes (DEGs) were identified by comparing male to resting gonads, consisting of 3499 which were comparatively upregulated in males and 1264 which were downregulated in males. Six hundred-ninteen DEGs between male and hermaphroditic gonads were identified, with 518 DEGs more strongly expressed in hermaphrodites and 101 more strongly expressed in males. GO (Gene Ontology) and KEGG pathway analyses revealed that various biological functions and processes, including functions related to the endocrine system, oocyte meiosis, carbon metabolism, and the cell cycle, were involved in regulating gonadal development and gametogenesis in T. squamosa. Testis-specific serine/threonine kinases 1 (TSSK1), TSSK4, TSSK5, Doublesex- and mab-3-related transcription factor 1 (DMRT1), SOX, Sperm surface protein 17 (SP17) and other genes were involved in male gonadal development in Tridacna squamosal. Both spermatogenesis- (TSSK4, spermatogenesis-associated protein 17, spermatogenesis-associated protein 8, sperm motility kinase X, SP17) and oogenesis-related genes (zona pellucida protein, Forkhead Box L2, Vitellogenin, Vitellogenin receptor, 5-hydroxytryptamine, 5-hydroxytryptamine receptor) were simultaneously highly expressed in the hermaphroditic gonad to maintain the hermaphroditism of T. squamosa.ConclusionAll these results from our study will facilitate better understanding of the molecular mechanisms underlying giant clam gonad development and gametogenesis, which can provided a base on obtaining excellent gametes during the seed production process for giant clams.

Molecular kinetics of proteins at the surface of porcine sperm before and during fertilization

Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.

Proteomic comparison of non-sexed and sexed (X-bearing) cryopreserved bull semen

It is widely recognized that quality of spermatozoa in sexed semen (SS) samples is not as great as for conventional, non-sexed semen (NS). There are differences in qualitative and biochemical variables between spermatozoa in NS and SS. Information, however, is lacking on molecular differences, especially concerning spermatozoa proteomic differences is SS and NS. The objective of this study was to compare commercially available NS and SS bull semen by evaluating sperm quality variables in conjunction with a proteomics approach. Results from flow cytometry and computer-assisted sperm analyses indicated there was less sperm motility, viability, mitochondrial potential and acrosome integrity in sperm from SS. Results from proteomic analysis indicated sperm from NS and SS samples were characterized by different protein profiles. There was identification of 70 sperm proteins that differed in abundance and six protein spots that were different in extent of carbonylation. Sperm from SS had altered structures of enzymes involved in glycolysis, oxidative phosphorylation and maintenance of a constant adenylate energy charge. Furthermore, sperm from SS had alterations of several flagella substructures, especially outer dense fiber proteins, which were less abundant and more carbonylated than in sperm from NS. In sperm of SS, compared with NS, there were differences in abundance of proteins involved in capacitation, acrosome reaction and sperm-egg fusion as well as lesser abundances of sperm surface proteins. Results enable a greater understanding of differences between sperm from NS and SS samples, thereby contributing to development of improved protocols for more effective protection of sexed spermatozoa during processing.

The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation.

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

Effect of seasonal infertility period on boar sperm proteins and quality characteristics

Swine seasonal infertility reduces the productivity and profitability of a pig farm. The main causes of this condition are elevated environmental temperatures and long photoperiod during the summer season. The aim of this study was to investigate which sperm proteins and parameters are affected during the period of seasonal infertility. Depending on the environmental temperatures, the period from October to June was considered as cold and the period from July to September as warm season. A total of 65 ejaculates from 18 boars were collected over a year. Each semen sample was evaluated for kinetics (Computer Assisted Semen Analyzer), morphology (Sperm Blue stain), viability (Propidium Iodide - Calcein AM stain), mitochondrial membrane potential (Rhodamine 123 – Propidium Iodide stain), membrane integrity and functionality (Hypo-osmotic swelling test) and sperm DNA integrity (Acridine Orange Test). Moreover, selected proteins (HSP90, GPX5, OPN) were detected and quantified. The kinetic parameters VSL, LIN and the midpiece abnormalities were significantly higher in the warm compared to the cold season (p&lt;0.05), while a strong tendency towards higher values for HSP90 and GPX5 was observed in warm compared to cold season (p=0.07and p=0.06, respectively). In conclusion, among the boar sperm characteristics tested in our study, seasonal infertility period negatively affected VSL and LIN kinetics, while GPX5 seminal plasma enzyme and HSP90 sperm surface protein increased their sperm protective effects.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment.

Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. Using the Pir Peptide Match program, three ovarian cancer biomarkers - namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance - were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

Growth factors and extracellular matrix proteins in interactions of cumulus–oocyte complex, spermatozoa and oviduct

The expression and localization of selected growth factor systems and extracellular matrix (ECM) components that may influence oocyte maturation and fertilization within the mammalian oviduct are reported. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) systems could be detected by use of RT–PCR, RNase protection assay (RPA) and immunohistochemistry in bovine follicles, bovine cumulus–oocyte complexes (COC) and bovine and marmoset oviducts. Two different subtypes of the FGF receptor (FGFR-1 and -2) were identified in distinct cell types, indicating a functional difference. A complete epidermal growth factor (EGF) system was found in the porcine, but not in the bovine, oviduct There were additional differences between bovine and primate oviducts: FGF-1/2 and FGFR were increased in the marmoset around ovulation, in contrast to an increase in FGF-1 in the cow. Immunohistochemistry revealed accumulation and storage of FGF and VEGF on the surface of the epithelium, possibly due to their binding property on heparan-glycoproteins. Other ECM components, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TTMP-1), were found to be modulated in the ovarian follicle, COC and oviduct during the cycle. An oviduct-mediated depletion of sperm surface proteins (BSP1–3) was discovered as well as a sperm-induced novel oviductal mRNA related to an anti-oxidant protein family. Associated systems of growth factors and ECM components can be suggested as paracrine or autocrine mediators during fertilization in a species-, cycle- and tissue-dependent manner.

Potential sexing of spermatozoa using new antibodies to sperm surface proteins

Potential sexing of spermatozoa using new antibodies to sperm surface proteins

Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation.

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality.

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.

Treatment of semen samples with α-chymotrypsin alters the expression pattern of sperm functional proteins-a pilot study.

Semen hyperviscosity delays the liquefaction of semen sample and is subjected to limited proteolysis by addition of α-chymotrypsin to reduce the viscosity. α-Chymotrypsin is a proteolytic enzyme involved in degradation of the proteins and polypeptides. Even though α-chymotrypsin improves the handling of hyperviscous samples, its effect on the sperm proteins is not clear. This study was aimed to evaluate the alteration in the expression of sperm functional proteins in samples treated with α-chymotrypsin. Among all the proteins examined in both donor and patient samples, HSPA2 (70 KDa), BAG6 (150 KDa), HIST1H2BA (14 KDa), SPA17 (17 KDa formed after cleavage of C-terminal calmodulin-binding domain), and OXPHOS complexes were undetectable in α-chymotrypsin-treated samples, while the expression of the native SPA17 (20 KDa) was significantly decreased in the α-chymotrypsin-treated samples in comparison with controls. The use of α-chymotrypsin for liquefaction of hyperviscous samples degrades functional proteins of spermatozoa. Intracellular proteins, such as OXPHOS complexes and HIST1H2BA, and sperm surface proteins (HSPA2, BAG6, and SPA17) were degraded in all treated samples. Whether treatment of samples with α-chymotrypsin affects the global proteomic outcome is unclear. More in-depth calibration studies are required to determine the appropriate concentration of α-chymotrypsin for processing hyperviscous semen samples without compromising its protein expression and function. Similarly, the effects of altered protein function on assisted reproductive techniques (ART), such as intrauterine insemination (IUI) and invitro fertilization (IVF) outcome, are not known and require further research.