Sperm carries a reservoir of proteins regulating the molecular functions to attain functional competence. Semen samples collected from buffalo bulls were assessed for sperm functional attributes (n = 11) and proteome profiling (n = 6). Sperm proteins were extracted and profiled by employing LC-MS/MS. Overall, the buffalo sperm contained 1365 proteins, of which 458 were common between the groups. The unique proteins were 477 and 430 in good and poor quality semen, respectively. In the whole proteome of buffalo sperm, sexual reproduction with phosphatidylethanolamine-binding protein1 (PEBP1), fetuin-B (FETUB) and acrosin (ACR) was the most enriched (p = 8.44E-19) biological process, also with thermogenesis (p = 0.003), oocyte meiosis (p = 0.007) and vascular smooth muscle contraction (p = 0.009) apart from metabolic pathways. In good quality semen, mesenchyme migration (p = 1.24E-07) and morphogenesis (p = 0.001) were abundant biological processes. In good quality semen, the fluid shear stress (p = 0.01) and, in poor quality semen, valine, leucine and isoleucine degradation (p = 3.8E-05) pathways were enriched. In good quality semen, 7 proteins were significantly (p < 0.05) upregulated and 33 proteins were significantly (p < 0.05) downregulated. On validating the abundantly expressed sperm proteins, serine protease inhibitor Kazal-type 2-like (SPINK2; 2.17-fold) and neddylin (NEDD8; 1.13-fold) were upregulated and YBX2 was downregulated (0.41-fold) in good quality semen as compared with poor quality semen (1-fold). The present findings revealed the importance of sperm proteins in oocyte maturation, fertilization process and early embryonic development. The variations in the proteomic composition can be used as potential markers for the selection of breeding bulls.

Sperm proteins play vital roles in fertilization, but little is known about their identities in free-spawning marine invertebrates. Here, 286 sperm proteins are reported from the Hong Kong oyster Crassostrea hongkongensis using label-free and semi-quantitative proteomics. Proteins extracted from three sperm samples are separated by SDS-PAGE, analyzed by LC-MS/MS, and identified using Mascot. Functional classification of the sperm proteome reveals energy metabolism (33%), signaling and binding (23%), and protein synthesis and degradation (12%) as the top functional categories. Comparison of orthologous sperm proteins between C. hongkongensis, Crassostrea gigas, Mytilus edulis, and M. galloprovincialis suggests that energy metabolism (48%) is the most conserved functional group. Sequence alignment of the C. hongkongensis bindin, an acrosomal protein that binds the sperm and the egg, with those of three other Crassostrea species, reveals several conserved motifs. The study has enriched the data of invertebrate sperm proteins and may contribute to studies of mechanisms of fertilization in free-spawning invertebrates. The proteomic data are available in ProteomeXchange with the identifier PXD018255.

Boar sperm quality is less during the summer as a result of the different photoperiod or ambient temperatures as compared with the winter. The present study was conducted to elucidate possible variations in proteomic profiles of boar spermatozoa collected during the summer and winter. Effects of season on sperm viability, total motility, progressive motility, acrosome status, mitochondrial membrane potential and plasma membrane lipid organization were also analyzed. Only sperm viability and mitochondrial membrane potential were less during the summer (P <  0.05). Spermatozoa were processed and evaluated using the nano LC-MS/MS QTof procedures. A total of 1028 characterized proteins were identified in sperm collected during both seasons of the year (False Discovery Rate < 0.01) and, among the total, 85 proteins differed in sperm collected in the winter and summer, with there being a lesser abundance of these proteins when there were ejaculate collections during the summer (q-value ≤ 0.05). The results from enrichment assessments for these protein networks utilizing UniProtKB procedures for determining reproductive processes indicates there were 23 proteins that were less abundant in the summer than winter. These proteins have essential functions in spermatogenesis, sperm motility, acrosome reaction and fertilization. These results are the first where there was ascertaining of proteomic differences in boar spermatozoa collected in the summer and winter. These results might help to explain the decreased sperm quality and prolificity when semen of boars is used for artificial insemination that is collected during the season of the year when ambient temperatures are relatively greater.

OBJECTIVE: Do alterations of human sperm protein profile affect embryo quality?
METHODS: Sperm proteins from 27 infertile couples undergoing intracytoplasmic sperm injection (ICSI) were extracted and digested. The resulting peptides were labelled using tandem mass tags, separated by two-dimensional liquid chromatography, and identified and quantified using tandem mass spectrometry. Subsequently, sperm protein and peptide abundance were statistically analysed for correlation with ICSI-derived embryo quality in the subset of idiopathic infertile couples. Detected correlations were further assessed in the subset of infertile patients with a known factor.
RESULTS: The abundance of 18 individual sperm proteins was found to correlate with embryo quality after ICSI. Of note, a high percentage of poor-quality ICSI-derived embryos was associated with alterations in several components of the eight-membered chaperonin-containing T-complex, which plays an important role in the folding of many essential proteins. Additionally, the abundance of sperm proteins with known functions in embryogenesis, such as RUBVL1, also correlated with early embryo quality (r = -0.547; P = 0.028). Some of the correlations found in this study were validated using either proteomic data from infertile patients with a known factor or data from similar published studies. Analysis at the peptide level revealed the association of some correlations with specific post-translational modifications or isoforms.
CONCLUSIONS: Our results support the hypothesis that the sperm proteome plays a role in early embryogenesis. Moreover, several sperm proteins have emerged as potential biomarkers that could predict the outcome of in-vitro assisted reproductive technologies, leading to the possibility of improved diagnosis of couples with idiopathic infertility.

Sperm proteins undergo post-translational modifications, such as phosphorylation, acetylation, and ubiquitination, which in turn play a key role in determining their fertilizing ability. In the current study, we examined the sperm proteome of men with unilateral and bilateral varicocele to identify the key proteins affected by acetylation to gain an insight into the difference in the severity of affected sperm function in the latter. An LTQ-Orbitrap Elite hybrid mass spectrometer system was used to profile the sperm proteome in pooled unilateral and bilateral varicocele patients. Bioinformatics database and tools, such as UniProtKB, Ingenuity Pathway Analysis Software (IPA) and Metacore, were used to identify the differentially expressed proteins (DEPs) involved in the acetylation process. A total of 135 DEPs in the spermatozoa of unilateral and bilateral varicocele patients were found to be affected by acetylation. The majority of these DEPs found were regulated by key transcription factors such as androgen receptor, p53, and NRF2. Furthermore, the DEPs predicted to be affected by the acetylation process were associated with fertilization, acrosome reaction, mitochondrial dysfunction and oxidative stress. Aberrant expression of proteins and their differential acetylation process may affect the normal physiological functions of spermatozoa. Protein-protein interactions identified dysregulation of the proteasome complex in the bilateral varicocele group. Damage to the proteasome complex may result in aggregation of the misfolded proteins, which in turn increase sperm DNA damage and apoptosis in patients with bilateral varicocele.

Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm‒oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility.

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.

BACKGROUND: Obesity is a worldwide crisis impairing human health. In this condition, declines in sperm quality stem from reductions in sperm concentration, motility and increase in sperm deformity. The mechanism underlying these alterations remains largely unknown. This study, determined if obesity-associated proteomic expression patterns in mice sperm parallel those in spermatozoa obtained from obese humans.
METHODS: An obese mouse model was established via feeding a high-fat diet (HFD). Histological analysis identified testicular morphology and a computer assisted semen analyzer (CASA) evaluated sperm parameters. Proteome analysis was performed using a label-free quantitative LC-MS/MS system. Western blot, immunohistochemical and immunofluorescent analyses characterized protein expression levels and localization in testis, sperm and clinical samples.
RESULTS: Bodyweight gains on the HFD induced hepatic steatosis. Declines in sperm motility accompanied sperm deformity development. Differential proteomic analysis identified reduced cytoskeletal proteins, centrosome and spindle pole associated protein 1 (CSPP1) and Centrin 1 (CETN1), in sperm from obese mice. In normal weight mice, both CSPP1 and CETN1 were localized in the spermatocytes and spermatids. Their expression was appreciable in the post-acrosomal region parallel to the microtubule tracks of the manchette structure in spermatids, which affects spermatid head shaping and morphological maintenance. Moreover, CSPP1 was localized in the head-tail coupling apparatus of the mature sperm, while CETN1 expression was delimited to the post-acrosomal region within the sperm head. Importantly, sperm CSPP1 and CETN1 abundance in both the overweight and obese males decreased in comparison with that in normal weight men.
CONCLUSIONS: These findings show that regionally distinct expression and localization of CETN1 and CSPP1 is strongly related to spermiogenesis and sperm morphology maintaining. Obesity is associated with declines in the CETN1 and CSPP1 abundance and compromise of both sperm morphology in mice and relevant clinical samples. This parallelism between altered protein expression in mice and humans suggests that these effects may contribute to poor sperm quality including increased deformity.

Antioxidant supplementation in idiopathic male infertility has a beneficial effect on semen parameters. However, the molecular mechanism behind this effect has not been reported. The objective of this study was to evaluate the sperm proteome of idiopathic infertile men pre- and post-antioxidant supplementation. Idiopathic infertile men were provided with oral antioxidant supplementation once daily for a period of 6 months. Of the 379 differentially expressed proteins (DEPs) between pre- and post-antioxidant treatment patients, the majority of the proteins (n = 274) were overexpressed following antioxidant treatment. Bioinformatic analysis revealed the activation of oxidative phosphorylation pathway and upregulation of key proteins involved in spermatogenesis, sperm maturation, binding of sperm, fertilization and normal reproductive function. In addition, the transcriptional factors associated with antioxidant defense system (PPARGC1A) and free radical scavenging (NFE2L2) were predicted to be functionally activated post-treatment. Key DEPs, namely, NDUFS1, CCT3, PRKARA1 and SPA17 validated by Western blot showed significant overexpression post-treatment. Our novel proteomic findings suggest that antioxidant supplementation in idiopathic infertile men improves sperm function at the molecular level by modulating proteins involved in CREM signaling, mitochondrial function and protein oxidation. Further, activation of TRiC complex helped in nuclear compaction, maintenance of telomere length, flagella function, and expression of zona pellucida receptors for sperm-oocyte interaction.

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.