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The coral Acropora spp., known for its reef-building abilities, is a simultaneous hermaphroditic broadcast spawning species. Acropora spp. release gametes into seawater, activating sperm motility. This activation is mediated by adenylyl cyclase (AC) and protein kinase A (PKA). Notably, membrane-permeable cAMP (8-bromo-cAMP) promotes sperm motility activation of Acropora florida. While the signal transduction for PKA-dependent motility activation is highly conserved among animals, the downstream signaling of PKA remains unclear. In this study, we used mass spectrometry (MS) analyses to identify sperm proteins in the coral Acropora digitifera, as well as the serine/threonine residues of potential PKA substrates, and then, we investigated the conservation of these proteins from corals to vertebrates. We identified 148 sperm proteins of A. digitifera with typical PKA recognition motifs, namely RRXT and RRXS. We subsequently used ORTHOSCOPE to screen for orthologs encoding these 148 proteins from corals to vertebrates. Among the isolated orthologs, we identified positive selection in 48 protein-encoding genes from 18 Acropora spp. Subsequently, we compared the conservation rates of the PKA phosphorylation motif residues between the orthologs under positive and purifying selections. Notably, the serine residues of the orthologs under positive selection were more conserved. Therefore, adaptive evolution might have occurred in the orthologs of PKA substrate candidates from corals to vertebrates, accompanied by phosphorylation residue conservation. Collectively, our findings suggest that while PKA signal transduction, including substrates in sperm, may have been conserved, the substrates may have evolved to adapt to diverse fertilization conditions, such as synchronous broadcast spawning. [ABSTRACT FROM AUTHOR]

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4-8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC-MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality. [ABSTRACT FROM AUTHOR]

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram– bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: Approximately 24% of men referred to assisted reproductive technology (ART) present with idiopathic male infertility. The current standard analysis of human semen does not allow for an accurate diagnosis of this infertility with unknown etiology. Spermatozoa cellular development and maturation, as well the acquisition of suitable motility and capacitation, are tightly coordinated by sperm protein phosphorylation, among other protein post-translational modifications. Extraordinary advances have been achieved in the field of spermatozoa using proteomics methodology in combination with bioinformatics. The aim of this work is to review, using a proteomic and phosphoproteomic analysis, the updated knowledge about proteins and phosphoproteins of spermatozoa that regulate cell processes necessary to achieve a proper fertilization. The phosphorylation of sperm proteins involved in spermatogenesis, in sperm capacitation, and in the maintenance of correct sperm motility, and consequently in sperm quality, is focused on in this review. Further investigations of protein spermatozoa in larger populations combined with other multi-omics technologies would offer a precise perspective of male fertility and would be especially relevant for those cases involving repeated failures in ART linked to idiopathic infertility. Infertility affects approximately 15% of couples worldwide of childbearing age, and in many cases the etiology of male infertility is unknown. The current standard evaluation of semen is insufficient to establish an accurate diagnosis. Proteomics techniques, such as phosphoproteomics, applied in this field are a powerful tool to understand the mechanisms that regulate sperm functions such as motility, which is essential for successful fertilization. Among the post-translational modifications of sperm proteins, this review summarizes, from a proteomic perspective, the updated knowledge of protein phosphorylation, in human spermatozoa, as a relevant molecular mechanism involved in the regulation of sperm physiology. Specifically, the role of sperm protein phosphorylation in motility and, consequently, in sperm quality is highlighted. Additionally, through the analysis of published comparative phosphoproteomic studies, some candidate human sperm phosphoproteins associated with low sperm motility are proposed. Despite the remarkable advances in phosphoproteomics technologies, the relatively low number of studies performed in human spermatozoa suggests that phosphoproteomics has not been applied to its full potential in studying male infertility yet. Therefore, further studies will improve the application of this procedure and overcome the limitations, increasing the understanding of regulatory mechanisms underlying protein phosphorylation in sperm motility and, consequently, in male fertility. [ABSTRACT FROM AUTHOR]

Study Question: Are epididymosomes implicated in protein transfer from the epididymis to spermatozoa?Summary Answer: We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes.What Is Known Already: Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract.Study Design, Size, Duration: We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy.Participants/materials, Setting, Methods: Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions.Main Results and the Role Of Chance: Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin.Large Scale Data: N/A.Limitations, Reasons For Caution: The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs.Wider Implications Of the Findings: Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility.Study Funding/competing Interest(s): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER 'una manera de hacer Europa' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

Background and Aim: Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels. Materials and Methods: The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman's correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands. Results: The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p

Fertility is the most important aspect in the efforts to increase livestock populations. Protamine and various proteins in sperm and seminal plasma are the results of the molecular analysis which can be used as a marker of fertility. Each of the proteins plays an important role in the normal function of sperm, starting from the formation of sperm structure, motility, capacitation, cell protection, acrosome reactions, successful fertilization, egg activation, and embryonic development. Finally, these molecular components can be a marker of fertility and can help to diagnose the cases of infertility/subfertility in livestock in the field.

Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.

Following an initial recovery, COVID-19 survivors struggle with a spectrum of persistent medical complications, including fatigue, breathlessness, weight loss, hair loss, and attention deficits. Additionally, there is growing evidence of adverse effects of COVID-19 on the male reproductive system. This investigation seeks to understand the long-term ramifications on male fertility by examining hormonal profiles, semen parameters, and sperm proteome of recovered COVID-19 patients compared to controls. The serum hormone profiles between the two groups showed minimal variations except for prolactin, cortisol, and testosterone levels. Testosterone levels were slightly lower, while prolactin and cortisol were elevated in COVID-19 cases compared to controls. Though semen parameters exhibited no significant disparities between the COVID-19 and control groups, quantitative proteomics analysis revealed changes in sperm proteins. It identified 190 differentially expressed proteins, of which 161 were upregulated and 29 downregulated in COVID-19 cases. Western blotting analysis validated the differential expression of serpin B4 and calpain 2. Bioinformatics analysis signifies cellular stress in the spermatozoa of COVID-19 recovered patients and thus, SOD and MDA levels in semen were measured. MDA levels were found to be significantly elevated, indicating lipid peroxidation in COVID-19 samples. While the effects of COVID-19 on semen parameters may exhibit a potential for reversal within a short duration, the alterations it inflicts on sperm proteome are persisting consequences on male fertility. This study paves the path for further research and emphasizes the significance of comprehending the complex molecular processes underlying the long-term consequences of COVID-19 on male reproductive health., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

During transit and storage in epididymis, spermatozoa undergo final maturation, acquire motility, functional competence and the ability to fertilise an oocyte. Epididymal secretions contain a complex biochemical milieu of diverse inorganic ions, proteins, metabolites and other molecules. Since it is believed that spermatozoa are translationally silent, proteins appearing in them are thought to be synthesised elsewhere, including epididymis, and then incorporated to the cells. One of the important mechanisms suggested to be involved in transfer of epididymal secretions to spermatozoa is through exosomes called epididymosomes. Epididymosomes released from the epididymal epithelium contain proteins, noncoding RNAs and distinct set of lipids that are transferred to spermatozoa while they pass through the different epididymal regions. Owing to the importance of these molecules for sperm maturation and fertilising ability, research on epididymosomes has gained increasing attention during the last decade. This review is focused on epididymosomes, with emphasis on recent advances in the understanding of mechanisms of epididymosomal cargo transfer to spermatozoa and potential roles of epididymosomes in sperm function and beyond. Possibilities of utilising the molecular signatures of epididymosomes as a tool for male fertility assessment are also discussed. [ABSTRACT FROM AUTHOR]

Infertility affects approximately 15% of couples worldwide of childbearing age, and in many cases the etiology of male infertility is unknown. The current standard evaluation of semen is insufficient to establish an accurate diagnosis. Proteomics techniques, such as phosphoproteomics, applied in this field are a powerful tool to understand the mechanisms that regulate sperm functions such as motility, which is essential for successful fertilization. Among the post-translational modifications of sperm proteins, this review summarizes, from a proteomic perspective, the updated knowledge of protein phosphorylation, in human spermatozoa, as a relevant molecular mechanism involved in the regulation of sperm physiology. Specifically, the role of sperm protein phosphorylation in motility and, consequently, in sperm quality is highlighted. Additionally, through the analysis of published comparative phosphoproteomic studies, some candidate human sperm phosphoproteins associated with low sperm motility are proposed. Despite the remarkable advances in phosphoproteomics technologies, the relatively low number of studies performed in human spermatozoa suggests that phosphoproteomics has not been applied to its full potential in studying male infertility yet. Therefore, further studies will improve the application of this procedure and overcome the limitations, increasing the understanding of regulatory mechanisms underlying protein phosphorylation in sperm motility and, consequently, in male fertility.

Abstract Background In the assisted reproduction, the infertile molecules of spermatozoa from normozoospermic men who underwent the unexplained failure of in vitro fertilization (IVF) due to the lack of sperm binding to the normal zona pellucida, and then achieved pregnancy with the rescue intracytoplasmic sperm injection (R-ICSI) remain unclear. More works are still necessary to explore this male infertile mechanism. Methods Normozoospermicmen with the IVF pregnancy and normozoospermic men with the R-ICSI pregnancy after the conventional IVF failure were collected. iTRAQ-based proteomic approach were performed to reveal the new infertile causes between the IVF pregnancy men and the R-ICSI pregnancy men. To validate the confidence of proteome data, the individual samples were analyzed by western blot and immunofluorescence. Further, the spontaneous acrosome reactions were measured to evaluate the sperm quality. Results Compared with IVF pregnancy group, 56 sperm proteins were differentially expressed in the R-ICSI pregnancy group. Bioinformatic analyses (PANTHER, DAVID, PubMed and STRING) indicated these altered sperm proteins were involved in various molecular functions: reproduction, chromosome organization, and sperm-oocyte interaction. Moreover, the confidence of proteome data was confirmed by western blot and immunofluorescence using the individual samples, which were consistent with our proteomic data. Additionally, an increased rate of the spontaneous acrosome reaction rate was found in the R-ICSI pregnancy group. Conclusions The sealtered sperm proteins and the increased spontaneous acrosome reaction rate might account for this unexplained male infertility in the R-ICSI pregnancy patients. The present proteomic results will throw light on the better understanding of the unexplained infertile mechanisms underlying these normozoospermic man who achieved R-ICSI pregnancy after IVF failure.

Abstract Background Protein-coding genes expressed in sperm evolve at different rates. To gain deeper insight into the factors underlying this heterogeneity we examined the relative importance of a diverse set of previously described rate correlates in determining the evolution of murine sperm proteins. Results Using partial rank correlations we detected several major rate indicators: Phyletic gene age, numbers of protein-protein interactions, and survival essentiality emerged as particularly important rate correlates in murine sperm proteins. Tissue specificity, numbers of paralogs, and untranslated region lengths also correlate significantly with sperm genes’ evolutionary rates, albeit to a lesser extent. Multifunctionality, coding sequence or average intron lengths, and mean expression level have insignificant or virtually no independent effects on evolutionary rates in murine sperm genes. Gene ontology enrichment analyses of three equally sized murine sperm protein groups classified based on their evolutionary rates indicate strongest sperm-specific functional specialization in the most quickly evolving gene class. Conclusions We propose a model according to which slowly evolving murine sperm proteins tend to be constrained by factors such as survival essentiality, network connectivity, and/or broad expression. In contrast, evolutionary change may arise especially in less constrained sperm proteins, which might, moreover, be prone to specialize to reproduction-related functions. Our results should be taken into account in future studies on rate variations of reproductive genes.

Background and Aim: Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels. Materials and Methods: The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman's correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands. Results: The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p<0.05). Five protein bands were found in the SDS-PAGE of sperm proteins of Holstein bulls with different concentrations. Sperm proteins with MWs of 17.51 kDa, 14.87 kDa, 33.71 kDa, and 41.97 kDa were abundant in the Holstein bulls with an HF level, while 55 kDa proteins were abundant in the LF level of Holstein bulls. The sperm of Holstein bulls in the HF level contained proteins of about 33.71 kDa that were not detected in the LF. Conclusion: The sperm protein with a molecular weight of 33.71 kDa was predicted to be a specific protein biomarker that influences bull fertility. Sperm fertilization abilities were also determined by the sperm proteins, the morphology of sperm acrosomes, and the quality of plasma membranes. This method can be used to select bulls with high fertility to increase the population of Holstein bulls. [ABSTRACT FROM AUTHOR]

Fertility is the most important aspect in the efforts to increase livestock populations. Protamine and various proteins in sperm and seminal plasma are the results of the molecular analysis which can be used as a marker of fertility. Each of the proteins plays an important role in the normal function of sperm, starting from the formation of sperm structure, motility, capacitation, cell protection, acrosome reactions, successful fertilization, egg activation, and embryonic development. Finally, these molecular components can be a marker of fertility and can help to diagnose the cases of infertility/subfertility in livestock in the field. [ABSTRACT FROM AUTHOR]

To unravel the toxic mechanism of phthalate ester plasticizer endocrine disruptor in spermatozoa, we examined the effect of dibutyl phthalate (DBP) on the stability and inhibitory phosphorylation of glycogen synthase kinase 3α (GSK3α), a protein kinase crucial for sperm motility in mice. In DBP-treated spermatozoa, reactive oxygen species (ROS) and lipid peroxide were significantly increased. In computer-assisted sperm analysis, DBP at concentrations of 10 - 100 μg/mL significantly decreased total motility and progressive motility of spermatozoa. On western blots, DBP decreased p-GSK3α(Ser21) and increased p-GSK3α(Tyr279) in spermatozoa. Similarly, hydrogen peroxide decreased p-GSK3α(Ser21) but not p-GSK3α(Tyr279) in spermatozoa. Immunofluorescent labeling demonstrated that DBP markedly decreased immunoreactivities of GSK3α and p-GSK3α(Ser21) but increased immunoreactivity of p-GSK3α(Tyr279) in spermatozoa. DBP at a concentration of 100 μg/mL significantly increased phosphatase activity in spermatozoa. Calyculin A, a protein phosphatase 1 and 2 A inhibitor, markedly increased p-GSK3α(Ser21) and sperm motility and attenuated a DBP-induced decrease of p-GSK3α(Ser21) and sperm motility. On western blot, 1-100 μg/mL DBP decreased GSK3α in spermatozoa. On immunoprecipitation western blot, DBP at 10 - 100 μg/mL increased polyubiquitinated sperm proteins including GSK3α. The MG115, proteasome inhibitor attenuated degradation of GSK3α in DBP-treated spermatozoa. Hydrogen peroxide at 10 μM increased polyubiquitinated sperm proteins, suggesting that DBP may increase ubiquitination of GSK3α via ROS induction. Together, DBP may decrease the cellular amount of GSK3α through the ubiquitin-proteasome pathway and p-GSK3α(Ser21) through ROS generation and activation of protein phosphatases, impairing sperm motility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract Spermatozoa exhibit remarkable variability in size, shape, and performance. Our understanding of the molecular basis of this variation, however, is limited, especially in avian taxa. The zebra finch (Taeniopygia guttata) is a model organism in the study of avian sperm biology and sperm competition. Using LC-MS based proteomics, we identify and describe 494 proteins of the zebra finch sperm proteome (ZfSP). Gene ontology and associated bioinformatics analyses revealed a rich repertoire of proteins essential to sperm structure and function, including proteins linked to metabolism and energetics, as well as tubulin binding and microtubule related functions. The ZfSP also contained a number of immunity and defense proteins and proteins linked to sperm motility and sperm-egg interactions. Additionally, while most proteins in the ZfSP appear to be evolutionarily constrained, a small subset of proteins are evolving rapidly. Finally, in a comparison with the sperm proteome of the domestic chicken, we found an enrichment of proteins linked to catalytic activity and cytoskeleton related processes. As the first described passerine sperm proteome, and one of only two characterized avian sperm proteomes, the ZfSP provides a significant step towards a platform for studies of the molecular basis of sperm function and evolution in birds. Significance Using highly purified spermatozoa and LC-MS proteomics, we characterise the sperm proteome of the Zebra finch; the main model species for the avian order Passeriformes, the largest and most diverse of the avian clades. As the first described passerine sperm proteome, and one of only two reported avian sperm proteomes, these results will facilitate studies of sperm biology and mechanisms of fertilisation in passerines, as well as comparative studies of sperm evolution and reproduction across birds and other vertebrates. Graphical abstract Unlabelled Image Highlights • The zebra finch sperm proteome (ZfSP) was characterized by LC-MS. • We report 494 proteins in the ZfSP, and compare these to the chicken sperm proteome. • The ZfSP includes a diversity of proteins essential to sperm structure and function. • The majority of proteins in the ZfSP exhibit low rates of evolution. • A small subset of proteins in the ZfSP appear to be more rapidly evolving. [ABSTRACT FROM AUTHOR]

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

In the recent years, great attention had been focused on cyanide toxicity because of its widespread use in industries and considered to be a ubiquitous pollutant in the environment. Therefore, the current study aimed to evaluate the toxic effect of cyanide on rat sperms at molecular level by using FT-IR technique. For this purpose, rats were randomly divided into four groups and treated with 0.0, 0.64, 1.2 and 3.2 mg kg−1 body weight (BW) for the period of 90 days. The group treated with lower dose (0.64 mg kg−1 BW) showed an insignificant change in all the peaks, except the peaks assigned to olefinic CH, CH2 asymmetric and CH2 symmetric stretching vibration in the lipids. While, the groups treated with higher doses (1.2 and 3.2 mg kg−1 BW) showed the significant decrease in the area under the peaks corresponds to different bio-molecules. In addition, spectral second derivative analysis showed the significant alteration in α-helix, turns, β-sheet, aggregated β-sheet and random coil structures in the proteins. In conclusion, the selected higher dosage of cyanide had caused significant decrease in the biochemical composition of rat sperms along with structural changes in the proteins. The FT-IR technique is an excellent tool used for the analysis of oxidative damage in the sperms.

In Brief: The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service., Abstract: Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P  > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation. [ABSTRACT FROM AUTHOR]

Summary: 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 in vitro fertilization (IVF) outcome, are not known and require further research. [ABSTRACT FROM AUTHOR]

Contents Antioxidants are known to prevent the reactive oxygen species ( ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll®. Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400 × 106 cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72 hr. Sperm motility, viability, lipid peroxidation ( LPO) and hypo-osmotic swelling test ( HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS- PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity ( VSL) were found significantly ( p < .05) higher in the antioxidant-treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower ( p < .05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase ( p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage. [ABSTRACT FROM AUTHOR]

This study aimed to identify sperm proteomic signatures regulating sperm functions and fertility by: (i) comparing the sperm electrophoretic protein profiles and identifying the differentially abundant proteins among breeding bulls differing in fertility status and (ii) elucidating the possible role of one of the identified novel proteins, PEBP4 on sperm function and fertility. The grouping of bulls as fertile ( n = 6) and low fertile ( n = 6) was performed based on bull fertility index and infertile ( n = 6) based on semen rejection rate (>33%). The sperm motility, fructolysis index, acrosomal reaction, intracellular calcium levels, and seminal plasma fructose and calcium levels were studied among fertility groups. The differentially expressed sperm proteins observed in single- and two-dimensional gel electrophoresis (2 DE) were identified using Nano- LC- MS/ MS. In the fertile bulls, the expression levels of calmodulin ( CALM1), spermadhesinZ13 ( SPADH2), and phosphatidylethanolamine-binding protein 4 ( PEBP4) were significantly ( p < 0.05) higher than in other fertility groups. In bovine, expression of PEBP4 a novel seminal protein was not observed in spermatozoa of infertile bulls. When the bulls were grouped based on the presence ( n = 8) or absence ( n = 10) of PEBP4 protein in spermatozoa, a positive significant ( p < 0.05) association of this protein with the percentage of motile, type-A spermatozoa, and sperm fructose uptake was observed. Further, PEBP4 was localized in elongated spermatids, Leydig cells, excurrent duct system, and principal piece of spermatozoa. These findings suggest a crucial role for the PEBP4 protein in spermiogenesis, epididymal sperm maturation, and sperm motility. This first study in bovine indicates the positive association of PEBP4 in regulating sperm maturation, functions, and fertility and could be a potential marker for predicting semen quality and fertility. [ABSTRACT FROM AUTHOR]

Bull fertility may be defined as the process by which spermatozoa fertilize and activate the ovum and then support embryonic development. Bull fertility is a complex trait having relatively low heritability and plays a vital role for efficient production and reproduction of bovine. Various mechanisms involved in regulating bull fertility associated phenotype and reliable biomarkers are poorly defined. Primary spermatozoa physiology indicators namely sperm molecules and epigenetic factors may play a vital role in predicting sperm physiology and fertility. Spermatozoa and their fingerprints or patterns can play a pivotal role in prediction of fertility in bovine. No reliable tests exist despite genomic selection for evaluating quality of semen and fertility of bull. This review focuses on various molecules related to bull fertility viz., sperm RNAs, seminal plasma proteins, sperm proteins and sperm epigenome. In combination with single-nucleotide polymorphism (SNP), microarrays or sperm molecules and epigenome markers can be used to determine sperm quality and to predict bull fertility. [ABSTRACT FROM AUTHOR]

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21 st to July 20 th ) and at the end (November 20 th to December 21 st ) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-). Rutinary assessments carried out in the ovine reproduction centers (testicular volume, libido, sperm production and mass motility) showed non-significant differences (P ≥ 0.05) between both studied times, as well as the ram ultrasonographic evaluation (Resistive and Pulsatility Index as Doppler parameters; and pixels mean gray level, and hypoechoic areas percentage and density as echotexture parameters). However, at level of sperm functionality, although sperm quality appeared non-significantly lower (P ≥ 0.05) in the EBS, we identified a significantly different (P < 0.05) sperm proteomic profile between the seasonality points. The following proteins were identified with the lowest abundance in the EBS with a fold change > 4, a P = 2.40e-07, and a q = 2.23e-06: Fibrous Sheath-Interacting Protein 2, Disintegrin and Metalloproteinase Domain-Containing Protein 20-like, Phosphoinositide-Specific Phospholipase C, Tektin 5, Armadillo Repeat-Containing Protein 12 Isoform X3, Solute Carrier Family 9B1, Radial Spoke Head Protein 3 Homolog, Pro-Interleukin-16, NADH Dehydrogenase [Ubiquinone] 1 Alpha Subcomplex Subunit 8, Testis, Prostate and Placenta-Expressed Protein, and Acyl Carrier Protein Mitochondrial. In conclusion, while our basic analyses on male and sperm quality showed similar results between the beginning and the end of the breeding season, on a proteomic level we detected a lower expression of sperm proteins linked to the energy metabolism, sperm-oocyte interactions, and flagellum structure in the EBS. Probably, this different protein expression could be related to the lower fertility rate of Assaf ewes after cervical artificial insemination at this time. More importantly, sperm proteins can be used as highly effective molecular markers in predicting sperm fertilization ability related to intraseasonal variations., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Identified through forward genetics, spe-9 was the first gene to be identified in C. elegans as necessary for fertilization. 1 Since then, genetic screens in C. elegans have led to the identification of nine additional sperm genes necessary for fertilization (including spe-51 reported by Mei et al. 2 and the spe-36 gene reported here). 3 , 4 , 5 , 6 , 7 , 8 , 9 This includes spe-45, which encodes an immunoglobulin-containing protein similar to the mammalian protein IZUMO1, and spe-42 and spe-49, which are homologous to vertebrate DCST2 and DCST1, respectively. 4 , 7 , 8 , 10 , 11 , 12 , 13 Mutations in any one of these genes result in healthy adult animals that are sterile. Sperm from these mutants have normal morphology, migrate to and maintain their position at the site of fertilization in the reproductive tract, and make contact with eggs but fail to fertilize the eggs. This same phenotype is observed in mammals lacking Izumo1, Spaca6, Tmem95, Sof1, FIMP, or Dcst1 and Dcst2. 10 , 14 , 15 , 16 , 17 , 18 , 19 Here we report the discovery of SPE-36 as a sperm-derived secreted protein that is necessary for fertilization. Mutations in the Caenorhabditis elegans spe-36 gene result in a sperm-specific fertilization defect. Sperm from spe-36 mutants look phenotypically normal, are motile, and can migrate to the site of fertilization. However, sperm that do not produce SPE-36 protein cannot fertilize. Surprisingly, spe-36 encodes a secreted EGF-motif-containing protein that functions cell autonomously. The genetic requirement for secreted sperm-derived proteins for fertilization sheds new light on the complex nature of fertilization and represents a paradigm-shifting discovery in the molecular understanding of fertilization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Fertilization is a fundamental process in sexual reproduction during which gametes fuse to combine their genetic material and start the next generation in their life cycle. Fertilization involves species-specific recognition, adhesion, and fusion between the gametes. 1 , 2 In mammals and other model species, some proteins are known to be required for gamete interactions and have been validated with loss-of-function fertility phenotypes. 3 , 4 Yet, the molecular basis of sperm-egg interaction is not well understood. In a forward genetic screen for fertility mutants in Caenorhabditis elegans, we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell autonomously and localizes to the surface of the spermatozoa. We further show that the gene product of the mammalian sperm function gene Sof1 is likewise secreted. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization in C. elegans (also see spe-36 5 ). This is also the first experimental evidence that mammalian SOF1 is secreted. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive-tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Infertility affects approximately 15% of couples of reproductive age, and 50% of the cases are directly related to men. The evaluation of male fertility is based on analyses of routine seminal parameters and the use of more advanced techniques can help identify fertility biomarkers. SP22 sperm protein is considered a biomarker in murine species since its concentration is highly correlated with sperm fertility. As the role of this protein as a biomarker is already well-established in other species, we hypothesized that this same correlation could apply to human. Thus, the present study aimed to investigate possible correlations between SP22 concentration and sperm parameters in fertile and infertile men. For this, a study was carried out on 21 volunteers' seminal samples who were grouped according to fertility as fertile (n = 10) or infertile (n = 11). Conventional and functional sperm analyses, membrane protein extraction, quantification and immunolocalization of SP22 were performed. The infertile volunteers showed an increase in the percentage of sperm with abnormalities in head morphology and a decrease in the percentage of sperm with intact plasma membrane and damaged acrosomal membrane. Serum concentration of the hormone SHBG was also decreased in infertile volunteers. The damage to the plasma membrane was positively correlated with the superoxide anion production. Although none of the functional parameters were correlated with SP22 concentration, type D sperm motility was negatively correlated and type A+B sperm motility was positively correlated. This preliminary study opens new paths in the characterization of SP22 as a non-invasive biomarker for predicting fertility/infertility., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Josiane de Lima Rosa reports financial support was provided by State of Sao Paulo Research Foundation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

It is widely established that proteins involved in reproduction diverge between species more quickly than other proteins. For male sperm proteins, rapid divergence is believed to be caused by postcopulatory sexual selection and/or sexual conflict. Here, we derive the expected levels of gene diversity within populations and divergence between them for male sperm protein genes evolving by postcopulatory, prezygotic fertility competition, i.e. the function imputed for some sperm and seminal fluid genes. We find that, at the mutation-selection equilibrium, both gene diversity within species and divergence between them are elevated relative to genes with similar selection coefficients expressed by both sexes. We show that their expected level of diversity is a function of the harmonic mean number of mates per female, which affects the strength of fertility selection stemming from male-male sperm competition. Our predictions provide a null hypothesis for distinguishing between other selective hypotheses accounting for the rapid evolution of male reproductive genes. [ABSTRACT FROM AUTHOR]

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins. [ABSTRACT FROM AUTHOR]

Arboviruses and symbiotic viruses can be paternally transmitted by male insects to their offspring for long-term viral persistence in nature, but the mechanism remains largely unknown. Here, we identify the sperm-specific serpin protein HongrES1 of leafhopper Recilia dorsalis as a mediator of paternal transmission of the reovirus Rice gall dwarf virus (RGDV) and a previously undescribed symbiotic virus of the Virgaviridae family, Recilia dorsalis filamentous virus (RdFV). We show that HongrES1 mediates the direct binding of virions to leafhopper sperm surfaces and subsequent paternal transmission via interaction with both viral capsid proteins. Direct interaction of viral capsid proteins mediates simultaneously invasion of two viruses into male reproductive organs. Moreover, arbovirus activates HongrES1 expression to suppress the conversion of prophenoloxidase to active phenoloxidase, potentially producing a mild antiviral melanization defense. Paternal virus transmission scarcely affects offspring fitness. These findings provide insights into how different viruses cooperatively hijack insect sperm-specific proteins for paternal transmission without disturbing sperm functions., (© 2023. The Author(s).)

Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species., Competing Interests: CC, IM, HM No competing interests declared, (© 2023, Chang et al.)

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility., (© 2023. The Author(s).)

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids 228 WEKHFLDAS 237 was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function., (© 2022 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1–5.6, 37%) and basic (pI; 7.9–10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility. [ABSTRACT FROM PUBLISHER]

To assess the relative impact of functional constraint and post-mating sexual selection on sequence evolution of reproductive proteins, we examined 169 primate sperm proteins. In order to recognize potential genome-wide trends, we additionally analysed a sample of altogether 318 non-reproductive (brain and postsynaptic) proteins. Based on cDNAs of eight primate species (Anthropoidea), we observed that pre-mating sperm proteins engaged in sperm composition and assembly show significantly lower incidence of site-specific positive selection and overall lower non-synonymous to synonymous substitution rates (dN/dS) across sites as compared with post-mating sperm proteins involved in capacitation, hyperactivation, acrosome reaction and fertilization. Moreover, database screening revealed overall more intracellular protein interaction partners in pre-mating than in post-mating sperm proteins. Finally, post-mating sperm proteins evolved at significantly higher evolutionary rates than pre-mating sperm and non-reproductive proteins on the branches to multi-male breeding species, while no such increase was observed on the branches to unimale and monogamous species. We conclude that less protein�protein interactions of post-mating sperm proteins account for lowered functional constraint, allowing for stronger impact of post-mating sexual selection, while the opposite holds true for pre-mating sperm proteins. This pattern is particularly strong in multi-male breeding species showing high female promiscuity. [ABSTRACT FROM AUTHOR]

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arunkumar, Kumaresan, Sinha, Elango, Ebenezer Samuel King, Nag, Karuthadurai, Baithalu, Mohanty, Kumar and Datta.)

The fertility of zebu cattle (Bos indicus) is higher than that of the European purebred (Bos taurus) and crossbred (Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility. [ABSTRACT FROM AUTHOR]

Abstract: Sperm proteins that interact with zona pellucida 3 (ZP3) have not been clearly identified in humans. In the present study, the yeast two-hybrid (Y2H) system was used to identify human sperm proteins that interact with human ZP3. Human ZP3 cDNA was cloned into pAS2-1 yeast vector and used as bait to find reactive proteins in the human testis cDNA library. Six specific clones were obtained that were further confirmed for interaction using the mammalian two-hybrid system. These six clones showed homologies with several proteins in the GenBank database. Of these, the strongest ZP3-interacting protein, that shows 97% homology with ubiquitin associated protein-2 like (UBAP2L), was tested in the hemizona assay. UBAP2L antibodies significantly (p <0.001) inhibited human sperm–zona binding in this assay. We conclude that the Y2H system is a useful strategy for identifying novel genes encoding proteins that interact with ZP proteins. To our knowledge, this is the first study using the Y2H system to identify sperm proteins that interact with human oocyte ZP3. Novel proteins identified using this system may find applications in elucidating the fertilization cascade, development of a new generation of non-steroidal contraceptives, and specific diagnosis and treatment of human infertility. [Copyright &y& Elsevier]

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10-13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24-29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39-44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved. [ABSTRACT FROM AUTHOR]

The availability of the amino acid sequence for nine different mammalian P1 family protamines and the revised amino acid sequence of the chicken protamine galline (Oliva and Dixon 1989) reveals a much close relationship between mammalian and avian protamines than was previously thought (Nakano et al. 1976). Dot matrix analysis of all protamine genes for which genomic DNA or cDNA sequence is available reveals both marked sequence similarities in the mammalian protamine gene family and internal repeated sequences in the chicken protamine gene. The detailed alignments of the cis-acting regulatory DNA sequences shows several consensus sequence patterns, particularly the conservation of a cAMP response element (CRE) in all the protamine genes and of the regions flanking the TATA box, CAP site, N-terminal coding region, and polyadenylation signal. In addition we have found a high frequency of the CA dinucleotide immediately adjacent to the CRE element of both the protamine genes and the testis transition proteins, a feature not present in other genes, which suggests the existence of an extended CRE motif involved in the coordinate expression of protamine and transition protein genes during spermatogenesis. Overall these findings suggest the existence of an avian-mammalian P1 protamine gene line and are discussed in the context of different hypotheses for protamine gene evolution and regulation. [ABSTRACT FROM AUTHOR]

Prolamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that prolamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically. [ABSTRACT FROM AUTHOR]