Phosphorylation Of Sperm Proteins Research Articles

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

Male infertility and perfluoroalkyl and poly-fluoroalkyl substances: evidence for alterations in phosphorylation of proteins and fertility-related functional attributes in bull spermatozoa

Abstract Background Perfluoroalkyl and poly-fluoroalkyl substances (PFAS) are pervasive environmental pollutants and potential threats to reproductive health. Epidemiological studies have established an association between PFAS and male infertility, but the underlying mechanisms are unclear. Objectives Investigate the effect of perfluorooctane sulfonic acid (PFOS), the most prevalent and representative PFAS, on bull sperm protein phosphorylation and function. Methods We exposed bull sperm to PFOS at 10 (average population exposure) and 100 μM (high-exposure scenario), and analyzed global proteomic and phosphoproteomic analysis by TMT labeling and Nano LC-MS/MS. We also measured sperm fertility functions by flow cytometry. Results PFOS at 10-μM altered sperm proteins linked to spermatogenesis and chromatin condensation, while at 100 μM, PFOS affected proteins associated with motility and fertility. We detected 299 phosphopeptides from 116 proteins, with 45 exhibiting differential expression between control and PFOS groups. PFOS dysregulated phosphorylation of key proteins (ACRBP, PRKAR2A, RAB2B, SPAG8, TUBB4B, ZPBP, and C2CD6) involved in sperm capacitation, acrosome reaction, sperm–egg interaction, and fertilization. PFOS also affected phosphorylation of other proteins (AQP7, HSBP9, IL4I1, PRKAR1A, and CCT8L2) related to sperm stress resistance and cryotolerance. Notably, four proteins (PRM1, ACRBP, TSSK1B, and CFAP45) exhibited differential regulation at both proteomic and phosphoproteomic levels. Flow cytometric analysis confirmed that PFOS increased protein phosphorylation in sperm and also decreased sperm motility, viability, calcium, and mitochondrial membrane potential and increased mitochondrial ROS in a dose-dependent manner. Conclusions This study demonstrates that PFOS exposure negatively affects phosphorylation of proteins vital for bull sperm function and fertilization.

Sperm Phosphoproteome: Unraveling Male Infertility.

Simple SummaryApproximately 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.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Exogenous gamma-aminobutyric acid addition enhances porcine sperm acrosome reaction.

The widely used porcine artificial insemination procedure involves the use of liquid‐stored semen because it is difficult to control the quality of frozen–thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma‐aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABAA receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABAA receptor antagonist. GABA binds to GABAA receptors, resulting in chloride ion influx. We found that treatment with 1 μM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function.

Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm

Simple SummaryPhosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during the epididymal transit. We demonstrated that 27 proteins underwent phosphorylation both in and out of the breeding season. Differences in the phosphorylation status were demonstrated in the case of endoplasmic reticulum chaperone BiP, albumin, protein disulfide-isomerase A3, nesprin-1, peroxiredoxin-5, and protein bicaudal D homolog.Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables.

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.

Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm.

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20-23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.

The Combined Effects of Recombinant Human Oviductin (rHuOVGP1) and Progesterone on Tyrosine Phosphorylation of Sperm Proteins

Worldwide, around 15% of couples who seek to conceive suffer from infertility. Assisted reproductive technology (ART) would have never been possible without research endeavors. However, to further improve the fertilization rates of ART procedures, there is still more to be done. The mammalian oviductal cells synthesize and secrete a major glycoprotein know as oviductin, or oviduct-specific glycoprotein (OVGP1). This protein has been implicated in enhancing sperm capacitation, sperm motility, sperm penetration and fertilization. Our lab has successfully produced recombinant human oviductin (rHuOVGP1), which has been shown to enhance tyrosine phosphorylation of sperm proteins, a biochemical hallmark of capacitation that takes place in the sperm tail. Additionally, the sex hormone progesterone (P4) has been implicated in increasing the influx of calcium ions through the CatSper channel which is similarly located in the sperm tail and is an important aspect of capacitation. Here we performed a study in the hope of gaining a better understanding of the mechanism of sperm capacitation by examining if rHuOVGP1 works synergistically with P4 to regulate tyrosine phosphorylation of sperm proteins during capacitation. Fresh sperm samples were processed and capacitated at different time points in the absence or presence of rHuOVGP1 (50 mg/mL) with or without P4. The results obtained indicate that both rHuOVGP1 and P4 can enhance tyrosine phosphorylation of sperm proteins, however, the best result was obtained when rHuOVGP1 was used in combination with P4. The addition of rHuOVGP1 with P4 to the capacitation medium may improve the fertilization rates of ART procedures.

Mercury-Induced Inhibition of Tyrosine Phosphorylation of Sperm Proteins and Altered Functional Dynamics of Buck Spermatozoa: an In Vitro Study.

Present study was undertaken on buck spermatozoa to investigate the effect of mercuric chloride on functional dynamics of buck spermatozoa. Four different concentrations (0.031, 0.125, 0.25 and 1.25μg/mL) of mercuric chloride, which were 1/40th, 1/10th, 1/5th and equivalent to the LC50 value of HgCl2, were selected for studying their effect following in vitro exposure for 15min and 3h. Exposure of spermatozoa to 0.031μg/mL mercuric chloride for 3h resulted in significant (p < 0.05) decrease in sperm motility, sperm having intact membrane, intact acrosome and high mitochondrial trans-membrane potential. However, following exposure to higher concentrations (0.25, 1.25μg/mL), similar results were observed even after 15min of exposure. HgCl2 significantly (p < 0.05) increased the levels of malondialdehyde and reactive oxygen species and significantly (p < 0.05) decreased total antioxidant capacity and superoxide dismutase activity in spermatozoa within 15min of exposure. Mercuric chloride-treated spermatozoa did not show capacitation, rather exhibited spontaneous acrosome reaction along with significant increase in intracellular Ca2+ and cAMP levels. Immuno-blotting of semen samples of control and 0.031μg/mL mercury-treated groups showed low intensity bands of p55, p70, p80, p105 and p190kDa tyrosine phosphorylation proteins while higher concentration-treated groups showed no such bands. Our findings evidently suggest that mercuric chloride even at 0.031μg/mL adversely affected sperm functions, inhibited tyrosine phosphorylation proteins and capacitation due to oxidative stress. Spontaneous acrosome reaction (AR) in mercury-treated spermatozoa may possibly be due to increase in intracellular Ca2+ and cAMP levels, and capacitation failure may be due to inhibition of tyrosine phosphorylation of proteins.

Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.

Capacitation of mouse sperm is modulated by gamma-aminobutyric acid (GABA) concentration.

In mammals, ejaculated sperm acquire their fertilizing ability during migration through the female reproductive tract, which secretes several factors that contribute to sperm capacitation. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter in the central nervous system, but additionally enhances the sperm acrosome reaction in the rat and cow. However, the detailed effects of GABA concentration on sperm function remain unclear. In this study, we detected the presence of the GABA type A receptor (GABA A) in mouse epididymal sperm by western blot analysis and in the sperm acrosome by immunocytochemistry. We also investigated the effects of GABA on sperm fertilizing ability. We found that GABA facilitated the tyrosine phosphorylation of sperm proteins, which is an index of sperm capacitation. GABA also promoted the acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. We then found that the effective GABA concentration for the acrosome reaction corresponds to sperm concentration, but we did not detect any marked effect of GABA on sperm motility using a computer-assisted sperm analysis system. Using immunohistochemistry, we also detected GABA expression in the epithelia of the mouse uterus and oviduct. Furthermore, we found that the mRNA levels of glutamate decarboxylase (Gad), which generates GABA from L-glutamate, were higher in the oviduct than in the uterus, and that Gad mRNA levels were higher at estrus than at the diestrus stage. These results indicate that the GABA concentration can act as a modulator of the acrosome reaction and sperm capacitation in the female reproductive tract.

Evaluation of protein phosphorylation in bull sperm during their maturation in the epididymis.

Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2±49.5 and 1252.6±86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction.

The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.

Lead Inhibits Human Sperm Functions by Reducing the Levels of Intracellular Calcium, cAMP, and Tyrosine Phosphorylation.

It is well known that there has been a worldwide decrease in human male fertility in recent years. One of the main factors affecting this is environmental pollution. Lead is one of the major heavy metal contaminants that threaten the health of animals and human beings in China. It preferentially accumulates in male reproductive organs and can be up to 10 µM in human seminal plasma. Lead impairs mammalian spermatogenesis and sperm quality in vivo. It also inhibits sperm functions in vitro but the underlying mechanisms remain unclear. Therefore, we aimed to investigate the in vitro toxicity of lead on human sperm functions and to elucidate the underlying mechanisms. Semen samples were collected from 20 healthy volunteers with different careers and backgrounds living in Nanchang, Jiangxi. Human sperm suspensions were treated with different concentrations of lead acetate (0, 0.5, 2.5, 10, 50, and 100 µM) and the viability, motility, capacitation and progesterone-induced acrosome reaction were examined. Treatment with 10-100 µM lead acetate dose-dependently inhibited total and progressive motility measures, capacitation and progesterone-induced acrosome reaction. It also dose-dependently decreased the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i), and reduced the tyrosine phosphorylation of sperm proteins, all of which are thought to be key factors in the regulation of sperm function. Our findings suggest that lead inhibits human sperm functions by reducing the levels of sperm intracellular cAMP, [Ca(2+)]i and tyrosine phosphorylation of sperm proteins in vitro.

Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction.

In a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm-zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0-100 μg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm-zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro.

Effect of sodium fluoride on male mouse fertility.

Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mm) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short-term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase-A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process ofCorynorhinus mexicanusbat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.