Sperm Acrosome Reaction Research Articles

Comparative proteomic analysis of seminal plasma exosomes in buffalo with high and low sperm motility

BackgroundExosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo.ResultsSPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis.ConclusionThis study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility.

Heat stress disrupts spermatogenesis via modulation of sperm-specific calcium channels in rats

Heat is a detrimental environmental stressor that disrupts spermatogenesis and results in male infertility. Previous investigations have shown that heat stress reduces the motility, number, and fertilization ability of living spermatozoa. Sperm hyperactivation, capacitation, acrosomal reaction, and chemotaxis towards the ova are regulated by the cation channel of sperm (CatSper). This sperm-specific ion channel triggers the influx of calcium ions into sperm cells. The aim of this study in rats was to investigate whether heat treatment affected the expression levels of CatSper-1 and -2, together with the sperm parameters, testicular histology and weight. The rats were exposed to heat stress for 6 days and the cauda epididymis and testis were collected 1, 14, and 35 days after heat treatment to measure sperm parameters, gene and protein expression, testicular weight, and histology. Interestingly, we found that heat treatment caused a notable downregulation of CatSper-1 and -2 expression at all three time points. In addition, there were significant reductions in sperm motility and number and an increase in the percentage of abnormal sperm at 1 and 14 days, with cessation of sperm production at 35 days. Furthermore, expression of the steroidogenesis regulator, 3 beta-hydroxysteroid dehydrogenase (3β-HSD) was upregulated in the 1-, 14- and 35-day samples. Heat treatment also upregulated the expression of the apoptosis regulator, BCL2-associated X protein (BAX), decreased testicular weight, and altered testicular histology. Therefore, our data showed for the first time that heat stress downregulated CatSper-1 and -2 in the rat testis, and that this may be a mechanism involved in heat stress-induced impairment of spermatogenesis.

The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.

Influence of hypoxanthine concentrations on capacitation, acrosome reaction and In vitro fertilization in Ram Spermatozoa

The aim of the study was to evaluate the effect of hypoxanthine concentrations and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) in ram sperm in vitro. Semen samples (n= 40) were collected using artificial vagina from 3 mature Barki rams. Freshly ejaculated spermatozoa were pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of hypoxanthine (5, 10, 15, 20 mg/ml). Motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation. They were examined for ability to fertilize oocyte in vitro. The results showed that hypoxanthine concentrations and incubation time for 2 h did not significantly affect the motility. However, increase in incubation time for >2 h significantly (P<0.05) decreased the motility of spermatozoa as compared to control. Treatment of spermatozoa with 20 mg/ml hypoxanthine significant (P<0.05) increased in HA after 2 h of incubation compared to control (5.2±0.3 vs. 1.0± 0.1%). Significant (P<0.05) increase in total AR was associated to the increase in concentration of hypoxanthine with maximum at 20 mg/ml at 3h of incubation (82.0±2.2%). The fertilization rate of oocytes treated with 20 mg/ml hypoxanthine was higher as compared with control (42.8 vs. 26.0%). In conclusion, treatment of ram spermatozoa with 20 mg/ml hypoxanthine for 2 to 3 h was considered the best concentration for in vitro fertilization in sheep.

Calcitriol induces post-thawed bovine sperm capacitation

<b>Background:</b> Capacitation is a set of physiological changes sperms undergo to acquire fertilizing capacity. <i>In vivo</i>, this process is directly associated with high calcium levels in sperm cytoplasm. Calcitriol, the vitamin D hypercalcemic metabolite, is related to human sperm motility, capacitation, and acrosome reaction. This work aimed to study the effect of calcitriol on bull sperm quality parameters and capacitation. <b>Methods:</b> One million freeze-thawed spermatozoa were obtained from different bulls and treated with 20 nM of calcitriol for 30 min. Untreated cells (negative control) and treated ones with calcitriol or heparin (100 µg/mL, positive capacitation control) were evaluated for motility, viability, and functional parameters. Menadione (70 µM, 30 min) treatment was included as a reactive oxygen species (ROS) positive sperm agent. <b>Results:</b> The results elucidated that sperm exposed to 20 nM calcitriol showed higher viability, vigor, and capacitation than their positive and negative controls. The percentage of sperm with intact plasma and acrosome membranes, mitochondrial membrane potential (ΔΨm), and phosphatidylserine externalization was similar in all the conditions evaluated, while ROS production was higher with heparin and menadione-treated groups than the calcitriol group or negative control. <b>Conclusion:</b> Our results indicate that calcitriol induces the capacitation of thawed bull spermatozoa and maintains acceptable values of progressive motility, viability, and vigor without altering key biological parameters such as redox status, ΔΨm, and cell death.

Extender Supplementation with Glutathione (GSH) and Taurine Improves In Vitro Sperm Quality and Antioxidant Status of New Zealand Rabbits during Chilled Storage for up to 72 hours.

This study assessed the influence of supplementing the rabbit semen extender with various concentrations of glutathione (GSH) and taurine at 24, 48, and 72 h postchilling at 5°C. Semen samples were collected from 20 New Zealand bucks, and ejaculates with standard color, motility (>85%), about 0.5 mL volume, and ∼400 × 106/mL concentration were used and diluted with extenders supplemented with 0.5, 1, and 2 mM of GSH and 1, 5, and 10 mM of taurine and chilled at 5°C. Nonsupplemented samples were used as a control. Sperm's progressive motility, acrosome reaction, and extracellular oxidative stress biomarkers such as MDA contents and GPx, SOD, and CAT concentrations and intracellular transcriptomic levels of SOD and CAT genes were assessed. GSH and taurine supplementation improved the sperm's kinetics by reducing cooling-associated stress, which was ascertained by lowering MDA concentration and increasing SOD, CAT, and GPx concentrations (P < 0.05). Increasing the levels of antioxidant enzymes in the extender was due to the increasing mRNA copies of the SOD and CAT genes (P < 0.05). Furthermore, GSH and taurine maintained the fructose levels in the extender and lowered the GPT levels, which implies sperm membrane stability is maintained through GSH and taurine supplementation. GSH and taurine supplementation to the extender had protective influences on the in vitro rabbit semen quality during chilled storage for up to 72 h, which were remarkable with increasing supplementation dose and cooling time at 5°C.

Effect of Antioxidant on Sperm Freezing

A major cause of male infertility, which includes 50 causes of infertility, is oxidative stress. Aerobic respiration in the sperm cell is associated with the production of reactive oxygen species (ROS). Some reactions, such as sperm capacitation and acrosome reactions require moderate levels of ROS. However, when the amount of ROS production surpasses the natural antioxidant defense of the cell, it causes a harmful effect called oxidative stress. This results in the oxidation of lipids, proteins, carbohydrates, and nucleotides. The production of ROS is increased by various factors, including the presence of leukocytes, sperms with abnormal and immature morphology, centrifugation of samples, and changes in oxygen concentration, pH, and temperature. Sperm freezing is a method widely used in assisted reproductive technology (ART) as well as infertility treatment. The process of freezing also contributes to increased oxidative stress because it alters the fluidity of the mitochondrial membrane, leading to increased production and release of ROS. Sperm have defense mechanisms against oxidative stress caused by increased ROS production, which includes the presence of enzymatic and non-enzymatic antioxidants in semen. The utilization of supplementary antioxidants in the freezing environment is a strategy to combat oxidative stress. This review aims to summarize the current evidence on the effect of antioxidants on sperm freezing.

Effects of capacitating media and incubation time on collared peccary sperm quality, acrosome reaction, and binding ability

Context Several assisted reproduction techniques have been proposed for collared peccaries due to the interest in its conservation. However, there is little information about the requirements for sperm capacitation, an initial step in in vitro fertilisation. Aims We aimed to determine the optimal conditions for collared peccary sperm capacitation by comparing Tyrode’s albumin lactate pyruvate (TALP) or the same media plus heparin, caffeine, or their combination at different exposure times (1, 3, and 6 h). Methods The samples were evaluated for kinetic parameters, membrane functionality and integrity, mitochondrial activity, morphology, DNA integrity, reactive oxygen species (ROS) expression levels, capacitation, and sperm binding ability using swine oocytes. Key results Samples incubated with caffeine or heparin had a higher percentage of capacitated spermatozoa. The maximum percentage of capacitation was achieved after 3 h of incubation with either agent. Moreover, spermatozoa subjected to heparin capacitation showed better motility than when subjected to caffeine, and lower ROS expression levels after 1 h. No differences were observed among incubation times for the binding ability. Conclusions In summary, collared peccary spermatozoa can be capacitated with caffeine or heparin; however, heparin better maintains sperm motility and ROS expression levels. The co-incubation of gametes in a medium with heparin for 3 h could be efficient for in vitro fertilisation in collared peccaries. Implications This result will contribute to the development of assisted reproduction techniques for conservation and productivity of collared peccaries.

Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

Biochemical metabolomic profiling of the Crown-of-Thorns Starfish (Acanthaster): New insight into its biology for improved pest management

The Crown-of-Thorns Starfish (COTS), Acanthaster species, is a voracious coral predator that destroys coral reefs when in outbreak status. The baseline metabolite and lipid biomolecules of 10 COTS tissues, including eggs from gravid females, were investigated in this study to provide insight into their biology and identify avenues for control. Targeted and untargeted metabolite- and lipidomics-based mass spectrometry approaches were used to obtain tissue-specific metabolite and lipid profiles. Across all COTS tissues, 410 metabolites and 367 lipids were identified. Most abundant were amino acids and peptides (18.7%) and wax esters (17%). There were 262 metabolites and 192 lipids identified in COTS eggs. Wax esters were more abundant in the eggs (30%) followed by triacylglycerols (TG), amino acids, and peptides. The diversity of asterosaponins in eggs (34) was higher than in tissues (2). Several asterosaponins known to modulate sperm acrosome reaction were putatively identified, including glycoside B, asterosaponin-4 (Co-Aris III), and regularoside B (asterosaponin A). The saponins saponin A, thornasteroside A, hillaside B, and non-saponins dictyol J and axinellamine B which have been shown to possess defensive properties, were found in abundance in gonads, skin, and radial nerve tissues. Inosine and 2-hexyldecanoic acid are the most abundant metabolites in tissues and eggs. As a secondary metabolite of purine degradation, inosine plays an important role in purine biosynthesis, while 2-hexyldecanoic acid is known to suppress side-chain crystallization during the synthesis of amphiphilic macromolecules (i.e., phospholipids). These significant spatial changes in metabolite, lipid, and asterosaponin profiles enabled unique insights into key biological tissue-specific processes that could be manipulated to better control COTS populations. Our findings highlight COTS as a novel source of molecules with therapeutic and cosmetic properties (ceramides, sphingolipids, carnosine, and inosine). These outcomes will be highly relevant for the development of strategies for COTS management including chemotaxis-based biocontrol and exploitation of COTS carcasses for the extraction of commercial molecules.

N-acetyl-l-cysteine and alpha lipoic acid are protective supplement on human sperm parameters in cryopreservation of asthenoteratozoospermia patients.

The aim of this study is to evaluate the beneficial effects of N-acetyl-cysteine (NAC), alpha lipoic acid (ALA) and combination of NAC + ALA supplement in freezing medium on Sperm structural and functional in asthenoteratozoospermia patients. Thirty freshly ejaculated semen samples were cryopreserved with sperm freezing medium (SFM) as control group and three group that SFM supplemented with NAC, ALA and their combination NAC+ ALA. The sperm samples were analysed according to WHO. Mitochondrial membrane potential (MMP), acrosome reaction (AR), antioxidant enzymes and DNA fragmentation were assessed using by Rhodamine123, PSA- FITC ELISA and TUNEL staining respectively. Expression level of NRF2 was assessed by real-time PCR assay. NAC and ALA alone significantly improved sperm motility, viability and DNA fragmentation (p< 0.05). MMP increased in NAC and ALA separately (p< 0.05). While did not affect the amount of sperm morphology and AR (p> 0.05). Antioxidant enzymes significantly difference in NAC and ALA groups (p< 0.05). In addition, NAC and ALA groups showed a significantly higher expression of NRF2 gene compared with other groups (p< 0.05). Our results revealed that the ALA and NAC supplements had a protective effect in cryopreservation process on the structural and functional characteristics of sperm.

Cortisol modulates Ca2+ signaling and acrosome reaction in human sperm.

Stress and elevated cortisol levels have negative effects on fertility, although there is controversy about the effect of cortisol on human sperm. One study reported that hydrocortisone (HC), the synthetic form of cortisol, does not activate CatSper channel but is able to inhibit its activation by progesterone (Pg). However, subsequent reports showed that HC has an agonist effect on CatSper, producing intracellular Ca2+ ([Ca2+ ]i ) increases. These studies were performed using only electrophysiological techniques and fluorometric measurements of Ca2+ . Therefore, it is important to evaluate the effects of different HC concentrations on human sperm physiology using other approaches and techniques. To analyze the effects of different HC concentrations on human sperm physiology and to evaluate the association of perceived stress, anxiety and depression with acrosome reaction (AR), and semen quality. In capacitated human sperm, we tested the effects of HC on [Ca2+ ]i mobilization and AR. Furthermore, the perceived stress and symptoms of anxiety and depression were evaluated with standardized psychometric tests. HC induces a low increase in [Ca2+ ]i compared to Pg response while, at higher concentrations, HC inhibits the Pg-induced Ca2+ response. Also, low concentrations of HC induce AR and when co-incubating it with Pg, the percentages of AR are decreased. Finally, the anxiety symptoms are negatively correlated with Pg induced AR and semen quality. HC has an agonist effect on [Ca2+ ]i mobilization and AR. However, our data also suggest that HC may compete for the binding site of Pg. Additionally, the association between anxiety and decreased AR could be explained by high cortisol levels. Our data showed that HC interferes with the Pg action. Therefore, the negative correlation between anxiety symptoms and Pg-induced AR is due to the accompanying high cortisol levels of this condition and its competitive action with Pg.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 μM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 μM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.

Identification and Validation of Yak (Bos grunniens) Frozen-Thawed Sperm Proteins Associated with Capacitation and the Acrosome Reaction.

To achieve fertilization, mammalian spermatozoa must undergo capacitation and the acrosome reaction (AR) within the female reproductive tract. However, the effects of cryopreservation on sperm maturation and fertilizing potential have yet to be established. To gain insight into changes in protein levels within sperm cells prepared for use in the context of fertilization, a comprehensive quantitative proteomic profiling approach was used to analyze frozen-thawed Ashidan yak spermatozoa under three sequential conditions: density gradient centrifugation-based purification, incubation in a capacitation medium, and treatment with the calcium ionophore A23187 to facilitate AR induction. In total, 3280 proteins were detected in these yak sperm samples, of which 3074 were quantified, with 68 and 32 being significantly altered following sperm capacitation and AR induction. Differentially abundant capacitation-related proteins were enriched in the metabolism and PPAR signaling pathways, while differentially abundant AR-related proteins were enriched in the AMPK signaling pathway. These data confirmed a role for superoxide dismutase 1 (SOD1) as a regulator of sperm capacitation while also offering indirect evidence that heat shock protein 90 alpha (HSP90AA1) regulates the AR. Together, these findings offer a means whereby sperm fertility-related marker proteins can be effectively identified. Data are available via Proteome Xchange with identifier PXD035038.

CRYOCAPACITATION AND ITS ASSOCIATION WITH OXIDATIVE FEATURES IN CRYOPRESERVED BOVINE SPERMATOZOA

The goal of our research was to evaluate the impact of cryopreservation on the antioxidant activity, lipid peroxidation and protein carbonylation as well as its connection with the progress of cryocapacitation in bovine spermatozoa. As biological material we used semen obtained from 20 sexually mature Holstein bulls. Each ejaculate was divided into three equal aliquots as follows: the first part or control (CTRL) was incubated in physiological saline solution while the second part (CAP) was incubated in a capacitation medium at specific conditions. The third part of each sample was cryopreserved (CRYO) and stored in liquid nitrogen at -196°C for further analysis. The motility of spermatozoa was assessed with CASA (computer assisted sperm analysis), while the capacitation status was evaluated by the chlortetracycline (CTC) fluorescent staining. Total antioxidant capacity (TAC) and the level of lipid peroxidation (LPO) was measured with a combined spectro-fluoro-luminometer. The presence of protein oxidation was detected by the traditional DNPH (2,4-dinitrophenylhydrazine) method and evaluated spectrophotometrically. Based on our data, the motility was significantly decreased (P&lt;0.0001; P&lt;0.01) in the CRYO group against CAP and CTRL. There was a significant increase (P&lt;0.01; P&lt;0.05) of acrosome-reacted spermatozoa (AR-pattern) in the CRYO group when compared to CTRL and CAP, while TAC was statistically decreased (P&lt;0.05) between the CRYO and CAP experimental group. In the case of protein oxidation and LPO, both parameters were significantly higher (P&lt;0.0001) in the CRYO group when compared to CAP or CTRL. In summary, cryopreservation induces capacitation-like changes and promotes oxidative damage in frozen-thawed bovine spermatozoa.

Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens).

Mammalian spermatozoa are not mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanism of protein phosphorylation during frozen-thawed sperm capacitation and AR has not been well studied. In this study, the phosphoproteomics approach was employed based on tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment). The identification of 1,377 proteins with 5,509 phosphorylation sites revealed changes in phosphorylation levels of sperm-specific proteins involved in regulation of spermatogenesis, sperm motility, energy metabolism, cilium movement, capacitation and AR. Some phosphorylated proteins, such as AKAP3, AKAP4, SPA17, PDMD11, CABYR, PRKAR1A, and PRKAR2A were found to regulate yak sperm capacitation and AR though the cAMP/PKA signaling pathway cascades. Notably, the phosphorylation level of SPA17 at Y156 increased in capacitated sperm, suggesting that it is also a novel functional protein besides AKAPs during sperm capacitation. Furthermore, the results of this study suggested that the phosphorylation of PRKAR1A and PRKAR2A, and the dephosphorylation of CABYR both play key regulatory role in yak sperm AR process. Protein-protein interaction analysis revealed that differentially phosphorylated proteins (AKAP3, AKAP4, FSIP2, PSMD11, CABYR, and TPPP2) related to capacitation and AR process played a key role in protein kinase A binding, sperm motility, reproductive process, cytoskeleton and sperm flagella function. Taken together, these data provide not only a solid foundation for further exploring phosphoproteome of sperm in yak, but an efficient way to identify sperm fertility-related marker phosphorylated proteins.

G-Protein Coupled Receptors in Human Sperm: An In Silico Approach to Identify Potential Modulatory Targets.

G protein-coupled receptors (GPCRs) are involved in several physiological processes, and they represent the largest family of drug targets to date. However, the presence and function of these receptors are poorly described in human spermatozoa. Here, we aimed to identify and characterize the GPCRs present in human spermatozoa and perform an in silico analysis to understand their potential role in sperm functions. The human sperm proteome, including proteomic studies in which the criteria used for protein identification was set as <5% FDR and a minimum of 2 peptides match per protein, was crossed with the list of GPCRs retrieved from GLASS and GPCRdb databases. A total of 71 GPCRs were identified in human spermatozoa, of which 7 had selective expression in male tissues (epididymis, seminal vesicles, and testis), and 9 were associated with male infertility defects in mice. Additionally, ADRA2A, AGTR1, AGTR2, FZD3, and GLP1R were already associated with sperm-specific functions such as sperm capacitation, acrosome reaction, and motility, representing potential targets to modulate and improve sperm function. Finally, the protein-protein interaction network for the human sperm GPCRs revealed that 24 GPCRs interact with 49 proteins involved in crucial processes for sperm formation, maturation, and fertilization. This approach allowed the identification of 8 relevant GPCRs (ADGRE5, ADGRL2, GLP1R, AGTR2, CELSR2, FZD3, CELSR3, and GABBR1) present in human spermatozoa that can be the subject of further investigation to be used even as potential modulatory targets to treat male infertility or to develop new non-hormonal male contraceptives.

Presence of leptin and its receptor in the ram reproductive system and in vitro effect of leptin on sperm quality.

Leptin is a 16 kDa hormone encoded by obese (OB) gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of mammals. Leptin and its receptor (OBR) have been found in male reproductive systems of human, bovine, equine and pig. The effects of leptin on sperm quality vary widely from different research findings. However, the presence of leptin and its receptor in the ram reproductive system and the in vitro effect of leptin on sperm quality have not reported yet. In the present study, we found that the OB was highly expressed in primary and secondary spermatocytes of the testes, OBR was highly expressed in secondary spermatocytes of the testes. The expressions of OB were in stereocilia of epididymis and in columnar cells of epididymal caput and cauda, the expressions of OBR were in columnar cells of epididymis and in stereocilia of epididymal corpus and cauda. The presence of both OB and OBR in testes, epididymis and sperm were confirmed through RT-PCR, immunolocalization and Western blot analyses. The RT-qPCR results indicated OB and OBR had higher expression levels in epididymal sperm than that of the ejaculated sperm in rams. When sperm were treated with 5 ng/mL leptin, the progressive motility (P < 0.01), straight-line velocity (VSL) (P < 0.05), average path velocity (VAP) (P < 0.05), membrane mitochondrial potential (MMP) (P < 0.01) and viability (P < 0.05) significantly increased, while DNA fragmentation index (DFI) and reactive oxygen species (ROS) significantly decreased compared to the control (P < 0.01), and the other semen parameters such as acrosome integrity and acrosome reaction rate had no significant changes between groups (P > 0.05). In conclusion, this is probably the first report describing localization of leptin and its receptors in the reproductive system of rams and their effects on sperm quality parameters. Our findings suggest that 5 ng/mL leptin treatment enhanced sperm motility, viability and MMP, and decrease DFI and ROS without obvious influence on the acrosome reaction in ram sperm. The potential mechanisms may be related to leptin’s ability to reduce the oxidative stress and apoptosis of sperms and improve their mitochondrial function and energy supply, therefore, to maintain the physiological homeostasis of the sperm.

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.