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The sperm ability to fertilize involves the regulation of ATP levels. Because inside cells, ATP is complexed with Mg 2+ ions, changes in ATP levels result in changes in intracellular Mg 2+ concentration ([Mg 2+ ] i ), which can be followed using intracellular Mg 2+ sensors such as Mag-520. In this work, we tested conditions known to decrease sperm ATP such as starvation and capacitation. As expected, in these conditions [Mg 2+ ] i increased in all cell compartments. In contrast, when ATP increases, such as adding nutrients to starved sperm, [Mg 2+ ] i significantly decreases in all compartments. On the other hand, when the acrosome reaction was induced, either with progesterone or with ionomycin, [Mg 2+ ] i was differentially regulated in the head and mid-piece. While Mag-520 fluorescence increased in the sperm mid-piece, it decreased in the head. These changes were observed in capacitated as well as in starved sperm but not in sperm incubated in conditions that do not support capacitation. Changes in [Mg 2+ ] i were still observed when the sperm were incubated in high extracellular Mg 2+ suggesting that this decrease is not due to Mg 2+ efflux. Interestingly, the progesterone and ionomycin effects on [Mg 2+ ] i were abolished on sperm incubated in Ca 2+ -free media. Altogether, these results indicate that [Mg 2+ ] i is regulated in sperm during capacitation and acrosomal reaction, and suggest that these measurements can serve to evaluate ATP levels in real time., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Sperm capacitation is a prerequisite for natural or in vitro fertilization. After capacitation, sperm become hyperactivated and undergo an acrosome reaction, which helps them penetrate the oocyte. Cordycepin, a bioactive compound first isolated from Cordyceps militaris, is an adenosine analog with numerous physiological activities. However, its effects on sperm capacitation remain unclear. This study aims to elucidate the effects and mechanisms of cordycepin on human sperm capacitation., Methods: During in vitro capacitation culture, healthy human sperm were treated with cordycepin (20, 100, 500 µM). Sperm motility and hyperactivation were detected using a computer-assisted sperm analyzer. Sperm acrosome reaction was measured using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. Sperm protein kinase A (PKA) activity was analyzed using an ELISA kit. The levels of sperm protein tyrosine phosphorylation were detected by western blotting. Sperm DNA damage was detected by a sperm chromatin dispersion assay. Reactive oxygen species (ROS) were measured using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate. The expression and localization of adenosine receptors were analyzed by western blotting and immunofluorescence. The specific inhibitors of adenosine receptors were used to confirm their effects on cordycepin-induced sperm capacitation. Finally, molecular docking was performed to analyze the interaction between cordycepin and adenosine receptors., Results: Cordycepin improved hyperactivated sperm motility, acrosome reaction, PKA activity, and protein tyrosine phosphorylation during capacitation while having no obvious effects on sperm ROS or DNA damage. Four adenosine receptor subtypes were expressed in human sperm, but their localizations differed. Inhibition of adenosine receptors significantly decreased cordycepin-induced sperm hyperactivation and the acrosome reaction. Molecular docking showed that cordycepin can bind to the four subtypes of adenosine receptors., Conclusion: Cordycepin may promote human sperm capacitation through adenosine receptor-mediated signaling pathways. These findings may be useful for assisted reproductive technology and animal breeding., Competing Interests: Declarations Ethics approval and consent to participate The study was performed in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Hangzhou Medical College (LL2022-16). All semen donors provided written informed consent. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Cryopreservation of porcine spermatozoa is detrimental due to their high sensitivity to cold shock, leading to changes akin to capacitation, known as cryocapacitation. These changes, including the acrosomal reaction, hypermotility induction, and protein phosphorylation, might be influenced by the presence of progesterone in seminal plasma and egg yolk, used in most freezing extenders. We tested the effect of various progesterone concentrations added to the freezing extenders (1, 10, and 100 μg/mL). At 100 μg/mL, progesterone decreased the proportion of straightness and tended to reduce viability and the proportion of progressive motility (p < 0.1). At 10 μg/mL, it increased reacted acrosomes in dead sperm (p < 0.05), protein phosphorylation rate (p < 0.05), and tended (p < 0.1) to enhance linear movement compared to the control. To counteract the capacitating effect of progesterone, we examined the effect of antiprogesterone mifepristone (RU 486) at concentrations of 5, 10, 20, 50, 100, and 200 μM, and co-incubated 10 μM of RU 486 with 10 μg/mL of progesterone. RU 486 maintained capacitation levels and motility parameters similar to the control, although high concentrations (100 μM) tended (p = 0.152) to increase protein phosphorylation. Co-incubation reduced the acrosome reaction in dead sperm, and RU 486 appeared to prevent hypermotility stabilizing motility and viability parameters compared to samples with progesterone alone. Protein phosphorylation increased and RU 486 could not restore capacitation to control levels due to its competitive antagonism for progesterone receptors, having less affinity than progesterone, which displaces RU 486 at high concentrations, allowing normal sperm capacitation., Competing Interests: Declaration of competing interest 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 © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study was aimed at evaluating the effects of the commercial formulation Roundup Ultramax® on sperm mortality and viability, in terms of the capacity of spermatozoids (spz) to trigger the acrosome reaction (AR), using the estuarine crab Neohelice granulata as a model. To this, an in vivo assay comprising 100 days duration was carried out, on a control group and two groups exposed to the formulation (0.01 mg/L and 0.2 mg/L of glyphosate) under controlled conditions of photoperiod, feeding, and temperature. At the end of the assay, the right vas deferens (VD, proximal and middle portion) was dissected, and after homogenizing it in calcium-free saline solution, the acrosome reaction was induced in the phase containing the spz. In each treatment, the percentage of spz with total and partial AR was calculated, as well as that of dead spz. Compared to the control, crabs exposed to the herbicide showed a significant decrease in spz with full AR, together with an increase in the percentage of spz with partial AR. Furthermore, spz mortality was significantly higher in both glyphosate concentrations compared to the control, in a concentration-dependent manner. On the other hand, abnormal spermatophores, showing expanded walls and coalescence, were observed in a significant percentage in the left VD of the groups treated with the herbicide. The results obtained are compared with those from other studies on several invertebrate and vertebrate species that found inhibition of the AR and abnormal sperm, together with inhibition of spermatogenesis, endocrine disruption, and reduced sperm motility by effect of pure glyphosate and/or different glyphosate formulations. In summary, the available evidence highlights the possible impact of glyphosate on sperm quality, in a wide variety of species., 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 Elsevier B.V. All rights reserved.)

Premature acrosomal exocytosis in cryopreserved semen is one of the reasons attributed to low fertility among livestock. In the present study, we attempted to enhance the cryopreserved semen quality by selective removal of acrosome-reacted spermatozoa using FITC-PNA conjugated iron magnetic nanoparticles (MNPs). Further, the effect of nano purification on other sperm functional attributes was also assessed. Iron MNPs were prepared using co-precipitation method and dextran-coated MNPs were conjugated with FITC-PNA (0.04 mg/mL). A preliminary experiment was conducted to standardise the dose of FITC-PNA conjugated iron MNPs (0.02, 0.05, 0.1, 0.15, 0.2, 0.4 and 0.6 mg). Among the different doses used, 0.6 mg FITC-PNA conjugated iron MNPs significantly (p < 0.05) removed higher acrosomal reacted spermatozoa from the semen, and therefore, this dose was used in further experiments. Cryopreserved semen from Holstein Friesian breeding bulls (n = 6) were thawed and washed using Sperm-TALP to remove residual extender. Washed spermatozoa (2 × 10 6 ) were exposed to 0.6 mg of FITC-PNA conjugated iron MNPs for 10 min at 37°C. The nano purified semen was assessed for various vital sperm parameters viz., viability, intracellular calcium, apoptosis, mitochondrial ROS and mitochondrial membrane potential using flow cytometry. We found that nanopurification using FITC-PNA conjugated iron MNPs significantly (p < 0.05) improved the sperm quality. The proportion of viable non-apoptotic spermatozoa with low intracellular calcium levels was significantly (p < 0.05) enriched in nano purified semen. Nano purification did not affect sperm mitochondrial membrane potential and ROS production. In conclusion, these preliminary findings indicate that FITC-PNA coated iron MNPs effectively removed acrosome reacted spermatozoa and significantly improved sperm functional attributes in the purified fraction., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

β-nerve growth factor (βNGF) plays a crucial role in reproductive physiology and sperm quality. Enzymatic activity of seminal plasma and vaginal fluids reduces available βNGF and it has been demonstrated that chitosan microspheres could protect rrβNGF from degradation. This study examined the effects of microencapsulated rrbNGF with chitosan on rabbit sperm viability, motility and capacitation status. Results showed that 0.5 and 1 μg/mL of microencapsulated rrβNGF, as well as free rrβNGF or empty microspheres, did not adversely affect sperm viability or total motility after 2 h of incubation. However, the highest progressivity kinetic parameters were observed with 1 μg/mL free rrβNGF, while the highest curvilinear velocity (VCL) occurred with 0.5 μg/mL microencapsulated rrβNGF. Empty chitosan microspheres did not induce acrosome reaction (AR), but both concentrations of free and rrβNGFch favoured AR during in vitro incubation. The study suggests that using chitosan spheres did not show any adverse effects on sperm traits, unlike free rβNGF and rrβNGFch promoted capacitation and AR. Further research is needed to explore the potential of rrβNGFch in modifying in vitro capacitation and inducing ovulation during artificial insemination., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na + /Ca 2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca 2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca 2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca 2+ , reactive oxygen species (ROS) and superoxides were evaluated., Results: Partial and complete blockage of Ca 2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation., Conclusions: Inhibition of outflux and influx of Ca 2+ triggered similar effects, thus indicating that both forward and reverse Ca 2+ transport through NCX exchangers are essential for sperm capacitation., (© 2024. The Author(s).)

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Exposure 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 (Pb 2+ ) 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., Results: Lead 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. Pb 2+ modulated intracellular cAMP and Ca 2+ 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 Pb 2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria., Conclusions: Pb 2+ not only mimics Ca 2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca 2+ 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 Ca 2+ 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., (© 2024. The Author(s).)

To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results., (© 2024. The Author(s).)

Phosphodiesterase (PDE) inhibitors can improve sperm motility in patients with asthenozoospermia. However, the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity. We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil. After seminal plasma was discarded, several semen samples were subjected to four treatments (control, PF-2545920, pentoxifylline, and sildenafil) to evaluate their ability to affect motility, viability, and spontaneous acrosome reactions. Intracellular calcium and adenosine triphosphate (ATP), mitochondrial membrane potential, and penetration through viscous medium were assessed by flow cytometry, luciferase, and hyaluronic acid after treatment with PF-2545920. Statistical analyses were performed using the analysis of variance statistical test. PF-2545920 elevated the percentage of motile spermatozoa compared to the control, pentoxifylline, and sildenafil groups at 10 µmol l -1 ( P < 0.01). It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions ( P < 0.05). PF-2545920 also increased mitochondrial membrane potential ( P < 0.001) and altered intracellular calcium ( P < 0.05) in a dose-dependent manner, including increasing sperm hyaluronic acid penetrating ability ( P < 0.05). Therefore, PF-2545920 might be an excellent choice for stimulating the sperm motility., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

Zinc ion (Zn2+) homeostasis is very important for sperm capacitation and hyperactivation. Zn2+ is a specific inhibitor of the voltage-dependent proton channel (Hv1). Intracellular alkalisation of human spermatozoa is mainly dependent on opening of Hv1. Anandamide may affect spermatozoa through activation of Hv1. An increase in intracellular pH and progesterone (P4) activate cation channels of spermatozoa (CatSper). This study was designed to elucidate the interaction between ZnCl2, P4 and anandamide on human sperm function and intracellular calcium concentrations ([Ca2+]i). Human normal semen samples (n = 30) were diluted (20 × 106 spermatozoa mL-1) and divided into control and ethanol (0.01%)-, anandamide (1 nM)-, ZnCl2 (1 mM)-, P4 (10µM)-, anandamide+ZnCl2- and P4+ZnCl2-treated groups. Sperm kinematics, viability, acrosome status and [Ca2+]i were assessed. The percentage of viable and motile spermatozoa and sperm velocity was reduced in the ZnCl2-treated groups. Anandamide and P4 attenuated the inhibitory effects of ZnCl2 on sperm kinematics. Loss of the acrosome membrane was observed in all experimental groups. P4 and anandamide are present naturally in secretions of the female reproductive tract and modulate the inhibitory effects of ZnCl2 on sperm kinematics. This attenuation is probably due to a change in [Ca2+]i and prevention of Hv1 inactivation by P4 and anandamide respectively.

Background: Bisphenol A (BPA), a widely used plastic monomer and plasticizer, is detectable in blood, urine and semen of a healthy people, with concentrations ranging from 0.1 nM to 10 nM. It has been shown that in vitro exposure of BPA as low as 0.001 nM could significantly inhibited mouse sperm motility and acrosome reaction. However, it is still unclear whether BPA at those physiologically detectable concentration affects human sperm., Methods: The effects of different concentrations of BPA (0, 10 -3 , 10 -2 , 10 -1 , 10, 10 3 nM) on sperm functions were examined, including human sperm viability, kinematic parameters, hyperactivation and capacitation., Results: BPA caused a remarkable decline in human sperm viability, motility and progressive motility, hyperactivation, capacitation and progesterone-induced acrosome reaction. Mechanism studies showed that BPA could suppress the protein tyrosine phosphorylation level of human sperm, but had no effect on sperm calcium signaling., Conclusions: Physiologically detectable concentrations of BPA may impair human sperm functions via suppressing protein tyrosine phosphorylation of human sperm, implying that environmental pollution of BPA might be a factor contributing to male infertility., (Copyright © 2021. Published by Elsevier Inc.)

Research Question: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa?, Design: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC)., Results: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group., Conclusions: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Lysozyme (LYZ) c-like proteins are primarily present in the testis and epididymis of male reproductive tissues. Here, we report a novel member of the c-type LYZ family, the seminal vesicle-secreted LYZ c-like protein (SVLLP). Three forms of SVLLP were purified from mouse seminal vesicle secretions and characterized as glycoproteins with the same protein core but different N-linked glycans. SVLLP is structurally similar to c-type LYZ proteins. Only one of the 20 invariant residues was altered in the consensus sequence of c-type LYZs; however, the changed residue (N53S) is one of two essential catalytic residues. LYZ activity assays demonstrated that the three glycoforms of SVLLP lacked enzyme activity. SVLLP is primarily expressed in seminal vesicles. Immunohistochemistry revealed that it occurs in the luminal fluid and mucosal epithelium of the seminal vesicles. Testosterone is not the primary regulator for its expression in the seminal vesicle. SVLLP binds to sperm and suppresses bovine serum albumin-induced sperm capacitation, inhibits the acrosome reaction, and blocks sperm-oocyte interactions in vitro, suggesting that SVLLP is a sperm capacitation inhibitor., (© 2021 Wiley Periodicals LLC.)

Purpose: The aim of this study was to examine the ability and safety of papaverine supplementation for in vitro sperm motility enhancement. In addition, sperm motility enhancement of papaverine was compared to pentoxifylline and theophylline. The post-thaw spermatozoa were used as an asthenozoospermia model., Methods: Post thaw sperm suspensions were divided into two groups: papaverine (100 μmol/L) and control, and each was investigated in two subgroups of 30- and 60-min exposure times. Detailed motility parameters were detected using a computerized sperm motility analyzer. Acrosomal status, viability, apoptosis, and DNA fragmentation were evaluated by flow cytometry. Furthermore, the motility-enhancing capacity of papaverine, pentoxifylline, and theophylline was compared., Results: Cryopreservation impaired sperm parameters dramatically but no significant changes occurred in acrosomal status and apoptosis. Supplementation of papaverine enhanced motility parameters consistently at all exposure intervals, significantly. However, viability was lower at the 60th minute compared to the 30th minute (p=0.019). Papaverine did not alter any acrosomal or apoptotic markers at any time points. All of the compounds compared in this study increased the motility parameters, where theophylline supplementation provided significantly better improvement in total motility compared to papaverine and pentoxifylline., Conclusion: Our results suggest that in vitro papaverine treatment for 30 min adequately improves motility of post-thaw sperm, without leading to acrosome reaction, DNA damage, and viability loss. Theophylline's potency on increasing the ratio of total motile spermatozoa was found significantly superior than the two tested compounds. Prospective clinical studies with embryo production, pregnancy, and live birth data should be undertaken.

Purpose: Phosphodiesterase type 5 (PDE5) inhibitors are effective treatments for erectile dysfunction, and several recent studies have reported positive effects of PDE5 inhibitors on semen parameters as well. However, the data are still controversial. We investigated the effect of PDE5 inhibitors on sperm function by analyzing sperm motility and acrosome reaction., Materials and Methods: This study included young healthy men who underwent fertility evaluation; 32 cases were finally included. Men were excluded if they used a PDE5 inhibitor within 2 weeks or if they had insufficient semen volume (≤2 mL), leukocytospermia, or a genitourinary infection. Changes in sperm motility and acrosome reaction were determined after in vitro exposure to the maximal semen concentration of oral intake of sildenafil (100 mg) or tadalafil (20 mg)., Results: Mean age of the participants was 35.4±4.9 years, mean sperm concentration was 68.7±32.4 ×10⁶/mL, and mean sperm motility was 50.38%±8.41%. All three groups (control, sildenafil, tadalafil) experienced trends of decreased average sperm motility over time, but these changes were not significant. There were no significant differences between the three groups in the acrosome reaction after 120 minutes of drug exposure, either. The maximal semen concentration of oral intake of sildenafil (100 mg) or tadalafil (20 mg) did not substantially affect sperm motility or acrosome reaction., Conclusions: Our results suggest that on-demand use of a PDE5 inhibitor is safe and useful for the male partner of an infertile couple; however, further studies are warranted for daily PDE5 inhibitor use., Competing Interests: The authors have nothing to disclose., (© The Korean Urological Association, 2021.)

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca 2+ ionophore A 23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca 2+ ([Ca 2+ ] i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca 2+ ] i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca 2+ ] i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features., (© 2021 Federation of American Societies for Experimental Biology.)

Context: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown., Objective: This work aimed to investigate the role of CaSR in human spermatozoa., Methods: We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and 3',5'-cyclic adenosine 5'-monophosphate (cAMP) in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors., Results: CaSR is expressed in human spermatozoa and is essential for sensing extracellular free ionized calcium (Ca2+) and Mg2+. Activators of CaSR augmented the effect of sperm-activating signals such as the response to HCO3- and the acrosome reaction, whereas spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca2+ or Mg2+ was indispensable for HCO3- activation of sAC. Two male patients with a CASR loss-of-function mutation in exon 3 presented with normal sperm counts and motility, whereas a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology., Conclusion: CaSR is important for the sensing of Ca2+, Mg2+, and HCO3- in spermatozoa, and loss-of-function may impair male sperm function., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A semi-synthetic methylenedioxyphenyl compound piperonyl butoxide (PBO) has been used as a ubiquitous synergist to increase the insecticidal effect of pesticides for agricultural and household use. Despite previously demonstrated effects of PBO, the detailed mechanism of PBO in spermatozoa and reproductive toxic effects on male germ cells have not been fully elucidated. Therefore, this study evaluated the effects of PBO on various sperm functions during capacitation and clarified the mechanisms of reproductive toxic effects on male fertility at different concentrations of PBO (0.1, 1, 10, and 100 μM). Sperm motility and kinematics were assessed using computer-assisted sperm analysis and the status of capacitation was evaluated using combined H33258/chlortetracycline (CTC) staining. Intracellular adenosine triphosphate (ATP) and cell viability levels were also measured. In addition, protein kinase A (PKA) activity and protein tyrosine phosphorylation were evaluated. In addition, in vitro fertilization was performed to determine the effects of PBO on cleavage and blastocyst formation rates. We found that PBO significantly decreased sperm motility, kinematics, and acrosome-reacted and capacitated spermatozoa. In addition, PBO suppressed the intracellular ATP levels and directly affected cell viability. Moreover, PBO detrimentally decreased the activation of PKA and altered the levels of tyrosine-phosphorylated proteins. Consequently, cleavage and blastocyst formation rates were significantly reduced in a dose-dependent manner. In line with our observations, the synergist of pesticides PBO may directly and/or indirectly cause disorder in male fertility. Hence, we suggest that careful attention is made to consider reproductive toxicity when using PBO as a synergist., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The steroid hormones 17-β estradiol (E2) and progesterone (P4) can regulate capacitation, hyperactive motility, and the acrosome reaction (AR) during the sperm transit through the female tract. Moreover, exogenous P4 and E2 can induce the AR in ovine spermatozoa, and progesterone receptor (PR) and estrogen receptors (ERα and ERβ) are present in these cells. Thus, to investigate whether the effects both steroid hormones in ram sperm capacitation and AR are receptor-mediated, we incubated them with receptor agonists (tanaproget 1 μM and 5 μM for PR or resveratrol 5 μM and 10 μM for ER) or antagonists (mifepristone 4 μM and 40 μM for PR or tamoxifen 5 μM and 10 μM for ER) in capacitating conditions. The addition of receptor modulators did not affect sperm viability or total motility, although changes in progressive motility were detected. The incubation with both receptor agonists increased the percentage of acrosome-reacted spermatozoa, evaluated by chlortetracycline staining, when compared with the capacitated nontreated sample (Cap-C, P < 0.001). Moreover, the ER agonist resveratrol 10 μM provoked a greater AR than E2 (P < 0.01). Furthermore, the incubation with the receptor antagonists prevented the induction of the AR by P4 or E2, as the antagonists-treated spermatozoa presented a similar CTC pattern to that of Cap-C. In conclusion, these results confirm that P4 and E2 can induce the AR in ram spermatozoa and that this effect is receptor-mediated., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pentachlorophenol (PCP), a highly toxic contaminant of chlorophenols, is common in a variety of environments and presents serious risks to animal and human health. However, the reproductive toxicity and potential actions of PCP have not been investigated thoroughly, especially in humans. Here, human spermatozoa were used to evaluate the effect of PCP on cell function and to explore the underlying mechanisms. PCP had no substantive effects on sperm viability or motility, nor on the ability to penetrate viscous medium, sperm hyperactivation or spontaneous acrosome reactions. However, PCP significantly inhibited these properties induced by progesterone (P4). Consistent with the functional observations, although PCP itself did not affect the basal intracellular Ca 2+ concentrations and CatSper current, PCP dose-dependently inhibited increases of intracellular Ca 2+ concentrations caused by P4. In addition, the activation of CatSper induced by P4 was largely suppressed by PCP. This is the first report showing that PCP may serves as an antagonist of the P4 membrane receptor to interfere with Ca 2+ signaling by compromising the action of P4 on regulating sperm function. These findings suggest that the reproductive toxicity of PCP should also be a matter of concern as a mammalian health risk., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Oxidative stress is a mechanism of cadmium-induced reproductive dysfunction. Carpolobia lutea is a free radical scavenger. Our study investigated the potential protective effects of Carpolobia lutea root methanol extract against cadmium-induced reproductive toxicity., Methods: We obtained the Carpolobia lutea root in Akure, and it was authenticated at the Forestry Research Institute of Nigeria (FRIN) herbarium, Ibadan, Nigeria, with FHI number 109784. We used Soxhlet extraction to obtain its methanol extract. We used thirty male Wistar rats (150-170g) in this study, (n=5 per group), and treated them as follows: Control (1 ml/kg normal saline), Cd (2 mg/kg), Cd+MCL (2 mg/kg+100 mg/kg), Cd+MCL (2 mg/kg+200 mg/kg), MCL (100 mg/kg), MCL (200 mg/kg). We administered Carpolobia lutea orally for 8 weeks. We administered a single dose of 2 mg/kg of cadmium intraperitoneally. We assessed the sperm profile using a computer-aided sperm analyzer. Under microscopy, we determined the sperm acrosome reaction and the DNA damage. We measured the seminal fructose level using spectrophotometry, and the data were analyzed using ANOVA at p<0.05., Results: Cd+MCL (2mg/kg+200 mg/kg) significantly increased sperm count (339.0±25.0 vs. 29.0±4.5 million/mL), motility (80.0±0.2 vs. 55.0±4.9%), viability (68.7±2.7 vs. 31.3±2.9%) and decreased abnormal sperm (28.3±1.7 vs. 43.3±2.5%), relative to the cadmium group. Cd+MCL (2mg/kg+200 mg/kg) significantly increased acrosome reaction (68.0±7.5 vs. 15.2±2.4%) and seminal fructose level (0.49±0.06 vs. 0.28±0.06 mmol/L) relative to the cadmium group. Cd+MCL (2mg/kg+200 mg/kg) significantly decreased sperm DNA damage (14.1±1.6 vs. 35.9±5.3%) in relation to the cadmium group., Conclusions: Carpolobia lutea root extract improves the sperm variables of rats exposed to cadmium.

Steroid hormones progesterone (P4) and 17β-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 μM in the media induced an increase in Ca 2+ i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H 2 DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Green tea is a popularly consumed beverage worldwide and contains polyphenols, whose antioxidant activities could improve sperm parameters and fertility thereof. We investigated the effect of green tea on the male rat reproductive system as well as its safety. Male Wistar rats were administered 2 and 5% aqueous extract of green tea for 52 days' ad libitum, while the control group received tap water. Total polyphenol, flavanol, flavonol and soluble solids significantly increased in a concentration-dependent manner in vitro (P < 0.01). Weights of body, testis, epididymis, prostate gland, seminal vesicles, and liver, serum levels of testosterone, ferric reducing antioxidant power, creatinine, and sperm motility, remained unchanged (P > 0.05). Kidney weight, sperm concentration and vitality, spontaneous acrosome reaction increased (P < 0.05), while alanine transaminase and aspartate transaminase levels decreased (P < 0.05). Catalase, superoxide dismutase, glutathione and lipid peroxidation remained unchanged in the testes, liver and kidney (P > 0.05). Histological sections of testis, epididymis, kidney and liver showed no conspicuous alteration. Diameter and epithelial height of seminiferous tubule decreased, while caudal epididymis epithelial height increased (P < 0.01). Consumption of green tea in the conditions used in the present study seems to be safe and improved sperm parameters. However, subtle structural changes observed in the decreased diameter and epithelial height of the seminiferous tubule and increased acrosome reaction needs further investigation.

Bacterial contamination in swine semen affects the quality and longevity of sperm and consequently fertility is reduced. Antibiotics have been used to prevent bacterial growth, but the frequency of bacterial resistance to various antibiotics are increasing. Silver nanoparticles (AgNPs) of 10-20 nm in size have shown a biocide effect in bacteria and fungi microorganisms without toxicity to certain mammalian cells. The goal of this study was to analyze both, antimicrobial activity against Staphylococcus aureus and toxicity in swine sperms after 10-20 nm AgNPs treatment. S. aureus proliferation decreased when concentrations from 0.4 to 10 mM AgNPs were assayed. Also, sperm viability measured by mitochondrial metabolism after AgNPs treatment up to a concentration of 10 mM, was viable. In addition, viability determined by membrane integrity of sperms showed that AgNPs treatment up to a concentration of 10 mM was safe. Sperm morphology was evaluated by automated quantification of proximal and distal drops and whiptails. Data indicated that AgNPs treatment up to a concentration of 4 mM were harmless. Finally, sperm capacitation and acrosome reactions were determined by (chlortetracycline) CTC assay. Data showed that no changes in sperm capacitation were observed when sperms were treated with 2 mM of AgNPs, but data showed increased calcium mobilization when treated with 10 mM AgNPs, which suggested sperm capacitation. Finally, there were no significant changes encountered on sperm acrosome reaction for any of the treatments after AgNPs treatment. Taken together, these results show the potential of AgNPs as an alternative to conventional antimicrobial agents that are currently used in extenders to preserve semen required for storage., Abbreviations: AgNPs: silver nanoparticles; AMK: amikacin; AMP: adenosine monophosphate; AR: acrosome reaction; C: capacitation; CF: cefallotin; CFU: colony-forming unit; CTC: chlortetracycline; CXM: cefuroxime; DMSO: dimethyl sulfoxide; NC: non-capacitation; NOM: Norma Oficial Mexicana; PBS: phosphate buffered saline; RLUs: relative light units; ROS: reactive oxygen species; SQS: Seminal Quality System.

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels. We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca 2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/β were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca 2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/β after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca 2+ and Tyr phosphorylation of GSK3α/β. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

Platelet-activating factor (PAF) affects capacitation, acrosome reaction and fertilisation potential of spermatozoa. This study investigated the underlying mechanism(s) through which PAF regulated sperm function. Our data demonstrated that PAF dose-dependently induced, whilst lyso-PAF (PAF precursor) showed no effect on acrosome reaction of capacitated human spermatozoa. Treatment with PAF for 90 min enhanced tyrosine phosphorylation and expression of extracellular signal-regulated protein kinases (ERK) 1 and 2 in human spermatozoa. Moreover, pre-treatment with the ERK inhibitor U0126 significantly and dose-dependently suppressed PAF-induced acrosome reaction. Therefore, PAF may be actively involved in the modulation of sperm acrosome reaction by interacting with ERK. The role of PAF in fertilisation warrants further investigation., (© 2020 Blackwell Verlag GmbH.)

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca 2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca 2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca 2+ entrance to the sperm head.

Background: Metformin, a drug used to treat type 2 diabetes, has gained attention for its multiple therapeutic applications. However, little is known about its effects on human sperm function at therapeutically relevant concentration., Objectives: The aim of this study was to elucidate the in vitro actions of metformin on human sperm function and explore the underlying mechanism of any effects., Materials and Methods: Human ejaculated spermatozoa were treated with therapeutically relevant concentrations (0.25, 5, 10, 20, 40, and 80 µM) of metformin in vitro. Fertilization-essential functions of spermatozoa were examined, including viability, motility, capacitation, acrosome reaction, hyperactivation, and penetration ability. The signaling pathways mediated by 5'-AMP-activated protein kinase (AMPK), intracellular calcium concentration ([Ca 2+ ] i ), and tyrosine phosphorylation of spermatozoa were also measured., Results: Although metformin did not affect sperm viability, motility, and [Ca 2+ ] i , it significantly increased the percentages of capacitated spermatozoa, acrosomal-reacted spermatozoa, and hyperactivated spermatozoa as well as penetration ability of human spermatozoa at the concentrations of 40 and 80 µM (P < .05). These concentrations of metformin also elevated the levels of phosphorylated AMPK and tyrosine phosphorylation in human spermatozoa. In addition, activation of AMPK by A769662 (an AMPK activator) had similar effects to metformin on human spermatozoa, while inhibition of AMPK by Compound C (an AMPK inhibitor) suppressed the enhancement of metformin on human spermatozoa., Conclusion: Our findings indicate that metformin activates human sperm function through an AMPK-related mechanism which increases tyrosine phosphorylation at therapeutically relevant concentrations, thereby suggesting its improvement on human sperm function when treating subfertile males of type 2 diabetes., (© 2020 American Society of Andrology and European Academy of Andrology.)

We previously reported that the administration of d-aspartate (D-Asp) in drinking water over a 2-4-week period to 7-week-old mice resulted in higher sperm quality and increased in vitro fertilisation (IVF) rates associated with a systemic increase of luteinizing hormone and testosterone levels in the serum. The goal of this study was to investigate the effects of in vitro treatment with D-Asp on the IVF rate, embryo transfer, and sperm parameters of cryopreserved-thawed C57BL/6NTacCnrm (B6N) spermatozoa derived from young and adult mice. In this study, cryopreserved-thawed B6N spermatozoa from males aging 9, 11, 13, and 16 weeks were treated for 1 h with 4 mM D-Asp during capacitation. Thereafter, the in vitro fertilisation ability and the embryo transfer efficiency were analysed. Also, the kinetic activity of the treated spermatozoa and the acrosome reaction were measured after 1 h, 2 h, and 5 h of incubation. The capacitation rate of spermatozoa was determined after 1 h of pre-incubation. Spermatozoa from 9- and 11-week-old mice, which were treated with D-Asp, led to significantly increased IVF rates. However, spermatozoa derived from 13- and 16-week-old mice did not lead to a significant improvement in the fertilisation rate. At all ages examined, no differences were observed in the birth rate and sperm kinetic parameters. After 1 h incubation under the same conditions as the IVF was performed, the capacitation rate and the acrosome reaction were significantly higher with the D-Asp-treated spermatozoa from 9-week-old (67.5% vs. 41% and 14.5% vs. 10.5%, respectively) and 11-week-old mice (78.5% vs. 41.1% and 21.0% vs. 3.8%, respectively), corresponding to the improved IVF results. Therefore, the present results demonstrate, for the first time, a direct role of D-Asp in the capacitation process and acrosome reaction., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study investigated the relationship between acrosome reactions and fatty acid composition with respect to fertility in boar sperm. The acrosome reaction was induced more than 85% by 60 mM methyl-beta-cyclodextrin (MBCD), and plasma membrane integrity was significantly reduced dependent on the MBCD level in boar sperm (p < .05). The acrosome-reacted sperm exhibited significantly higher saturated fatty acids (SFAs) and lower polyunsaturated fatty acids (PUFAs) composition compared to the non-acrosome reaction group (p < .0001). In addition, the PUFAs, C22:5n-6 (docosapentaenoic acid [DPA]; p < .01) and C22:6n-3 (docosahexaenoic acid [DHA]; p < .0001) were significantly decreased, and cleavage and blastocyst formation of oocytes were significantly (p < .0001) decreased in acrosome-reacted sperm relative to non-acrosome-reacted sperm. Moreover, acrosome reaction was positively correlated with SFAs, whereas negatively correlated with PUFAs, of the PUFAs, the DPA (p = .0005) and DHA (p = <.0001) were negatively correlated with the acrosome reaction. Therefore, these results suggest that the PUFAs composition of sperm is closely involved in acrosome reaction in pigs., (© 2020 Blackwell Verlag GmbH.)

During sperm capacitation, intracellular signaling leads to protein tyrosine phosphorylation (PTP) of multiple cellular structures. However, the connection of this molecular signaling to the physiology of capacitated spermatozoa is not completely understood. This is the case of the short lifespan of capacitated spermatozoa and their increased susceptibility to initiate acrosomal exocytosis (AE) during incubation. Herein, by employing frozen/thawed bull spermatozoa, we aimed to study the relationship between PTP with AE and with plasma membrane integrity (PMI) at the cellular level. For this, we employed double staining following immunofluorescence for PTP combined with fluorescence probes for the acrosome (PNA-FITC) and PMI (LIVE/DEAD Fixable Dead Cell Stain Kit). Our results revealed that the presence of PTP at sperm head was less abundant in the sperm fraction that triggered the AE after 3 h of incubation under capacitating conditions, or by its induction with calcium ionophore, compared to the unreacted fraction. Furthermore, PTP at the equatorial region of the head (PTP-EQ) was enriched in the fraction showing damaged membrane while induction of AE with calcium ionophore did not alter the PMI and its relation to PTP-EQ. These results suggest that spontaneous AE and induced AE trigger similar cellular events regarding PTP and the spermatozoa showing PTP-EQ are more prone to suffer plasma membrane damage.

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER ® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER ® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER ® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway., Competing Interests: None

We conducted this study for the purpose of evaluating the protective mechanisms of curcumin against oxidative stress in asthenozoospermic individuals. Asthenozoospermic individuals were grouped into AS group, curcumin treatment group and brusatol + curcumin treatment group. The sperm motility was measured by computer-aided sperm analysis. We conducted flow cytometry and spectrophotometry to assess the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Chlortetracycline (CTC) was used to examine the acrosomal reaction of spermatozoa. Also, Western blotting was carried to measure antioxidant gene Nrf2 (nuclear factor erythroid 2-related factor) expression level. As our results shown, treatment with curcumin significantly decreased ROS formation and MDA production, compared with spermatozoa of AS group; however, Nrf2 inhibitor, Brusatol, inhibited Nrf2 expression and sperm function. Our results have shown that curcumin might protect spermatozoa by regulating Nrf2 level., (© 2019 The Authors. Andrologia published by Blackwell Verlag GmbH.)

Di-2-ethylhexyl phthalate (DEHP), a plastic-derived, endocrine-disrupting chemical, has been shown to exhibit male reproductive toxicity. However, its effects on human mature spermatozoa are largely unknown. In this study we investigated the invitro effects of DEHP and mono-2-ethylhexyl phthalate (MEHP; the main metabolite of DEHP) on sperm function and the mechanisms involved. Human spermatozoa were exposed to phthalates invitro at the doses that cover the concentrations detected in human semen: 20nM-8 μM DEHP, 1nM-20 μM MEHP or a mixture of 20nM-8 μM DEHP and 1nM-20 μM MEHP. DEHP and MEHP, alone or in combination, had no effect on the viability, membrane integrity, motility, homeostasis of reactive oxygen species or mitochondrial activity of human spermatozoa. Interestingly, 1nM-20 μM MEHP and combinations of 20nM-8 μM DEHP and 1nM-20 μM MEHP enhanced penetration ability, hyperactivation and the spontaneous acrosome reaction of human spermatozoa, and increased intracellular free Ca2+ concentrations ([Ca2+]i) and tyrosine phosphorylation, two key signalling pathways that regulate sperm function. The findings of this study suggest that invitro exposure to MEHP metabolised from DEHP affects human sperm function by inducing increases in sperm [Ca2+]i and tyrosine phosphorylation, which adds to our understanding of the effects of DEHP on male reproduction.

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of "sperm maturation" during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is "β-defensin 1" which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO 2 atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition., (© 2019 Blackwell Verlag GmbH.)

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K + -channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa., Competing Interests: The authors declare no conflict of interest.

Energy balance is an important feature for spermatozoa functions. The 5'-AMP-activated protein kinase (AMPK), a sensor of cell energy, has been implicated as a mediator between spermatozoa functions and energy balance. We recently identified and localized the AMPK protein in chicken spermatozoa and showed that its activation with non-specific activators significantly modified spermatozoa quality. The aim of the present study was to determine more directly the role of AMPK activation induced by the specific activator A-769662 and the interaction between AMPK and another pathway, the protein kinase A (PKA) signaling pathway in bird spermatozoa. The results showed that A-769662 induced at the low dose 50 μM an increase in spermatozoa motility, viability, and acrosome reaction through AMPK activation. Furthermore, phospho-Thr172-AMPK levels were greatly decreased by the PKA inhibitor H89 that also decreased spermatozoa quality. The inhibitory action of H89 was also efficient on A-769662 AMPK phosphorylation. We conclude that AMPK activity in bird spermatozoa is stimulated by low dose of A-769662 with consequent increase in spermatozoa quality, and that AMPK is upstream regulated by PKA pathway in this model., (Copyright © 2019. Published by Elsevier Inc.)

Different antioxidants have been introduced to reduce oxidative stress during the cryopreservation. The main goal of this study was to evaluate the effects of canthaxanthin on human sperm parameters during the freeze-thaw process. This study was performed on 25 normozoospermic semen samples dividing into five groups including 0, 0.1, 1, 10, and 25 µM of canthaxanthin. The prepared spermatozoa were cryopreserved by rapid freezing technique. Sperm motility, viability (eosin-nigrosin), morphology (Papanicolaou), acrosome reaction (double staining), DNA denaturation (acridine orange), chromatin packaging (aniline blue and toluidine blue), and DNA fragmentation (sperm chromatin dispersion test) were evaluated before freezing and after thawing. All sperm parameters after thawing significantly were decreased compared to before freezing. Twenty-five micromolar canthaxanthin could significantly improve the progressive and total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Ten micromolar canthaxanthin significantly improved total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Whereas, in 1 µM group, there were significant differences only in improvement of acrosome integrity, chromatin packaging (toluidine blue) and DNA denaturation and fragmentation. But, in 0.1 µM group, there were no significant differences in any of measured parameters. It seems that canthaxanthin ameliorates detrimental effects of cryopreservation on human sperm parameters., (© 2019 Blackwell Verlag GmbH.)

When nonhuman primate sperm undergoes cryopreservation in an egg yolk medium there is an increased risk that the egg yolk might adversely affect the sperm due to containing of avian pathogens. Although commercial egg-yolk-free medium for human sperm cryopreservation has been used for macaque sperm, the cryo-survival remains less than optimal. The present study, therefore, was conducted to determine the optimal concentration of antifreeze protein (AFP) III supplemented in a commercial egg-yolk-free medium for cynomolgus macaque (Macaca fascicularis) sperm cryo-survival. The function of frozen-thawed sperm was evaluated by post-thaw sperm motility, acrosome integrity, and mitochondrial function. Results indicate that the sperm motilities were greater when 0.1, 1, and 10 μg/ml of AFP III were supplemented into the sperm freezing medium (P < 0.05). In addition, the mitochondrial membrane potential was greater in the sperm cryopreserved with the medium that was supplemented with 0.1 μg/ml of AFP III (P < 0.05). The addition of AFP III at any of the concentrations, however, did not have any cryoprotection effect on the sperm acrosome, and the greatest concentrations of AFP III at 100 and 200 μg/ml had detrimental effects on acrosomal integrity (P < 0.05). Results of the present study indicated the methods used are effective for the cryopreservation of cynomolgus monkey sperm while reducing associated health risks due to avian pathogens being present in egg yolk-based extenders., (Copyright © 2019 Elsevier B.V. All rights reserved.)

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,β-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.

Effect of sericin pretreatment of sperm cells on the osmotic tolerance, ability to undergo acrosome reaction induced by calcium ionophore (CI), heparin (H) or lysophosphatidylcholine (LPC), post-thaw sperm quality and in vivo fertility was evaluated in four successive experiments in rabbit. In experiment 1, fresh semen was pretreated with sericin (0, 0.1% or 0.5% w/v) before exposing to fructose solutions adjusted to either 50, 100, 290, 500 or 1000 mOsm/L. Sericin pretreatment increased sperm livability in addition to live-membrane intact and total membrane intact sperm rates (P < 0.05) in 50 and 290 mOsm/L groups. In experiment 2, sperm samples were pretreated by either 0.1 or 0.5% sericin after removal of the semen plasma. CI, H or LPC were used to induce acrosome reaction in pretreated sperm samples. Sericin pretreatment, reduced the ability of sperm cells to undergo acrosome reaction (P < 0.05) in vitro. In experiment 3, ejaculates were frozen with or without sericin pretreatment in DMSO-sucrose extender. In post-thaw samples sericin pretreatment improved total and progressive motility, livability, membrane and acrosome integrity in a dose dependent manner (P < 0.05). In vivo fertility trials by artificial inseminations revealed contradictory results in experiment 4. Although 0.5% sericin pretreatment totally inhibited fertility, 0.1% sericin provided high pregnancy rates. In conclusion; sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality, but reduces the ability of rabbit sperm cells to undergo in vitro induced acrosome reaction, but this effect is restored in vivo by dose dependent manner.

Pyrethroid toxicity using dosages that are relevant to the human settings are not reported. Male Wistar rats were treated for 9 or 12 months daily with a mixture of pyrethroids equivalent to a fifth or a twenty-fifth of that is present in cereals and vegetables consumed by an average Indian adult. Altered oxidant and antioxidant status, severe anatomical damage in the testis, caput, cauda, prostate, liver, lung and kidney and increased serum SGPT, SGOT and ALP activity was evident in all the treatment groups. Decreased levels of 3β- and 17β-HSD activity, litter size and impaired acrosome reaction was observed in all the treatment groups. To the best of our knowledge, for the first time, we demonstrate that exposure to even very low levels of pyrethroids (relevant to human consumption) for longer durations could cause damage to tissues that are important in general and male reproductive physiology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05). Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [ 35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [ 35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment., (© 2019 Wiley Periodicals, Inc.)