Human Sperm Acrosome Reaction Research Articles

Lipidomics profiles of human spermatozoa: insights into capacitation and acrosome reaction using UPLC-MS-based approach.

Lipidomics elucidates the roles of lipids in both physiological and pathological processes, intersecting with many diseases and cellular functions. The maintenance of lipid homeostasis, essential for cell health, significantly influences the survival, maturation, and functionality of sperm during fertilization. While capacitation and the acrosome reaction, key processes before fertilization, involve substantial lipidomic alterations, a comprehensive understanding of the changes in human spermatozoa's lipidomic profiles during these processes remains unknown. This study aims to explicate global lipidomic changes during capacitation and the acrosome reaction in human sperm, employing an untargeted lipidomic strategy using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Twelve semen specimens, exceeding the WHO reference values for semen parameters, were collected. After discontinuous density gradient separation, sperm concentration was adjusted to 2 x 106 cells/ml and divided into three groups: uncapacitated, capacitated, and acrosome-reacted. UPLC-MS analysis was performed after lipid extraction from these groups. Spectral peak alignment and statistical analysis, using unsupervised principal component analysis (PCA), bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) analysis, and supervised partial least-squares-latent structure discriminate analysis (PLS-DA), were employed to identify the most discriminative lipids. The 1176 lipid peaks overlapped across the twelve individuals in the uncapacitated, capacitated, and acrosome-reacted groups: 1180 peaks between the uncapacitated and capacitated groups, 1184 peaks between the uncapacitated and acrosome-reacted groups, and 1178 peaks between the capacitated and acrosome-reacted groups. The count of overlapping peaks varied among individuals, ranging from 739 to 963 across sperm samples. Moreover, 137 lipids had VIP values > 1.0 and twenty-two lipids had VIP > 1.5, based on the O2PLS-DA model. Furthermore, the identified twelve lipids encompassed increases in PI 44:10, LPS 20:4, LPA 20:5, and LPE 20:4, and decreases in 16-phenyl-tetranor-PGE2, PC 40:6, PS 35:4, PA 29:1, 20-carboxy-LTB4, and 2-oxo-4-methylthio-butanoic acid. This study has been the first time to investigate the lipidomics profiles associated with acrosome reaction and capacitation in human sperm, utilizing UPLC-MS in conjunction with multivariate data analysis. These findings corroborate earlier discoveries on lipids during the acrosome reaction and unveil new metabolites. Furthermore, this research highlights the effective utility of UPLC-MS-based lipidomics for exploring diverse physiological states in sperm. This study offers novel insights into lipidomic changes associated with capacitation and the acrosome reaction in human sperm, which are closely related to male reproduction.

Editor's note on 'Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif'.

Editor's note on 'Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif'.

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.

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.

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.

P-077 Human sperm acrosome becomes alkalinized during capacitation

Abstract Study question Does during the human sperm capacitation the acrosomal pH (pHa) increases naturally and how it change can be modulate? Summary answer Sperm acrosome is progressively alkalinized during capacitation in a HCO3- and Ca2+ dependent manner. pHa is regulated by V-ATPase and involving the sAC-PKA signaling pathway. What is known already Capacitation involves biochemical and physiological modifications undergone by sperm as they travel through the female reproductive tract. These modifications prepare the sperm to enact the acrosome reaction (AR), an essential step for egg fertilization. Capacitation requires [Ca2+]i and pHi increases. Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the pHa is sufficient to trigger the AR in mouse and human sperm, but it is unknown if the pHa increases physiologically during human sperm capacitation. Study design, size, duration In this work we followed pHa changes during human sperm capacitation (6 h) and evaluated the mechanisms of its regulation using pharmacological tools. Sperm were stained with LysoSensor green and the relative fluorescence measurements for pHa kinetics during capacitation were performed at 15 min of incubation (initial time: 0 h) and after 3 and 6 h of incubation. Independent experiments of at least 3 donors were performed per condition. Participants/materials, setting, methods We used semen samples from 11 healthy donors fulfilled the requirements defined by the OMS (OMS, 2010). Sperm recovered by swim-up were incubated in a capacitating or non-capacitating HTF medium. LysoSensor green fluorescence dye was used to evaluate pHa dynamic during capacitation using single cell epifluorescence microscopy and image-based flow cytometry. Additionally, pHa was evaluated in the presence of specific inhibitors to assess regulatory mechanisms. A linear model was used to study the pHa kinetics. Main results and the role of chance Human sperm pHa is alkalinized progressively at 3 and 6 h of the capacitation (p &amp;lt; 0.05). V-ATPase activity mainly responsible for the acrosome acidity and can modulate at least in part acrosome alkalinization during capacitation (***p&amp;lt;0.001, *p&amp;lt;0.05). V-ATPase is immunolocalized to the acrosome and equatorial segment of human sperm. Bicarbonate (HCO3-) was essential for the pHa increase observed during capacitation and its absence inhibits completely the acrosome alkalinization (**p&amp;lt;0.01, *p&amp;lt;0.05). Inhibition of the Cystic Fibrosis Transmembrane Regulator (CFTR) or the Na+/HCO3- Cotransporter (NBC) partially blocked the pHa changes during capacitation. Extracelular Ca2+ is involved in pHa regulation during capacitation. Under low external Ca2+ the pHa regulation is altered and occurred delay in acrosome alkalinization (*p &amp;lt; 0.05 and **p &amp;lt; 0.001). Finally, we tested a pharmacological blockade of sAC and PKA during capacitation. sAC inhibition did not prevent acrosome alkalinization, but it significantly reduced the rate of the pHa changes as compared to the capacitation control (***p=0.0009), revealing a certain influence. On the other hand, PKA inhibition induced acrosomal acidification at the start of capacitation and accelerated significantly the alkalinization (*p&amp;lt;0.05). Together these results allow us to assume that a signaling pathway mediated to sAC is participating in the pHa regulation. Limitations, reasons for caution Experiments were performed in vitro and due to model limitations is difficult to establish strategies that allow us to know more directly, other molecular mechanisms that regulate the pHa. Wider implications of the findings Our findings show that pHa is subject of regulation during human sperm capacitation. The results provide new insights regarding the molecular mechanisms involved in human sperm capacitation and this knowledge results interesting for the study of specific molecules that inhibit capacitation or AR. Trial registration number Not applicable

Acrosomal alkalinization occurs during human sperm capacitation.

Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR.

Complex combined steroid mix of the female tract modulates human sperm

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca2+]i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, ‘physiological standard’ steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca2+]i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca2+]i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.

Discrete Dynamic Model of the Mammalian Sperm Acrosome Reaction: The Influence of Acrosomal pH and Physiological Heterogeneity.

The acrosome reaction (AR) is an exocytotic process essential for mammalian fertilization. It involves diverse physiological changes (biochemical, biophysical, and morphological) that culminate in the release of the acrosomal content to the extracellular medium as well as a reorganization of the plasma membrane (PM) that allows sperm to interact and fuse with the egg. In spite of many efforts, there are still important pending questions regarding the molecular mechanism regulating the AR. Particularly, the contribution of acrosomal alkalinization to AR triggering physiological conditions is not well understood. Also, the dependence of the proportion of sperm capable of undergoing AR on the physiological heterogeneity within a sperm population has not been studied. Here, we present a discrete mathematical model for the human sperm AR based on the physiological interactions among some of the main components of this complex exocytotic process. We show that this model can qualitatively reproduce diverse experimental results, and that it can be used to analyze how acrosomal pH (pHa) and cell heterogeneity regulate AR. Our results confirm that a pHa increase can on its own trigger AR in a subpopulation of sperm, and furthermore, it indicates that this is a necessary step to trigger acrosomal exocytosis through progesterone, a known natural inducer of AR. Most importantly, we show that the proportion of sperm undergoing AR is directly related to the detailed structure of the population physiological heterogeneity.

Epac activation induces an extracellular Ca2+ -independent Ca2+ wave that triggers acrosome reaction in human spermatozoa.

The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways

BackgroundHeat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.MethodsHuman sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation.ResultsHuman sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation.ConclusionsTaken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

Aqueous leaf extract of Moringa oleifera reduced intracellular ROS production, DNA fragmentation and acrosome reaction in Human spermatozoa in vitro.

The effects of aqueous leaf extract of Moringa oleifera (MO) on human sperm functions and integrity was studied in vitro. Semen was obtained by masturbation after 3-5days' abstinence from 34 healthy donors in Western Cape, South Africa. Liquefied semen was washed in human tubular fluid supplemented with 1% bovine serum albumin (HTF-BSA;1:5) with 10min centrifugation at 300g. Sperm suspensions were subsequently incubated with MO extract (0.625, 6.25, 62.5 and 625µg/ml) for 1hr, where HTF-BSA served as control. Sperm motility, vitality, DNA fragmentation, reactive oxygen species production, mitochondrial membrane potential, capacitation and acrosome reaction were assessed. Sperm motility, vitality, mitochondrial membrane potential and capacitation remained unchanged (p>.05). A dose-dependent decrease in sperm reactive oxygen species production (p<.0001), DNA fragmentation (p<.0001) and acrosome reaction (p<.001) was observed. An increase in the percentage of non-capacitated sperm (p<.01) was noted at 625µg/ml. The antioxidant properties of MO actively maintained basic sperm functions, inhibited excess sperm free superoxide production and preserved acrosome reaction and DNA integrity. Further studies are needed to confirm the effect of aqueous MO leaf extract on fertility potential.

Neurotensin stimulates the sperm acrosome reaction and reduces percentages of fertilization in vitro

To determine the impact of neurotensin (NTS), a naturally occurring peptide, on the function of human and nonhuman primate sperm. Experimental study. University-based research laboratory. Consenting normozoospermic human donors and cynomolgus macaques. None. Sperm acrosome status was assessed. Computer-assisted semen analysis assessed sperm motility, progression, and velocity. Immunocytochemistry and receptor selective agonists were used to identify specific NTS receptors on sperm. Monkey oocytes were obtained after ovarian stimulation, and NTS-treated monkey sperm were used for invitro fertilization. Neurotensin treatment of human sperm stimulated the acrosome reaction in both a dose-dependent (0.1-10 μmol/L) and time-dependent (5-30 minutes) manner. Neurotensin treatment did not alter sperm motility or progression. Both a general NTS receptor antagonist (SR142948) and a NTSR1 selective antagonist (SR48692) reduced the ability of NTS to stimulate the acrosome reaction. The neurotensin receptor NTSR1, but not NTSR2 or SORT1, was detected in monkey sperm using immunostaining. Neurotensin treatment also compromised the ability of sperm to fertilize an oocyte. Percentage of fertilization with untreated monkey sperm and monkey oocytes was 72%. Sperm pre-treated with NTS yielded a significantly lower fertilization rate of 18%. Neurotensin effectively stimulates the acrosome reaction in human and monkey sperm. Neurotensin produced by the oviduct or cumulus cells may promote natural fertilization. Pretreatment of sperm with NTS significantly reduces fertilization. Exposure of sperm to NTS prior to reaching the oviduct has the potential for contraceptive development. Identification of NTSR1 as the mediator of NTS action provides a specific target for future studies.

Role of Angiotensin-(1-7) via MAS receptor in human sperm motility and acrosome reaction.

Rennin-angiotensin system (RAS) has been involved in sperm function, even so, little is known about the implication of one of the RAS axis formed by Ang-(1-7) (angiotensin-(1-7)) and MAS receptor. Hence, in the present work, we focused on elucidating the function of the MAS receptor in human spermatozoa. We analyzed the expression and localization of MAS receptor in human spermatozoa and we observed if its activation is able to modulate the sperm motility of normal motility and/or asthenozoospermic patients, as well as, the acrosome reaction of the spermatozoa. MAS receptor is present in human mature spermatozoa, not only at the mRNA level but also at protein level. MAS is localized at the acrosome region, as well as, in the tail of spermatozoa. The sperm incubation with MAS agonist Ang-(1-7) activates at dose-dependent manner the PI3K/AKT pathway (P < 0.01 vs control) and improves the motility of asthenozoospermic patients (P < 0.01 vs control), which is blocked by the specific antagonist (A779) (P < 0.01), but it do not modulate the acrosome reaction. These findings suggest that the ACE2/Ang-(1-7)/Mas axis may be a useful biochemical tool for the treatment of male infertility related to sperm mobility.

Effects of the environmental endocrine disruptors di-2-ethylhexyl phthalate and mono-2-ethylhexyl phthalate on human sperm function in vitro.

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.

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa.

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.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Effect of Lepidium meyenii on in vitro fertilization via improvement in acrosome reaction and motility of mouse and human sperm.

PurposeThe direct effects of Lepidium meyenii (Maca) on sperm remain unclear. Herein, we examined the direct effect of Maca on in vitro fertilization.MethodsWe examined the fertilization rate in a mouse model and the rate of acrosome reaction in sperm from transgenic mice expressing enhanced green fluorescent protein (EGFP) in a Maca extract‐containing human tubal fluid (HTF) medium. Using human sperm, we assessed acrosome status via fluorescein isothiocyanate‐conjugated peanut agglutinin (FITC‐PNA) staining and performed detailed analysis using a sperm motility analysis system (SMAS).ResultsIn the mouse model, the fertilization rate in the Maca extract‐containing HTF was significantly higher than that in the control medium. The acrosome reaction rate in sperm from transgenic mice expressing EGFP was also significantly higher in the Maca extract‐containing HTF than that in the control medium. Similarly, a high acrosome reaction rate, identified via FITC‐PNA staining of human sperm samples, was found in the Maca extract‐containing HTF compared with that in the control medium. Human sperm motility in the Maca extract‐containing HTF was also increased compared with that in the control medium as measured using an SMAS.ConclusionsMaca improved in vitro fertilization rates by inducing an acrosome reaction and increasing sperm motility.

Paraoxonase 1 (PON1) attenuates sperm hyperactivity and spontaneous acrosome reaction.

Sperm capacitation is essential for proper fertilization and is associated with increased sperm hyperactivity (HA) and acrosome reaction (AR). For successful fertilization, AR timing is critical; accordingly, early spontaneous AR may not facilitate fertilization. Paraoxonase 1 (PON1) possesses antioxidant properties which affect sperm capacitation. The association between PON1, semen parameters, and capacitation is not fully understood. To study PON1 activity in relation to human sperm hyperactivity and AR. Semen samples were collected, and parameters were determined (volume, concentration, total sperm count, percentage total motility, and percentage normal morphology) according to World Health Organization (WHO) guidelines. AR and hyperactivity were evaluated using FITC-PSA, staining, and computer-aided sperm analysis (CASA). PON1 activity was assessed using arylesterase activity assay. Purified PON1 inhibited both sperm hyperactivity and AR in a dose-dependent manner. Native semen PON1 activity was positively associated with higher sperm concentration and negatively associated with spontaneous acrosome reaction (sAR). PON1 may have a positive effect on fertility via its ability to prevent early spontaneous sperm capacitation and AR before reaching the female genital tract.

CD9 and CD81 Interactions and Their Structural Modelling in Sperm Prior to Fertilization.

Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm–egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm–egg membrane fusion.