Showing papers on "Acrosome reaction published in 2021"

Causes and consequences of sperm mitochondrial dysfunction.

Abstract: Mitochondria have multiple functions, including synthesis of adenine triphosphate, production of reactive oxygen species, calcium signalling, thermogenesis and apoptosis. Mitochondria have a significant contribution in regulating the various physiological aspects of reproductive function, from spermatogenesis up to fertilisation. Mitochondrial functionality and intact mitochondrial membrane potential are a pre-requisite for sperm motility, hyperactivation, capacitation, acrosin activity, acrosome reaction and DNA integrity. Optimal mitochondrial activity is therefore crucial for human sperm function and semen quality. However, the precise role of mitochondria in spermatozoa remains to be fully explored. Defects in sperm mitochondrial function severely impair the maintenance of energy production required for sperm motility and may be an underlying cause of asthenozoospermia. Sperm mtDNA is susceptible to oxidative damage and mutations that could compromise sperm function leading to infertility. Males with abnormal semen parameters have increased mtDNA copy number and reduced mtDNA integrity. This review discusses the role of mitochondria in sperm function, along with the causes and impact of its dysfunction on male fertility. Greater understanding of sperm mitochondrial function and its correlation with sperm quality could provide further insights into their contribution in the assessment of the infertile male.

Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development.

Abstract: Reactive oxygen species (ROS) generated at low levels during mitochondrial respiration have key roles in several signaling pathways. Oxidative stress (OS) arises when the generation of ROS exceeds the cell's antioxidant scavenging ability and leads to cell damage. Physiological ROS production in spermatozoa regulates essential functional characteristics such as motility, capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion. OS can have detrimental effects on sperm function through lipid peroxidation, protein damage, and DNA strand breakage, which can eventually affect the fertility of an individual. Substantial evidence in the literature indicates that spermatozoa experiencing OS during in vitro manipulation procedures in human- and animal-assisted reproduction are increasingly associated with iatrogenic ROS production and eventual impairment of sperm function. Although a direct association between sperm OS and human assisted reproductive techniques (ART) outcomes after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) is still a matter of debate, studies in animal models provide enough evidence on the adverse effects of sperm OS in vitro and defective fertilization and embryo development. This review summarized the literature on sperm OS in vitro, its effects on functional ability and embryo development, and the approaches that have been proposed to reduce iatrogenic sperm damage and altered embryonic development.

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

Abstract: Heat 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 Human 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 Human 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 Taken 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

Proteomic Profiling of Seminal Plasma Proteins in Varicocele Patients

Abstract: Purpose Seminal plasma provides a nutritive and protective milieu for spermatozoa. It contains factors/proteins required for sperm maturation, hyperactivation, capacitation and acrosome reaction. Alteration in the expression levels of seminal plasma proteins affect the fertilization process. The main objective of this study is to compare the seminal plasma proteome of healthy fertile men (control group) with varicocele patients in order to identify the differentially expressed seminal plasma proteins. Materials and methods Pooled seminal plasma samples from control (n=5) and varicocele (unilateral: n=5 and bilateral: n=5) subjects were used for proteomic profiling and functional bioinformatic analysis. Key differentially expressed proteins (DEPs) associated with binding of zona pellucida (acrosin; ACR), protein folding (heat shock related 70 kDa protein 2; HSPA2), oxidative stress (peroxiredoxin 2; PRDX2), lipid peroxidation and DNA fragmentation (apolipoprotein A2; APOA2) were validated by Western blot. Statistical analysis was conducted using Mann-Whitney test. Results A total of 412 and 486 proteins were detected in seminal plasma of control group and varicocele patients respectively. Twenty-eight proteins were identified as DEPs between varicocele and control group. Validation of DEPs revealed downregulation of HSPA2 (p=0.0037) as well as APOA2 (p=0.0373), and upregulation of PRDX2 (p=0.0474). Conclusions The seminal plasma protein profile of varicocele patients differ from healthy fertile men. Aberrant expression of seminal plasma proteins serve as an indicator of sperm pathology in varicocele patients.

Human Zona Pellucida Glycoproteins: Binding Characteristics With Human Spermatozoa and Induction of Acrosome Reaction

Abstract: Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes encoding human ZP glycoproteins are one of the causative factors leading to abnormal ZP matrix and infertility in women. Relevance of the human ZP glycoproteins in 'sperm-oocyte' binding has been delineated by using either transgenic animal models expressing human zona proteins or purified native/recombinant human zona proteins. Studies based on the purified native/recombinant human zona proteins revealed that ZP1, ZP3, and ZP4 primarily bind to the capacitated acrosome-intact human spermatozoa whereas ZP2 binds to acrosome-reacted spermatozoa. On the contrary, human spermatozoa binds to the eggs obtained from transgenic mouse lines expressing human ZP2 but not to those expressing human ZP1, ZP3, and ZP4 suggesting that ZP2 has an important role in human 'sperm-oocyte' binding. Further studies using transgenic mouse lines showed that the N-terminus of human ZP2 mediate the taxon-specific human sperm-oocyte binding. Both glycans and protein-protein interactions have a role in human gamete interaction. Further studies have revealed that the purified native/recombinant human ZP1, ZP3, and ZP4 are competent to induce acrosome reaction. Human sperm binds to the mouse transgenic eggs expressing human ZP1-4 instead of mouse ZP1-3 proteins, penetrated the ZP matrix and accumulated in the perivitelline space, which were acrosome-reacted suggesting that human ZP2 in transgenic mouse model also induce acrosome reaction. In humans N-linked glycosylation of zona proteins have been shown to play an important role in induction of the acrosome reaction. Hence in humans, based on studies using transgenic mouse model as well as purified native/recombinant zona proteins, it is likely that more than one zona protein is involved in the 'sperm-oocyte' binding and induction of the acrosome reaction.

The cell biology of fertilization: Gamete attachment and fusion.

Abstract: Fertilization is defined as the union of two gametes. During fertilization, sperm and egg fuse to form a diploid zygote to initiate prenatal development. In mammals, fertilization involves multiple ordered steps, including the acrosome reaction, zona pellucida penetration, sperm-egg attachment, and membrane fusion. Given the success of in vitro fertilization, one would think that the mechanisms of fertilization are understood; however, the precise details for many of the steps in fertilization remain a mystery. Recent studies using genetic knockout mouse models and structural biology are providing valuable insight into the molecular basis of sperm-egg attachment and fusion. Here, we review the cell biology of fertilization, specifically summarizing data from recent structural and functional studies that provide insights into the interactions involved in human gamete attachment and fusion.

Roles of CatSper channels in the pathogenesis of asthenozoospermia and the therapeutic effects of acupuncture-like treatment on asthenozoospermia.

Abstract: Rationale: Idiopathic asthenozoospermia (iAZS) is one of the major causes of male infertility and has no effective therapeutic treatment. Understanding the potential mechanisms that cause it may be helpful in seeking novel targets and treatment strategies for overcoming the problem of low sperm motility in iAZS individuals. Methods: Computer-assisted semen analysis (CASA) was utilized to assess the sperm motility. RT-qPCR, Western blot, immunofluorescence staining, and calcium imaging analysis were performed to examine the expression and function of CatSper channels. Hyperactivation and acrosome reaction were used to evaluate the functional characteristics of epididymal sperm. In vivo fertility assay was applied to determine the fertility of rats. CatSper1 knockdown and overexpression experiments were performed to confirm the roles of CatSper channels in the pathogenesis of iAZS and the therapeutic effects of electroacupuncture (EA) treatment on AZS model rats. Results: Here, we reported a functional down-regulation of CatSper channel from CatSper1 to CatSper 4 in the sperm of both iAZS patients and ornidazole (ORN)-induced AZS model rats, and an impaired sperm function characterized by a reduction of protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in the epididymal sperm of AZS rats. Knockdown of CatSper1 in the testis tissues is sufficient to induce AZS in normal rats, and this action was validated by the reversal effects of CatSper1 overexpression. Transcutaneous electrical acupoint stimulation (TEAS) and electroacupuncture (EA) at 2 Hz frequency improve the sperm motility via enhancing the functional expression of CatSper channels in the sperm. Gene silencing CatSper1 in the sperm abolishes the therapeutic effects of 2 Hz-EA treatment on AZS rats. Conclusions: We conclude that a functional down-regulation of CatSper channel in the sperm may be a contributor or a downstream indicator for a portion of AZS, especially iAZS, while 2 Hz-TEAS or EA treatment has a therapeutic effect on iAZS through inducing the functional up-regulation of CatSper channels in the sperm. This study provides a novel mechanism for the pathogenesis of some AZS especially iAZS, and presents a potential therapeutic target of CatSper for iAZS treatment. Acupuncture treatment like TEAS may be used as a promising complementary and alternative medicine (CAM) therapy for male infertility caused by iAZS in clinical practice.

Sperm Lipid Markers of Male Fertility in Mammals.

Abstract: Sperm plasma membrane lipids are essential for the function and integrity of mammalian spermatozoa. Various lipid types are involved in each key step within the fertilization process in their own yet coordinated way. The balance between lipid metabolism is tightly regulated to ensure physiological cellular processes, especially referring to crucial steps such as sperm motility, capacitation, acrosome reaction or fusion. At the same time, it has been shown that male reproductive function depends on the homeostasis of sperm lipids. Here, we review the effects of phospholipid, neutral lipid and glycolipid homeostasis on sperm fertilization function and male fertility in mammals.

Molecular mechanisms and evolution of fertilization proteins.

Abstract: Sexual reproduction involves a cascade of molecular interactions between the sperm and the egg culminating in cell-cell fusion. Vital steps mediating fertilization include chemoattraction of the sperm to the egg, induction of the sperm acrosome reaction, dissolution of the egg coat, and sperm-egg plasma membrane binding and fusion. Despite decades of research, only a handful of interacting gamete recognition proteins (GRPs) have been identified across taxa mediating each of these steps, most notably in abalone, sea urchins, and mammals. This review outlines and compares notable GRP pairs mediating sperm-egg recognition in these three significant model systems and discusses the molecular basis of species-specific fertilization driven by GRP function. In addition, we explore the evolutionary theory behind the rapid diversification of GRPs between species. In particular, we focus on how the coevolution between interacting sperm and egg proteins may contribute to the formation of boundaries to hybridization. Finally, we discuss how pairing structural information with evolutionary insights can improve our understanding of mechanisms of fertilization and their origins.

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization.

Abstract: Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

Role of calcium oscillations in sperm physiology

Abstract: Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells often use cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations as a means to decodify external and internal information. [Ca2+]i oscillations faster than those usually found in other cells and correlated with flagellar beat were the first to be described in sperm in 1993 by Susan Suarez, in the boar. More than 20 years passed before similar [Ca2+]i oscillations were documented in human sperm, simultaneously examining their flagellar beat in three dimensions by Corkidi et al. 2017. On the other hand, 10 years after the discovery of the fast boar [Ca2+]i oscillations, slower ones triggered by compounds from the egg external envelope were found to regulate cell motility and chemotaxis in sperm from marine organisms. Today it is known that sperm display fast and slow spontaneous and agonist triggered [Ca2+]i oscillations. In mammalian sperm these Ca2+ transients may act like a multifaceted tool that regulates fundamental functions such as motility and acrosome reaction. This review covers the main sperm species and experimental conditions where [Ca2+]i oscillations have been described and discusses what is known about the transporters involved, their regulation and the physiological purpose of these oscillations. There is a lot to be learned regarding the origin, regulation and physiological relevance of these Ca2+ oscillations.

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model

Abstract: In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm However, the complete biological role of ACRBP has not yet been elucidated In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<005) Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine

Differential impact of four sperm preparation techniques on sperm motility, morphology, DNA fragmentation, acrosome status, oxidative stress and mitochondrial activity: a prospective study

Abstract: BACKGROUND Suboptimal human semen handling in vitro may induce sperm damage. However, the effects of semen swim-up, pellet swim-up, density gradient, and density gradient followed by SU on sperm motility, morphology, DNA fragmentation, acrosome reaction, intracellular reactive oxygen species, and mitochondrial activity were not fully understood. OBJECTIVES To study the impact of four sperm preparation techniques on sperm functional parameters. MATERIALS AND METHODS This study was conducted on 60 infertile men with a minimum sperm concentration of 20 × 106 /ml and total sperm motility of ≥30%. Each raw semen sample was divided into four aliquots. Each aliquot was prepared by one of the tested techniques. Various sperm characteristics were assessed before and after sperm preparation. RESULTS Density gradient and density gradient followed by SU resulted in significantly higher DNA fragmentation percentages compared with semen swim-up (p < 0.001 and p < 0.001, respectively) and pellet swim-up (p < 0.001 and p < 0.001, respectively). Significantly higher percentages of spermatozoa with intact acrosome were detected in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen. The percentage of reactive oxygen species-positive spermatozoa was significantly higher after pellet swim-up (p < 0.001), density gradient (p < 0.001), and density gradient followed by SU (p < 0.001) than raw semen. In addition, the percentages of 100% stained midpiece (active mitochondria) were significantly higher in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen. DISCUSSION AND CONCLUSION To the best of our knowledge, this is the first report comparing the impact of these techniques on various sperm functional parameters. Semen swim-up was more effective than density gradient in selecting better spermatozoa in terms of DNA integrity, reactive oxygen species levels, acrosome status, and mitochondrial activity. Randomized clinical trials comparing these four techniques are required to test their impact on embryo development and pregnancy outcomes.

A Simple, Centrifugation-Free, Sperm-Sorting Device Eliminates the Risks of Centrifugation in the Swim-Up Method While Maintaining Functional Competence and DNA Integrity of Selected Spermatozoa

Abstract: This pilot study was conducted to explore the benefits of using a centrifugation-free device based on the migration–sedimentation (MS) technique over centrifugation-based techniques in selecting competent spermatozoa, as compared with using split human semen samples. Ejaculates from 35 men undergoing semen analysis were split into four parts where one part was retained as the neat (NE) and the other three parts were subjected to sperm selection by using migration–sedimentation (MS), density gradient (DG) separation, and swim-up (SU) techniques. Sperm functional characteristics along with mitochondrial integrity, tyrosine phosphorylation, acrosome reaction, and ultrastructure were measured. The ability of selection techniques in reducing spontaneous and radiation-induced sperm DNA lesions was assessed by the TUNEL assay. In results, MS-selected spermatozoa had higher viability (P < 0.001), longevity in terms of total motility at the end of 6 and 18 h post-extraction (P < 0.001), and mitochondrial integrity (P < 0.001) compared with those selected by DG. Furthermore, spontaneous DNA lesions were significantly reduced in MS and SU fractions compared with NE (P < 0.001). Similarly, radiation-induced sperm DNA lesions were significantly lower in MS and SU fractions (P < 0.001) compared with DG. Ultrastructural analysis using scanning electron microscopy suggested a moderate, non-significant increase in the number of spermatozoa with normal head and mid-piece in MS fraction compared with other methods. In conclusion, the MS-based device offers a centrifugation-free, efficient, and reliable sperm selection method, making it suitable for partially equipped intra-uterine insemination (IUI) laboratories or office IUI programmes. Further research should focus on the safety and clinical usefulness of the device in assisted conception programmes in general and IUI in specific.

Bovine Follicular Fluid Derived Extracellular Vesicles Modulate the Viability, Capacitation and Acrosome Reaction of Bull Spermatozoa.

Abstract: While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.

A decrease of docosahexaenoic acid in testes of mice fed a high-fat diet is associated with impaired sperm acrosome reaction and fertility.

Abstract: Obesity is a major worldwide health problem that is related to most chronic diseases, including male infertility. Owing to its wide impact on health, mechanisms underlying obesity-related infertility remain unknown. In this study, we report that mice fed a high-fat diet (HFD) for over 2 months showed reduced fertility rates and increased germ cell apoptosis, seminiferous tubule degeneration, and decreased intratesticular estradiol (E2) and E2-to-testosterone ratio. Interestingly, we also detected a decrease in testicular fatty acid levels, behenic acid (C22:0), and docosahexaenoic acid (DHA, 22:6n-3), which may be related to the production of dysfunctional spermatozoa. Overall, we did not detect any changes in the frequency of seminiferous tubule stages, sperm count, or rate of in vitro capacitation. However, there was an increase in spontaneous and progesterone-induced acrosomal exocytosis (acrosome reaction) in spermatozoa from HFD-fed mice. These data suggest that a decrease in E2 and fatty acid levels influences spermatogenesis and some steps of acrosome biogenesis that will have consequences for fertilization. Thus, our results add new evidence about the adverse effect of obesity in male reproduction and suggest that the acrosomal reaction can also be affected under this condition.

Role and Modulation of TRPV1 in Mammalian Spermatozoa: An Updated Review.

Abstract: Based on the abundance of scientific publications, the polymodal sensor TRPV1 is known as one of the most studied proteins within the TRP channel family. This receptor has been found in numerous cell types from different species as well as in spermatozoa. The present review is focused on analyzing the role played by this important channel in the post-ejaculatory life of spermatozoa, where it has been described to be involved in events such as capacitation, acrosome reaction, calcium trafficking, sperm migration, and fertilization. By performing an exhaustive bibliographic search, this review gathers, for the first time, all the modulators of the TRPV1 function that, to our knowledge, were described to date in different species and cell types. Moreover, all those modulators with a relationship with the reproductive process, either found in the female tract, seminal plasma, or spermatozoa, are presented here. Since the sperm migration through the female reproductive tract is one of the most intriguing and less understood events of the fertilization process, in the present work, chemotaxis, thermotaxis, and rheotaxis guiding mechanisms and their relationship with TRPV1 receptor are deeply analyzed, hypothesizing its (in)direct participation during the sperm migration. Last, TRPV1 is presented as a pharmacological target, with a special focus on humans and some pathologies in mammals strictly related to the male reproductive system.

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

Abstract: 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 3-5 days' 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 10 min centrifugation at 300 g. Sperm suspensions were subsequently incubated with MO extract (0.625, 6.25, 62.5 and 625 µg/ml) for 1 hr, 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 625 µg/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.

Effects of Semen Processing on Sperm Function: Differences between Swim-Up and Density Gradient Centrifugation.

Abstract: Purpose Andrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories. Materials and methods To compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions. Results Semen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation. Conclusions Our results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.

Sperm membrane proteins DCST1 and DCST2 are required for the sperm-egg fusion process in mice and fish

Abstract: The process of sperm-egg fusion is critical for successful fertilization, yet the underpinning mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) spermatozoa are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO spermatozoa can bind to the oolemma, they rarely fuse with oocytes, resulting in male sterility. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO spermatozoa are motile and can approach the egg, but rarely bind to the oolemma. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species highlighting their crucial role as conserved factors in fertilization.

Pharmacological Inactivation of CatSper Blocks Sperm Fertilizing Ability Independently of the Capacitation Status of the Cells: Implications for Non-hormonal Contraception.

Abstract: Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1-20 μM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm's ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm-egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.

An update on the techniques used to measure oxidative stress in seminal plasma

Abstract: Reactive oxygen species (ROS) are produced in significant amounts by spermatozoa and leucocytes. They are necessary to carry out various physiological functions such as sperm capacitation, hyperactivation, acrosome reaction, sperm-zona binding and cellular signalling pathways. Oxidative stress (OS) results when the ROS levels overwhelm the available antioxidant reserve. A number of direct and indirect tests have been developed to assess oxidative stress. In this manuscript, we discuss these common direct and indirect tests as well as their advantages and disadvantages. Tests measuring sperm dysfunction secondary to oxidative stress such as lipid peroxidation, DNA fragmentation and protein alterations are also described.

Collapsed mitochondrial cristae in goat spermatozoa due to mercury result in lethality and compromised motility along with altered kinematic patterns.

Abstract: Earlier we have reported mercury-induced alterations in functional dynamics of buck spermatozoa through free radicals-mediated oxidative stress and spontaneous acrosome reaction. Based on our earlier findings, we aimed to investigate the effect of mercury exposure on motility, kinematic patterns, DNA damage, apoptosis and ultra-structural alterations in goat spermatozoa following in vitro exposure to different concentrations (0.031-1.25 µg/ml) of mercuric chloride for 15 min and 3 h. Following exposure of sperm cells to 0.031 µg/ml of mercuric chloride for 3 h, livability and motility of sperms was significantly reduced along with altered kinematic patterns, significant increase in per cent necrotic sperm cells and number of cells showing DNA damage; and this effect was dose- and time-dependent. Contrary to up-regulation of Bax gene after 3 h in control group, there was significant increase in expression of Bcl-2 in mercury-treated groups. Transmission electron microscopy studies revealed rifts and nicks in plasma and acrosomal membrane, mitochondrial sheath, and collapsed mitochondria with loss of helical organization of mitochondria in the middle piece of spermatozoa. Our findings evidently suggest that mercury induces necrosis instead of apoptosis and targets the membrane, acrosome, mid piece of sperms; and the damage to mitochondria seems to be responsible for alterations in functional and kinematic attributes of spermatozoa.

Role of Integrins in Sperm Activation and Fertilization.

Abstract: In mammals, integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrin receptors are an important part of signalization pathways and have an ability to transmit signals into and out of cells and participate in cell activation. In addition to somatic cells, integrins have also been detected on germ cells and are known to play a crucial role in complex gamete-specific physiological events, resulting in sperm-oocyte fusion. The main aim of this review is to summarize the current knowledge on integrins in reproduction and deliver novel perspectives and graphical interpretations presenting integrin subunits localization and their dynamic relocation during sperm maturation in comparison to the oocyte. A significant part of this review is devoted to discussing the existing view of the role of integrins during sperm migration through the female reproductive tract; oviductal reservoir formation; sperm maturation processes ensuing capacitation and the acrosome reaction, and their direct and indirect involvement in gamete membrane adhesion and fusion leading to fertilization.

Arrdc4-dependent extracellular vesicle biogenesis is required for sperm maturation

Abstract: Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4-/- mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4-/- epididymal epithelial cells, and further, supplementation of Arrdc4-/- sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

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

Abstract: 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 (pH a ) and cell heterogeneity regulate AR. Our results confirm that a pH a 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.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Abstract: Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

The molecular mechanisms underlying acrosome biogenesis elucidated by gene-manipulated mice†.

Abstract: Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-Golgi network, and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasing number of genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation. Summary sentence The acrosome is generated from a combination of many membrane trafficking systems and the morphology and integrity are maintained by the coordination of the cytoskeletal structures during spermiogenesis.