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Sperm selection in the female reproductive tract (FRT) is sophisticated. Only about 1,000 sperm out of millions in an ejaculate reach the fallopian tube and thus have a chance of fertilizing an oocyte. In assisted reproduction techniques, sperm are usually selected using their density or motility, characteristics that do not reflect their fertilization competence and, therefore, might result in failure to fertilize the oocyte. Although sperm processing in in vitro fertilization (IVF) and intrauterine insemination (IUI) bypasses many of the selection processes in the FRT, selection by the cumulus mass and the zona pellucida remain intact. By contrast, the direct injection of a sperm into an oocyte in intracytoplasmic sperm injection (ICSI) bypasses all natural selection barriers and, therefore, increases the risk of transferring paternal defects such as fragmented DNA and genomic abnormalities in sperm to the resulting child. Research into surrogate markers of fertilization potential and into simulating the natural sperm selection processes has progressed. However, methods of sperm isolation - such as hyaluronic acid-based selection and microfluidic isolation based on sperm tactic responses - use only one or two parameters and are not comparable with the multistep sperm selection processes naturally occurring within the FRT. Fertilization-competent sperm require a panel of molecules, including zona pellucida-binding proteins and ion channel proteins, that enable them to progress through the FRT to achieve fertilization. The optimal artificial sperm selection method will, therefore, probably need to use a multiparameter tool that incorporates the molecular signature of sperm with high fertilization potential, and their responses to external cues, within a microfluidic system that can replicate the physiological processes of the FRT in vitro., (© 2021. Springer Nature Limited.)

Mammalian oocytes are surrounded by an acellular zona pellucida (ZP). Fertilization begins when a capacitated spermatozoon binds to the ZP. Defective sperm-ZP interaction is a cause of male infertility and reduced fertilization rates in clinical assisted reproduction treatment. Despite the importance of spermatozoa-ZP binding, the mechanisms and regulation of the interaction are unclear partly due to the failure in the identification of ZP receptor on spermatozoa. Most of the previous studies assumed that the sperm ZP receptor is a single molecular species, and a number of potential candidates had been suggested. Yet none of them can be considered as the sole sperm ZP receptor. Accumulated evidence suggested that the sperm ZP receptor is a dynamic multi-molecular structure requiring coordinated action of different proteins that are assembled into a functional complex during post-testicular maturation and capacitation. The complex components may include carbohydrate-binding, protein-binding and acrosomal matrix proteins which work as a suite to mediate spermatozoa-ZP interaction. This article aims to review the latest insights in the identification of the sperm ZP receptor. Continued investigation of the area will provide considerable understanding of the regulation of fertilization that will be useful for practical application in human contraception and reproductive medicine., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To study the potential protective action in vitro of oviductal epithelial cell membrane proteins against oxidative damage in human spermatozoa., Design: Prospective in vitro study., Setting: University research laboratory and infertility clinic., Patient(s): Semen from men attending the infertility clinic at the Queen Mary Hospital with normal semen parameters (World Health Organization, 2010)., Intervention(s): We studied the effect of oviductal epithelial cell membrane proteins on the sperm functions and endogenous antioxidant enzyme activities., Main Outcome Measure(s): Sperm motility, lipid peroxidation, DNA fragmentation, intracellular reactive oxygen species (ROS) level, superoxide dismutase, and glutathione peroxidase activities., Result(s): Oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. Spermatozoa-oviduct epithelial cell interaction also enhances the antioxidant defenses in spermatozoa., Conclusion(s): Our results demonstrated the protective effects of spermatozoon-oviductal epithelial cell interaction against oxidative stress in human spermatozoa. The results enhance our understanding of the protective mechanism of oviduct on sperm functions., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from α-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' UTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3'UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression.

Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewis(x) sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewis(x) sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewis(x) sequence represents the major carbohydrate ligand for human sperm-egg binding.

Background: Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction., Methods: Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining., Results: Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction., Conclusions: Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

Context: Adrenomedullin (ADM) has been found expressed in the mouse oviduct and might play a role in reproduction., Objective: The objective of the study was to demonstrate the expression of ADM in the human oviduct, investigate its regulation by steroidal hormones and spermatozoa contact, and study its effect on ciliary beat frequency (CBF) in the human oviduct. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS: Oviducts from women undergoing hysterectomy for benign diseases in a university hospital were collected. The oviducts were treated with estradiol and/or progesterone to simulate different phases of the ovarian cycle. ADM expression was studied at the peptide and mRNA levels by immunohistochemistry and RT-PCR, respectively. CBF was measured after treatment with graded concentrations of ADM and its antagonists. Cells from OE-E6/E7, an immortalized oviductal cell line, as well as oviductal tissue were cocultured with and without direct contact with capacitated human spermatozoa to compare oviductal cell ADM expression levels. CBF was also analyzed in oviductal tissue after spermatozoa-oviduct coincubation., Results: ADM expression was the highest in the isthmic region and in a hormonal environment simulating the early luteal phase. CBF was increased by ADM in a dose-dependent manner, which was blocked by ADM and calcitonin-gene-related peptide receptor antagonists. Direct contact with spermatozoa in coculture resulted in higher ADM expression in OE-E6/E7 cell line and oviductal tissue and higher CBF in oviductal epithelium., Conclusions: ADM expression in the human oviduct is hormone dependent and is up-regulated by sperm contact. ADM stimulates ciliary motility of the human oviduct.

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.

Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca2+ channels, and extracellular Ca2+ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca2+ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding.

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.

Glycodelin is a glycoprotein with three isoforms, namely glycodelin-S, glycodelin-A and glycodelin-F. They have similar protein core but different glycan side chains. Spermatozoa are exposed to these isoforms during their passage to the fertilization site. They first encounter glycodelin-S in the seminal plasma. Data suggest that glycodelin-S suppresses albumin-induced cholesterol loss and maintains the spermatozoa in an uncapacitated state before they enter into the cervical canal where glycodelin-S is removed. This allows albumin in the uterine cavity to initiate capacitation. In the fallopian tube, the spermatozoa are exposed to glycodelin-A and -F produced by the fallopian tube. Glycodelin-A is an endogenous glycoprotein that inhibits the binding of spermatozoa to the zona pellucida. Glycodelin-A may protect the spermatozoa from maternal immune attack by its immunosuppressive activity. Glycodelin-F is the main glycodelin isoform in the follicular fluid. Similar to glycodelin-A, it inhibits spermatozoa-zona pellucida binding. The biological significance of the anti-fertilization activity of glycodelin-A and -F remains to be established. Glycodelin-F also suppresses progesterone-induced acrosome reaction. This is important to prevent premature acrosome reaction when the spermatozoa are swimming through the cumulus mass towards the oocyte and become exposed to progesterone produced by the cumulus cells. In summary, different isoforms of glycodelin act in succession to modulate different aspects of sperm function, and thereby, contribute to the success of fertilization.

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

Glycodelin is a glycoprotein with three well-defined isoforms. They are named as glycodelin-S, glycodelin-A and glycodelin-F. The three isoforms have similar protein core but different carbohydrate moieties. Glycodelin-S is abundant in the human seminal plasma. It suppresses sperm capacitation and in doing so, it maintains the spermatozoa in an uncapacitated state before they enter into the uterine cavity. Glycodelin-A is abundant in the amniotic fluid. It is also secreted from endometrial glands into uterine fluid and is produced by the fallopian tube. Glycodelin-A is the first endogenous glycoprotein that was found to inhibit the binding of spermatozoa to the zona pellucida. The immunosuppressive properties of glycodelin-A suggest that the molecule may protect the spermatozoa from immune attack in the maternal reproductive tract. Glycodelin-F was first found in the follicular fluid, hence its name. It also inhibits spermatozoa-zona pellucida binding. In addition, glycodelin-F suppresses progesterone-induced acrosome reaction, and may serve to prevent premature acrosome reaction. Preliminary findings suggest possible presence of yet another glycodelin isoform in the extracellular matrix of cumulus oophorus. Unlike glycodelin-A and -F, it stimulates spermatozoa-zona pellucida binding. In summary, different isoforms of glycodelin have different biological roles on sperm function, and they act in succession to contribute to the success of fertilization.

Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Two doses of 750-microg levonorgestrel at 12 h apart is one of the regimens for emergency contraception. The mechanism of action of this regimen is not fully known. We investigated whether levonorgestrel influences sperm functions and thereby, exerts contraceptive activity. The motility, acrosome reaction, zona binding capacity, and oocyte fusion capacity of human spermatozoa treated with 1, 10, and 100 ng/mL levonorgestrel for 3 h were evaluated. Levonorgestrel decreased the curvilinear velocity of the treated spermatozoa in a dose-dependent manner. A significant decrease in straight-line velocity, average path velocity and linearity were also found with 100 ng/mL levonorgestrel treatment. This concentration of levonorgestrel, but not others, also marginally decreased (p = 0.045) the zona binding capacity of the treated spermatozoa. The steroid had no effect on acrosome reaction but had a dose-dependent inhibition on spermatozoa-oocyte fusion. These data show that levonorgestrel affects sperm function only at high concentration and the contribution of these effects to emergency contraception is unlikely to be significant., (Copyright 2002 Elsevier Science Inc.)

Human fertilization begins when a capacitated spermatozoon binds to the zona pellucida (ZP) surrounding a mature oocyte. Defective spermatozoa-ZP interaction contributes to male infertility and is a leading cause of reduced fertilization rates in assisted reproduction treatments (ARTs). Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. This observation suggests that human ZP selectively interacts with competitively superior spermatozoa characterized by high fertilizing capability and genetic integrity. However, direct evidence for ZPmediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilizationcompetent spermatozoa in humans. ZP-bound and unbound spermatozoa were respectively collected by a spermatozoa-ZP coincubation assay. The timecourse data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZPbinding ability. Immunofluorescent staining indicated that the ZP-bound spermatozoa had significantly higher expression levels of HSPA2 and SPACA3 than the unbound spermatozoa. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound spermatozoa. The results validated the possibility of applying spermatozoa-ZP interaction to select fertilization-competent spermatozoa in ART. This highly selective interaction might also provide diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis. [ABSTRACT FROM AUTHOR]

The aim of the randomised trial was to compare conventional semen parameters, sperm DNA fragmentation levels and satisfaction levels between semen samples collected at home and at the clinic. We recruited 110 men with a history of infertility for at least 1 year from the outpatient andrology clinic. Each man collected two semen samples, one at home and one at the clinic. Men were randomly assigned into the home first (n = 55) or clinic first (n = 55) groups. The primary outcome was sperm concentration. There was no significant difference in sperm concentration, sperm DNA fragmentation levels or other conventional semen parameters between home first and clinic first samples (p >.05), while satisfaction levels were significantly higher for home first samples (p <.01). Consistent results were obtained when comparing home‐collected and clinic‐collected samples within individuals. Men can be offered the option to collect semen samples at home for examination or assisted reproduction without compromising semen quality, especially for those with difficulty in producing semen samples at the clinic. [ABSTRACT FROM AUTHOR]

Study question: Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction?Summary answer: ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content.What is known already: A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins.Study design, size, duration: The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed.Participants/materials, setting, methods: Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays.Main results and the role of chance: Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa.Large scale data: N/A.Limitations, reasons for caution: The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted.Wider implications of the findings: Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction.Study funding/competing interest(s): This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests. [ABSTRACT FROM AUTHOR]

In mammals, the sperm deliver mRNA of unknown function into the oocytes during fertilization. The role of sperm microRNAs (miRNAs) in preimplantation development is unknown. miRNA profiling identified six miRNAs expressed in the sperm and the zygotes but not in the oocytes or preimplantation embryos. Sperm contained both the precursor and the mature form of one of these miRNAs, miR-34c. The absence of an increased level of miR-34c in zygotes derived from a-amanitin-treated oocytes and in parthenogenetic oocytes supported a sperm origin of zygotic miR-34c. Injection of miR-34c inhibitor into zygotes inhibited DNA synthesis and significantly suppressed first cleavage division. A 3' IJTR luciferase assay and Western blotting demonstrated that miR-34c regulates B-cell leukemia/lymphoma 2 (Bcl-2) expression in the zygotes. Coinjection of anti-Bcl-2 antibody in zygotes partially reversed but injection of Bcl-2 protein mimicked the effect of miR-34c inhibition. Oocyte activation is essential for the miR-34c action in zygotes, as demonstrated by a decrease in 3' UTR luciferase reporter activity and Bcl-2 expression after injection of precursor miR-34c into parthenogenetic oocytes. Our findings provide evidence that sperm-borne miR-34c is important for the first cell division via modulation of Bcl-2 expression. [ABSTRACT FROM AUTHOR]

Spermatogenesis is regulated by a cascade of steroid regulated genes in the testis. Recent studies suggested that acupuncture may improve fertility in men with abnormal semen parameters. Yet, the underlying mechanisms in which acupuncture enhances spermatogenesis remain largely unknown. Here we used a scrotal heat-treated rat model to study the effect of electroacupuncture (EA) on recovery of spermatogenesis. In this model, spermatogenesis was disrupted by 30 min scrotal heat treatment at 43°C. Ten sessions of EA were given at Baihui (GV20), Guanyuan (CV4), Zusanli (ST36) and Sanyinjiao (SP6) from day 9 to day 36 post-treatment. Sperm motility and production, morphology of the germinal epithelium by Johnsen's scoring, germ cell apoptosis by TUNEL staining, proliferation by proliferating cell nuclear antigen (PCNA) staining, as well as serum testosterone and inhibin B levels by immunoassays were evaluated on day 0, 1, 9, 25, 37, 46, 56 and 79. When compared with the heat-treated (H) group, the heat-treated plus EA (H+EA) group showed a significant increase (p, 0.05) in PCNA-positive cells and inhibin B levels on days 37 and 46, and a higher Johnsen's score till day 56. On day 79, motile spermatozoa could be found in the vas deferens of H+EA group only. Consistently, there was a trend of improved motility and increased number of motile epididymal spermatozoa in the H+EA group than the H group; while apoptosis of germ cells and serum testosterone levels were similar between the two groups. Taken together, EA enhanced germ cell proliferation through improvement of Sertoli cell functions. This may facilitate the recovery of spermatogenesis and may restore normal semen parameters in subfertile patients. [ABSTRACT FROM AUTHOR]

Background: Glycodelin-C is a glycodelin isoform isolated from the cumulus matrix. It stimulates spermatozoa-zona pellucida binding. Here, we report the isolation and characterization of a novel glycodelin interacting protein (GIP) from human cumulus matrix. Methods: GIP was purified by liquid chromatograph and identified by mass spectrometry. The interaction of GIP with glycodelin, matrix molecule and spermatozoa were investigated. Results: Mass spectrometry analysis suggested that GIP contained the N-terminal region of alpha2-macroglobulin, confirmed by western blot with anti-alpha2-macroglobulin antibody. GIP bound to native but not deglycosylated glycodelin-C in native gel electrophoresis, suggesting that the binding was glycosylation-dependent. GIP did not bind to capacitated and uncapacitated human spermatozoa. The cumulus cells could convert exogenous labeled alpha2-macroglobulin into GIP in vitro. GIP interacted with hyaluronic acid, a major component of the cumulus matrix. Glycodelin-C bound to hyaluronic acid-coated agarose beads in the presence of GIP. Human spermatozoa acquired the hyaluronic acid-GIP-bound glycodelin-C during incubation in vitro. Conclusion: The hyaluronic acid-GIP complex formed in the cumulus matrix retains and concentrates glycodelin-C in the cumulus matrix for displacing sperm-bound glycodelin-A and -F and stimulating the zona binding activity of the spermatozoa traversing through the cumulus mass. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome-specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger-like motif, coiled-coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT-PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry-fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late-staged germ cells in rat testis, and co-localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and β-actin was associated together in complex by co-immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary-conserved acrosome-specific gene and likely functions in acrosome-cap formation. J. Cell. Physiol. 209: 755–766, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]