Changes in hyaluronidase, acrosin, and N-acetylhexosaminidase activities of dog sperm after incubation. (1/514)

Hyaluronidase, acrosin and N-acetylhexosaminidase activities were examined in sperm collected from 12 beagle dogs and in culture medium after 0.5 hr and 7 hr of sperm incubation. The activities of the three enzymes were significantly higher at 7 hr than at 0.5 hr (P < 0.05, 0.01), and the increases were associated with sperm capacitation. It was considered that the three enzymes in the dog sperm are related to fertilization by reason of the findings of the release of these enzymes from the sperm into the medium after 7 hr of incubation.  (+info)

Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do? (2/514)

Evidence from pharmacological studies suggests that induction of the acrosome reaction of mammalian spermatozoa by solubilized zona pellucida, and possibly by progesterone, is dependent upon Ca2+ influx through voltage-operated Ca2+ channels. Studies on Ca2+ accumulation and membrane potential in ligand-stimulated or artificially depolarized spermatozoa support such a conclusion. Electrophysiological studies on rodent spermatogenic cells have revealed the presence of a 'T' type voltage-operated Ca2+ current. This current has pharmacological attributes consistent with those of the putative channel responsible for Ca2+ influx mediating the acrosome reaction. However, use of molecular techniques to study human and rodent testis and spermatogenic cells has detected the presence of three different voltage-operated Ca2+ channel subunits. One of these (alpha lE) may generate T-currents, though this is currently disputed. Voltage-operated Ca2+ channel structure and the relationship between channel subunit expression and the characteristics of consequent Ca2+ currents is briefly reviewed. The nature and function of T-channel-mediated Ca2+ influx is examined in the context of the time-course of ligand- and depolarization-induced elevation of [Ca2+]i in mammalian spermatozoa. It is likely that a secondary Ca2+ response (mobilization of stored Ca2+ or activation of a second Ca(2+)-influx pathway) is required for the acrosome reaction. Evidence for the existence and participation of various candidates is discussed (including voltage-operated Ca2+ channels, which may be functionally expressed only in mature spermatozoa), the available evidence favouring a secondary Ca(2+)-influx pathway. Immediate priorities for future research in this area are proposed.  (+info)

Ion channels in sperm physiology. (3/514)

Fertilization is a matter of life or death. In animals of sexual reproduction, the appropriate communication between mature and competent male and female gametes determines the generation of a new individual. Ion channels are key elements in the dialogue between sperm, its environment, and the egg. Components from the outer layer of the egg induce ion permeability changes in sperm that regulate sperm motility, chemotaxis, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize protein and difficult to study electrophysiologically. Thus understanding how sperm ion channels participate in fertilization requires combining planar bilayer techniques, in vivo measurements of membrane potential, intracellular Ca2+ and intracellular pH using fluorescent probes, patch-clamp recordings, and molecular cloning and heterologous expression. Spermatogenic cells are larger than sperm and synthesize the ion channels that will end up in mature sperm. Correlating the presence and cellular distribution of various ion channels with their functional status at different stages of spermatogenesis is contributing to understand their participation in differentiation and in sperm physiology. The multi-faceted approach being used to unravel sperm ion channel function and regulation is yielding valuable information about the finely orchestrated events that lead to sperm activation, induction of the acrosome reaction, and in the end to the miracle of life.  (+info)

Detection of partial and complete acrosome reaction in human spermatozoa: which inducers and probes to use? (4/514)

The acrosome reaction (AR), an essential step for achieving mammalian fertilization, was recently introduced as a means of clinical evaluation of male fertility. However, most of the available techniques for acrosomal status assessment (except those employing electron microscopy) do not define whether the measurements represent partial or complete AR. We, therefore, performed a crossover investigation of the types of inducers and probes required for detecting partial or complete AR in human spermatozoa. The acrosomal status before and after stimulation with four AR inducers was evaluated after incubation for 3 h in capacitating conditions. We used a fluorescence-activated cell sorter with fluorescein isothiocyanate-conjugated monoclonal antibody CD46 (FITC-CD46) targeting the inner acrosomal membrane for detecting a complete AR, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) targeting the acrosomal content for detection of both partial and complete AR. Without stimulation or following stimulation with progesterone, follicular fluid (FF) or phorbol myristate ester (PMA), the AR could be detected with FITC-PSA but not with FITC-CD46. Following stimulation with the calcium ionophore A23187, the AR could be detected by both FITC-PSA and FITC-CD46. These results suggest that spontaneous AR as well as AR induced by progesterone, PMA and FF are partial. In contrast, the AR induced by A23187 is total, i.e. both partial and complete. These findings are valuable for both research and clinical purposes and are a step towards an international agreement on a standard test for human sperm AR, for which there is an urgent need.  (+info)

Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities. (5/514)

In a systematic effort to develop a microbicide contraceptive capable of preventing transmission of human immunodeficiency virus (HIV), as well as providing fertility control, we have previously identified novel phenyl phosphate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in the thymine ring and halo substitution in the phenyl moiety respectively. Here, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent anti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 was tested by measuring viral p24 antigen production and reverse transcriptase activity as markers of viral replication in HIV-1 infected human peripheral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects of WHI-07 on human sperm motion kinematics were analysed by computer-assisted sperm analysis (CASA), and its effects on sperm membrane integrity were examined by confocal laser scanning microscopy (CLSM), and high-resolution low-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion. The in-vitro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using normal human ectocervical and endocervical epithelial cells by the MTT cell viability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acrosomal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively spermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-microemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizing activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 day period, there was no irritation or local toxicity to the vaginal epithelia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have the clinical potential to become the active ingredient of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.  (+info)

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining. (6/514)

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.  (+info)

Characterization of human zona pellucida glycoproteins. (7/514)

The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy.  (+info)

Identification of Rab3A GTPase as an acrosome-associated small GTP-binding protein in rat sperm. (8/514)

The acrosome reaction is a membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion, the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm.  (+info)

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