Regulation Of Sperm Function Research Articles

A role for diacylglycerol in human sperm acrosomal exocytosis.

Using human spermatozoa stimulated with either progesterone or the Ca2+ ionophore A23187 to undergo acrosomal exocytosis, we have investigated potential pathways for generation of diacylglycerol (DAG) and have examined the possibility that DAG plays an important role in the exocytotic response. Both treatments resulted in rapid and considerable generation of DAG, followed by a limited rise in phosphatidic acid (PA). Further experiments indicated that phospholipase C (PLC) activity is important in this generation of DAG, but phospholipase D activity probably is not. In addition, polyphosphoinositide-specific phosphoinositidase C activation and hydrolysis, of phosphatidylinositol 4,5-bisphosphate appears to be a necessary prerequisite for activation of the PLC pathway. Finally the DAG formed appears to be important in acrosomal exocytosis: (i) blocking DAG metabolism with a DAG kinase inhibitor resulted in both increased endogenous levels of DAG and a significantly increased exocytotic response in stimulated cells and (ii) exogenous DAG induced exocytosis in capacitated spermatozoa whereas PA did not. Taken together, these results suggest that DAG plays a key role in events leading to membrane fusion during human sperm acrosomal exocytosis stimulated by natural agonists.

Detection of mRNA transcripts of beta 1 integrins in ejaculated human spermatozoa by nested reverse transcription-polymerase chain reaction.

Matrix proteins (e.g. fibronectin, laminin and vitronectin) and their receptors, especially beta 1 integrins, may play a role in sperm-oocyte interactions. These proteins are well described on spermatogenic cells but especially in ejaculated spermatozoa, the presence of mRNA as a necessity for their synthesis is not clear. Therefore the mRNA synthesis of the beta 1, alpha 3, alpha 4, alpha 5 and alpha 6 chains (fibronectin and laminin receptors) of the beta 1 integrins was investigated by nested reverse transcription-polymerase chain reaction (RT-PCR) in spermatozoa of human semen samples (n = 18) which showed normal classical semen parameters after swim-up preparation. After the second amplification, we obtained specific signals for mRNA transcripts of the beta 1, alpha 3, alpha 4 and alpha 6 chains in 17 of 18 semen samples investigated. All control PCRs were negative for somatic cell transcripts and contamination by somatic cells was excluded. These results demonstrate for the first time that human ejaculated spermatozoa contain the mRNA transcripts of beta 1 integrins and support the role of these proteins in adhesive mechanisms in the fertilization process.

Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa.

The involvement of protein kinases in platelet activating factor (PAF)-induced acrosome reaction of human spermatozoa was investigated using specific inhibitors of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). PAF (10(-9)-10(-11) M) treatment of spermatozoa enhanced the acrosome reaction in a dose-dependent manner (32 +/- 4% at 10(-9) M, 28 +/- 4% at 10(-10) M and 24 +/- 3% at 10(-11) M respectively). When spermatozoa were preincubated with PKA, PKC and PTK inhibitor (KT5720, calphostin C and genistein) for 15 min prior to addition of PAF, there was a significantly reduced acrosome reaction induced by PAF, but complete inhibition was not observed. On the other hand, combined use of three inhibitors completely inhibited PAF-induced acrosome reaction to levels of non-treated samples. These results suggest that the induction of the acrosome reaction by PAF treatment may involve the activation of PKA, PKC and PTK signalling pathways, and that interaction between these pathways may regulate complex mechanisms of PAF-induced acrosome reaction.

Measurement of intracellular pH in human spermatozoa by flow cytometry with the benzo[c]xanthene dye SNAFL-1: a novel, single excitation, dual emission, molecular probe.

In numerous animal species the acrosome reaction of spermatozoa has been linked to elevations in intracellular pH (pHi). However, whether or not this is merely a passive consequence of calcium ion influx is not known. Studies into the fluctuations in pHi in sperm cells have been hampered by the lack of a pH-sensitive probe that could be used in conjunction with flow cytometry. In this study, flow cytometric analysis of pHi in human spermatozoa was accomplished by using one of the new benzo[c]xanthene dyes (SNAFL-1). SNAFL-1 was then observed in situ with conventional fluorescent microscopy and was found to be located in the post-acrosomal cytoplasm of the head. It was then used to measure the differences in pHi between acrosome intact populations of spermatozoa, and populations that had been induced to acrosome-react with human follicular fluid or the calcium ionophore A23187 to mimic the calcium influx. It was concluded that the human sperm acrosome reaction is also accompanied by a rise in pHi and the natural agonist-induced rise could not be accounted for by calcium ion influx alone.

Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility.

The aim of this study was to investigate the presence of nitric oxide synthase (NOS) and the production of nitric oxide (NO) by human spermatozoa. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase and monoclonal endothelial (eNOS) and brain (bNOS) antibodies. Using each antibody, NOS was observed localized in the head and midpiece regions of the spermatozoon. Immunofluorescence observed for eNOS and bNOS was more intense in normozoospermic samples. Sperm motility was assessed by computer-assisted semen analysis (CASA) in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME; 10(-5)M), and NO synthesis inhibitor or tumour necrosis factor (TNF)-alpha (20 IU/ml), a superoxide inducer. In the presence of L-NAME, percentage progressive motility, average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were significantly reduced after 30 min. Sperm viability was not decreased by TNF alpha or L-NAME. The accumulation of nitrite (the stable end-product of the NOS/NO pathway) by spermatozoa was measured using the Griess reaction. After 8 h, nitrite concentrations were lower in asthenozoospermic compared to normozoospermic samples. In the presence of TNF alpha, nitrite accumulation was significantly reduced in normozoospermic samples. We conclude that NOS is present in human spermatozoa and that eNOS and bNOS are abundant in normozoospermic samples. Nitric oxide (at endogenous concentrations) appears to be necessary for adequate sperm motility.

Effects of gamma-aminobutyric acid on human sperm motility and hyperactivation.

This study was undertaken to evaluate the effects of gamma-aminobutyric acid (GABA) and GABAergic agonists and antagonists on sperm kinematic parameters and hyperactivation, evaluated by a computer-assisted semen analysis (CASA) system, and intracellular cAMP content in 22 normozoospermic semen samples. Because of the possible interaction of progesterone with the GABAA receptor, we also evaluated the effects of progesterone on these parameters. GABA increased beat cross frequency, curvilinear velocity (VCL), the percentage of spermatozoa moving with an average path velocity > 10 microns/s (active) and hyperactivation, and decreased linearity and straightness. Bicuculline, a GABAA receptor antagonist, antagonized the effects of GABA on all these parameters except the percentage of active spermatozoa. Muscimol, a GABAA receptor agonist, increased VCL, the percentage of active spermatozoa, and hyperactivation by about the same extent as GABA, suggesting the involvement of the GABAA receptor. However, the GABAB receptor also seems to mediate some of the effects of GABA, because baclofen, a selective agonist for this receptor, increased significantly the percentage of active spermatozoa and hyperactivation. The effect of baclofen on this latter parameter was, however, less pronounced than that obtained with GABA or muscimol. Progesterone had the same effects as GABA on sperm kinematic parameters and hyperactivation and the simultaneous presence of both compounds was not more effective than each single one. GABA and progesterone did not have any effect on intracellular cAMP content. In conclusion, GABA modulated sperm kinematic parameters and increased hyperactivation. These effects have the same magnitude of those produced by progesterone and seem to be mediated mainly by the GABAA receptor. We speculate that GABA may be a physiological regulator of sperm function.

Composition of the human zona pellucida and modifications following fertilization.

The composition of individual human zonae pellucidae and modifications to this extracellular coat both before and after fertilization were analysed using a rapid, sensitive, non-radioactive biotinylation- or lectin-based detection system; these assays use commercially available reagents and can be performed on fragments of individual zonae pellucidae. The zona pellucida from unfertilized eggs is composed of three glycoprotein species designated as huZP1, huZP2 and huZP3. Under non-reducing conditions, the molecular weights of these proteins are approximately 150 kDa, approximately 100 kDa, and approximately 55-65 kDa respectively. Following fertilization, huZP1 was not detected under either non-reducing or reducing conditions. In contrast, after fertilization huZP2 was detected under non-reducing conditions, but not under reducing conditions. The ability to detect pre- and post-fertilization changes in a single human zona pellucida is discussed in relation to its value in assessing deficiencies in clinical and laboratory protocols used for in-vitro fertilization.

Mechanisms of Signal Transduction in Mouse Spermatozoaa

Sperm from all species studied thus far contain G-proteins. The presence of such signal-transducing proteins in these cells suggests that the regulation of sperm function might have control elements that are similar to ligand:receptor:G-protein:second messenger systems common to many somatic cells. This hypothesis is supported by experiments that demonstrate a potential intermediary role for the mouse sperm Gi-like protein in the acrosome reaction induced by ZP3. The specific function of this Gi-like protein in this important physiologic event is not known at this time, although possible roles in regulating ionic movements, cyclic nucleotide metabolism, and polyphosphoinositide turnover are possible candidates. Studies directed at the localization and biochemical identity of the mouse sperm Gi-like protein, as well as the nature of the second messenger system(s) modulated by this protein, are in progress and should help to delineate the sequence of events involved in some of the early steps of sperm-ZP interaction.