RESUMEN
The role of Na(+) fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na(+) channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 µM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca(2+)-containing or Ca(2+)-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na(+), [Na(+)]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na(+) channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , ARN Mensajero/metabolismo , Espermatozoides/metabolismo , Análisis de Varianza , Compuestos de Anilina , Western Blotting , Cartilla de ADN/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Furanos , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.8/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/metabolismo , Capacitación Espermática/fisiología , Motilidad Espermática/efectos de los fármacos , Veratridina/farmacologíaRESUMEN
BACKGROUND: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. METHODS: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). RESULTS: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). CONCLUSION: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.
Asunto(s)
Comunicación Autocrina/genética , Motilidad Espermática/genética , Taquicininas/fisiología , Adolescente , Adulto , Antidepresivos/farmacología , Antipsicóticos/farmacología , Comunicación Autocrina/efectos de los fármacos , Benzamidas/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Neprilisina/genética , Neprilisina/metabolismo , Neuroquinina A/genética , Neuroquinina A/metabolismo , Neuroquinina B/genética , Neuroquinina B/metabolismo , Piperidinas/farmacología , ARN Mensajero/análisis , Receptores de Taquicininas/antagonistas & inhibidores , Receptores de Taquicininas/genética , Receptores de Taquicininas/fisiología , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/química , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Taquicininas/genética , Taquicininas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility. METHODS: Freshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2. RESULTS: The mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels. CONCLUSION: This research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.
Asunto(s)
Canales de Sodio/fisiología , Espermatozoides/fisiología , Adolescente , Adulto , Calcio/metabolismo , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.1 , Canal de Sodio Activado por Voltaje NAV1.3 , Proteínas del Tejido Nervioso/biosíntesis , ARN Mensajero/metabolismo , Canales de Sodio/biosíntesis , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Veratridina/farmacologíaRESUMEN
Algae and vascular plants are cysteine (Cys) prototrophs. They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds. Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway. The derived amino acid sequences of the C. reinhardtii genes encoding 5'-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants. The Cys biosynthetic pathway of C. reinhardtii is regulated by sulfate availability. The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate. The sac1 mutation, which impairs C. reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase. The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity. These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process.