RESUMEN
Numerous sperm functions including the acrosome reaction (AR) are associated with Ca(2+) influx through voltage-gated Ca(2+) (Ca(V)) channels. Although the electrophysiological characterization of Ca(2+) currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (Ca(V)3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of Ca(V)3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca(V)3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca(V)3.1 and Ca(V)3.2 were detected in the head, suggesting its participation in the AR. Ca(V)3.1 and Ca(V)3.3 were found in the principal and the midpiece of the flagella. All Ca(V)3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca(V)3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca(V)3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca(V) channels caused small but significant motility alterations. Antibodies to HVA channels detected Ca(V)1.3 and Ca(V)2.3 in human sperm flagella.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Células Germinativas/metabolismo , Espermatozoides/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/efectos de los fármacos , Canales de Calcio Tipo T/genética , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente Indirecta , Células Germinativas/efectos de los fármacos , Células Germinativas/ultraestructura , Humanos , Activación del Canal Iónico/fisiología , Masculino , Mibefradil/farmacología , Ratones , Ratones Endogámicos , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructuraRESUMEN
Capacitative Ca(2+) entry is a process whereby the activation of Ca(2+) influx through the plasma membrane is triggered by depletion of intracellular Ca(2+) stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca(2+) channels. Recent evidence indicates that capacitative Ca(2+) entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer-assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.