RESUMEN
Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR.
Asunto(s)
Reacción Acrosómica , Calcio , Capacitación Espermática , Acrosoma/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Calcio/metabolismo , Humanos , Masculino , Mamíferos , Ratones , Capacitación Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²âº imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²âº indicator. These AR and intracellular Ca²âº ([Ca²âº]i) recordings revealed that the presence of [Ca²âº]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²âº]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²âº]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²âº]i.
Asunto(s)
Reacción Acrosómica , Señalización del Calcio , Análisis de Semen/métodos , Espermatozoides/fisiología , Reacción Acrosómica/efectos de los fármacos , Compuestos de Anilina/química , Ionóforos de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/fisiología , Extensiones de la Superficie Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Femenino , Colorantes Fluorescentes/química , Humanos , Ionomicina/farmacología , Cinética , Masculino , Microscopía Electrónica de Rastreo , Progesterona/metabolismo , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Análisis de la Célula Individual , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Xantenos/químicaRESUMEN
Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca(2+) ([Ca(2+)]i); however, very few studies exist on [Ca(2+)]i dynamics in these cells. Other tissues display Ca(2+) oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca(2+) imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca(2+)]i changes of living germ cells in situ within the SST preparation. Ca(2+) imaging revealed that a subpopulation of male germ cells display spontaneous [Ca(2+)]i fluctuations resulting from Ca(2+) entry possibly throughout Ca(V)3 channels. These [Ca(2+)]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca(2+) fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca(2+) oscillations for at least 10 min. Synchronous Ca(2+) oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca(2+)]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca(2+) signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.