Separation of acrosome reacted living human sperm based on the use of PSA-FITC and FACS

ZOPPINO FCM, HALÓN ND, BUSTOS MA, MAYORGA LS

Potrero de los Funes

Congreso; SAIB; 2011

SAIB

The acrosome reaction (AR) in mammalian sperm is a regulated secretion absolutely necessary for fertilization. In this study we evaluated the possibility of assessing AR in living human sperm by using Pisum sativum agglutinin (PSA) conjugated with fluorescein isothiocyanate (FITC). This lectin binds with high affinity glycoproteins present in the lumen of the acrosome. Capacitated sperms were stimulated with the calcium ionophore A23187 in the presence of PSA-FITC to detect the accumulation of the lectin in the interior of the granule as the fusion pores opened. Propidium iodide was included in the assay to label dead sperms. We observed by confocal microscopy that the acrosome of living sperm became suddenly fluorescent. Surprisingly, the label was not lost with time, as it would be expected because of the dispersion of the acrosome content after exocytosis. By electron microscopy we corroborated that in the presence of the lectin, the hybrid vesicles formed during exocytosis and the acrosomal content remain attached to the cells. With this procedure, we have generated a population of fluorescent live reacted sperm. Next, we succeeded in separating this population by fluorescence-activated cell sorting (FACS). Few techniques to assess AR in living cells have been developed, so we expect that these two new techniques will contribute to unveil new aspects of the AR.