ACTIN LOCALIZATION IN RAM SPERMATOZOA - EFFECT OF FREEZING THAWING, CAPACITATION AND CALCIUM IONOPHORE-INDUCED ACROSOMAL EXOCYTOSIS/

We have analyzed, by immunofluorescence, the localization of actin in ram spermatozoa, its colocalization with the actin-binding protein, ge lsolin, and the effect of freeze/thawing, in vitro capacitation, and i nduced acrosomal exocytosis on its distribution. The monoclonal anti-a ctin and anti-gelsolin antibodies used recognized single bands at 43 0 00 and 90 000 kDa, respectively. In all spermatozoa, intense actin sta ining was observed in the whole length of the flagellum and, depending on the protocol used, in the neck and postacrosomal region of the hea d. Comparison of three staining methods, together with the use of NBD- phallacidin, allowed us to characterize ram sperm actin as a monomeric , intracellular, membrane-associated protein. Gelsolin was also presen t in ram spermatozoa and precisely colocalized with actin. Processes i nvolving alterations in membrane structure such as freezing/thawing, i n vitro capacitation, and calcium ionophore-induced acrosomal exocytos is provoked changes in the exposure of actin to the antibody. This str ongly suggests a physical association of this protein to the plasma me mbrane, most likely by its intracellular side. The possible role of ac tin in sperm function is discussed.