F‐actin in guinea pig spermatozoa: Its role in calmodulin translocation during acrosome reaction

Abstract

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.

ADP‐ribosylation of Rho proteins inhibits sperm motility

“…Actin has been detected immunologically also in spermatozoa from various species, including bovine spermatozoa [11]. It has been proposed that actin could play a role in the stabilization of the acrosome structure [12] but no direct involvement of actin in sperm motility has been offered. To assess the occurrance of Rho proteins in bovine spermatozoa, spermaozoal membrane proteins and cytosolic proteins were ADP-ribosylated in the presence of [32p]NAD, and Rho proteins were visualized by autoradiography after SDS-PAGE.…”

Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction

“…This cytoskeleton is responsible for the clearance of integral membrane proteins from the peri-acrosomal plasma membrane, an event which seems critical for acrosomal exocytosis by mammalian spermatozoa (e.g. Moreno-Fierros et al, 1992) including those of man (Feuchter et al, 1986;Ochs et al, 1986;Benoff et al, 1996Benoff et al, , 1997aBenoff, 1997b). Subsequent conversion of polymerized actin (F-actin) to monomer actin (G-actin) apparently facilitates fusion of sperm plasma and outer acrosomal membranes.…”

The role of F‐actin cytoskeleton‐associated gelsolin in the guinea pig capacitation and acrosome reaction

“…3, lane 2). Regarding the presence of gelsolin in the postacrosomal region and flagellum, our group has detected F-actin in both regions [Moreno-Fierros et al, 1992]. How gelsolin is involved in F-actin regulation in these regions will be the subject of future work.…”

“…Similarly, the location of actin in the acrosomal region has been reported in the spermatozoa of several mammalian species [Talbot and Kleve, 1978;Camatini et al, 1986;Flaherty et al, 1988;Olson and Winfrey, 1991;Moreno-Fierros et al, 1992;Yagi and Paranko, 1995]. It has also been suggested that sperm cells must undergo actin filament depolymerization for AR to occur, due to the fact that, when actin depolymerization is inhibited by adding phalloidin to an isolated sperm membrane model [Spungin et al, 1995], membrane fusion is also hindered.…”

“…Gelsolin was detected by IIF, using a previous protocol [Moreno-Fierros et al, 1992], in formaldehydefixed, acetone permeabilized spermatozoa. For gelsolin detection, antibodies were diluted in blocking solution (1% BSA in PBS): an anti-gelsolin monoclonal antibody (1:100) was used, and then a TRITC-labeled rabbit antimouse second antibody (1:25).…”