Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

Abstract

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39°C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (± 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by >0.025% Triton X-100 before immunofluorescence, actin was localized in the post-acrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15°C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10°C markedly increased the proportion of immunoreactive cells (mean ± s.e.m.; 12 ± 4.5% at 15°C; 27 ± 4.5% at 10°C; n = 4 ejaculates). Further cooling to 5°C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfils a stabilizing function in spermatozoa.