Holt W V, North R D
Institute of Zoology, Zoological Society of London, UK.
J Reprod Fertil. 1991 Mar;91(2):451-61. doi: 10.1530/jrf.0.0910451.
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 degrees 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 greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal 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 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees 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 fulfills a stabilizing function in spermatozoa.
研究了可控应激即冷却对公羊精子中肌动蛋白分布的影响，以探究细胞骨架蛋白参与维持精子质膜完整性这一假说。首先确定了肌动蛋白在精子上的正常分布。一种单克隆抗体（IgM）仅与顶体后区域和鞭毛的主段相互作用。通过使用多克隆抗体，在顶体（不包括赤道段）、顶体后区域和整个鞭毛上检测到了肌动蛋白。肌动蛋白以非丝状形式存在。在39℃固定精子，然后用单克隆抗体进行肌动蛋白的免疫荧光检测，大多数精子未染色（染色比例=4.4%（±1.6；标准误）；n=8次射精）。如果在免疫荧光之前精子用大于0.025%的 Triton X-100 进行通透处理，肌动蛋白定位于所有精子头部的顶体后区域，在鞭毛主段上也有少量定位。使用多克隆抗体证实，去污剂处理可使顶体后抗原暴露。在2小时内缓慢冷却至5至15℃之间的不同温度，也会导致显示顶体后肌动蛋白免疫反应性的细胞比例增加。从15℃冷却至10℃显著增加了免疫反应性细胞的比例（平均值±标准误；15℃时为12±4.5%；10℃时为27±4.5%；n=4次射精）。进一步冷却至5℃未能引起染色增加。对冷却精子的超微结构检查证实，冷却后有一部分精子表现出顶体后肌动蛋白免疫反应性。这些结果与肌动蛋白在精子中发挥稳定功能的观点相符。
