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LOCALIZATION OF CALMODULIN IN MAMMALIAN SPERMATOZOA BY IMMUNOFLUORESCENCE METHODS *

Prior to fertilization the mammalian spermatozoon undergoes an activation process comprised of two distinct events. The first of these processes, capacitation, results in increased permeability of the plasma membrane to metal ions including calcium. In response to this influx of calcium ions, a second process termed the acrosome reaction is initiated. This reaction results in the vesiculation and eventual loss of plasma and outer acrosomal membranes terminating in the release of the hydrolytic contents of the acrosome. The possible role of calmodulin as a coupling factor in these processes has been explored by surveying the content of calmodulin in sperm isolated from a variety of species and by examining the localization of calmodulin in mammalian sperm by indirect immunofluorescence techniques. Calmodulin, as measured by its ability to activate porcine brain phosphodiesterase, was found to be present in surprisingly high levels in human, rabbit, hamster, rooster, and sea urchin spermatozoa. Subcellular fractionation of rabbit spermatozoa and subsequent assay of these fractions demonstrated that greater than 99% of the measurable calmodulin activity was associated with the sperm head. while no detectable activity was present within the tail and midpiece fraction. In order to more properly define the subcellular localization of calmodulin in mammalian sperm, sperm isolated from rabbits and guinea pigs were treated with goat or sheep antibody directed against rat testes calmodulin followed by fluorescein or rhodamine-tagged second antibody, and viewed with videoenhanced fluorescence microscopy. In both species fluorescence was observed in four distinct areas including the anterior portion of the head corresponding to the acrosome, a band in the posterior portion of the equatorial segment, and spots at the base and tip of the flagella. Although fluorescence was seen along the length of the flagellum as well, it was usually weak compared to that in the four distinct regions mentioned above. In sperm that had undergone the acrosome reaction, fluorescence was absent from the acrosomal portion of the head but remained in the other three regions. In sperm exhibiting various degrees of acrosomal shedding, the fluorescence associated with the anterior portion of the head corresponded with the partially lifted membrane as viewed by phase contrast. When “free” acrosome caps were found, they were also fluorescent, though their fluorescence was often weaker than that observed in intact sperm.