Leclerc P, Goupil S
Endocrinologie de la Reproduction, Centre de Recherche du CHUQ and Centre de Recherche en Biologie de la Reproduction, Quebec, Quebec, Canada G1L 3L5.
Biol Reprod. 2000 Jun;62(6):1875-81. doi: 10.1095/biolreprod62.6.1875.
Previous studies from our laboratory have shown that a decrease in the calmodulin binding properties of a few sperm proteins occurs during the capacitation process, an effect associated with a decrease in intracellular calmodulin concentrations. Using biotinylated-calmodulin nitrocellulose overlay assay on protein extracts of subcellular fractions of bull spermatozoa, one of these proteins (p32) is detected in the flagellar-enriched fractions, whereas p30 is found in the fraction enriched with sperm heads. This latter calmodulin binding protein, p30, appears to be associated with the perinuclear theca. None of these binding proteins was solubilized by nonionic detergents. Sodium dodecyl sulfate was effective solubilizing p32, whereas p30 was extracted only in conditions reported to isolate the perinuclear theca. Cellular localization of calmodulin binding proteins was also achieved by incubating spermatozoa fixed on slides with biotinylated calmodulin and revealed in a further step by fluorescein-conjugated streptavidin. Using this procedure, it was found that calmodulin binds to the sub- and postacrosomal areas of the sperm head along with the midpiece in the presence of Ca(2+). Only a sharp band of fluorescence at the subacrosomal area was observed when this procedure was performed in the absence of Ca(2+) in the presence of EGTA. The pattern of cellular calmodulin binding was highly decreased when spermatozoa were incubated under capacitating conditions, in the presence of heparin, in agreement with the published effect of capacitation on calmodulin binding proteins.
我们实验室之前的研究表明，在获能过程中，一些精子蛋白的钙调蛋白结合特性会降低，这种效应与细胞内钙调蛋白浓度的降低有关。通过对公牛精子亚细胞组分的蛋白质提取物进行生物素化钙调蛋白硝酸纤维素覆盖分析，在富含鞭毛的组分中检测到其中一种蛋白质（p32），而p30则存在于富含精子头部的组分中。后一种钙调蛋白结合蛋白p30似乎与核周膜有关。这些结合蛋白均不能被非离子去污剂溶解。十二烷基硫酸钠能有效溶解p32，而p30仅在据报道用于分离核周膜的条件下才能被提取出来。钙调蛋白结合蛋白的细胞定位也可通过将固定在载玻片上的精子与生物素化钙调蛋白孵育来实现，并在下一步用荧光素偶联的链霉亲和素进行显示。使用该方法发现，在Ca(2+)存在的情况下，钙调蛋白与精子头部的顶体下和顶体后区域以及线粒体鞘结合。当在EGTA存在且无Ca(2+)的情况下进行该操作时，仅在顶体下区域观察到一条清晰的荧光带。当精子在获能条件下、在肝素存在的情况下孵育时，细胞钙调蛋白结合模式显著降低，这与已发表的获能对钙调蛋白结合蛋白的影响一致。
