Forms of Acrosin during Guinea Pig Spermatogenesis
Forms of Acrosin during Guinea Pig Spermatogenesis
复制标题
豚鼠精子发生过程中顶体素的形式
DOI：
10.1111/j.1749-6632.1987.tb25024.x
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发表时间：
1987
影响因子：
5.2
通讯作者：
G. Gerton
中科院分区：
文献类型：
--
作者：
C. Arboleda;G. Gerton
本实验室的工作是为了了解成熟精子的特定成分何时合成，以及它们在精子发生过程中如何插入各自的细胞器。作为这些研究的一部分，我们正在研究顶体的发展，使用顶体酶作为这个细胞器的标记。在这份报告中，我们的特点顶体酶的形式存在于不同的生精细胞的豚鼠。通过单位重力沉降制备生精细胞。从输精管和附睾尾制备豚鼠精子。通过离子载体A23 187激活精子制备可溶性顶体内容物（SAC）。2用含10 pg/ml抑肽酶、10 pg/ ml亮抑酶肽和1 mM对氨基苯甲脒的磷酸盐缓冲盐水中的1.0% Triton X-100、1.0%脱氧胆酸钠提取细胞。使用含有0.1%明胶的SDS-聚丙烯酰胺凝胶进行蛋白酶的酶谱分析。3除了使用乙醇胺转移缓冲液（pH 9.5）外，4免疫印迹使用标准程序。纯化的猪顶体酶的多克隆兔抗血清是Jerry L.赫德里克。该抗体通过免疫荧光与完整的、固定的、透化的豚鼠精子的顶体交叉反应。精子提取物和SAC含有三种主要的蛋白水解活性，分子量（MW）分别为32，000、34，000和47，000。这些蛋白酶都被胰蛋白酶样蛋白酶的传统抑制剂抑制，并且最适pH在7.0-8.5的范围内。在从常规凝胶进行免疫印迹后，在SAC中仅识别出单个宽带（MW 35，000 - 47，000）。当SAC在免疫印迹之前在含有0.1%明胶的凝胶中分离时，交叉反应抗原以更紧密的条带（MW = 40，000 - 47，000）运行。在32，000 - 34，000 MW区域未观察到反应。精子提取物的免疫印迹是相同的，除了检测到低于MW = 26，000的条带，并且在47，000 - 50，000 MW区域发现额外的交叉反应性。当检测生精细胞提取物时，发现粗线期精母细胞（纯度>75%）缺乏显著的蛋白水解活性。圆形精子细胞（纯度>75%）含有主要的蛋白水解活性（MW = 55，000）和次要活性（MW = 50，000）。凝聚精子细胞和残留体的细胞群含有四种蛋白水解活性（MW = 51，000、50，000、40，000和36，000）。抗顶体酶抗体的免疫反应模式与蛋白水解活性非常相似。
The work of this laboratory is directed toward understanding when specific components of mature spermatozoa are synthesized and how they are inserted into their respective organelles during spermiogenesis. As part of these studies, we are investigating the development of the acrosome using acrosin as a marker for this organelle. In this report, we characterize the forms of acrosin present in different spermatogenic cells of the guinea pig. Spermatogenic cells were prepared by sedimentation at unit gravity. Guinea pig spermatozoa were prepared from the vas deferens and cauda epididymidis. Soluble acrosomal contents (SAC) were prepared by ionophore A23 187 activation of spermatozoa.2 Cells were extracted with 1.0% Triton X-100, 1.0% sodium deoxycholate in phosphate-buffered saline containing 10 pg/ml aprotinin, 10 pg/ ml leupeptin, and 1 mM p-aminobenzamidine. Enzymography of proteases was performed using SDS-polyacrylamide gels containing 0.1% gelatin.3 With the exception of using an ethanolamine transfer buffer (pH 9.5),4 immunoblotting used standard procedure^.^ A polyclonal rabbit antiserum to purified boar acrosin was a gift from Dr. Jerry L. Hedrick. This antibody cross-reacted by immunofluorescence with the acrosomes of intact, fixed, permeabilized guinea pig spermatozoa. Sperm extracts and SAC contained three major proteolytic activities with molecular weights (MW) of 32,000, 34,000, and 47,000. These proteases were all inhibited by traditional inhibitors of trypsin-like proteases and had pH optima in the range of 7.0-8.5. After immunoblotting from a regular gel, only a single broad band was recognized in SAC (MW 35,000-47,000). When SAC was separated in a gel containing 0.1% gelatin prior to immunoblotting, the cross-reacting antigen ran as a tighter band (MW = 40,000-47,000). No reaction was seen in the 32,00034,000 MW region. Immunoblots of sperm extracts were the same except that a band just below MW = 26,000 was detected, and additional cross-reactivity was found in the 47,000-50,000 MW region. When extracts of spermatogenic cells were examined, pachytene spermatocytes (>75% pure) were found to be devoid of significant proteolytic activity. Round spermatids (>75% pure) contained a major proteolytic activity (MW = 55,000) and a minor activity (MW = 50,000). A cell population of condensing spermatids and residual bodies contained four proteolytic activities (MWs = 51,000, 50,000, 40,000, and 36,000). Patterns of immunological reactivity with antiacrosin antibody were very similar to those seen for proteolytic activities.
一种将十二烷基硫酸钠-聚丙烯酰胺凝胶中的强碱性蛋白质有效印迹到硝酸纤维素上的方法。
影响因子：
2.9
作者：
Szewczyk,B;Kozloff,LM
通讯作者：
Kozloff,LM