Nakamura M, Moriya M, Baba T, Michikawa Y, Yamanobe T, Arai K, Okinaga S, Kobayashi T
Department of Obstetrics and Gynecology, School of Medicine, Teikyo University, Tokyo, Japan.
Exp Cell Res. 1993 Mar;205(1):101-10. doi: 10.1006/excr.1993.1063.
Recently, we purified a Ca(2+)-binding protein from rat spermatogenic cells [Biochem. Biophys. Res. Commun. 176, 135-1364, 1991]. In the present study, this protein was identified as calreticulin, which is a resident protein of the endoplasmic reticulum (ER). Immunohistochemical studies revealed that calreticulin was present in the acrosome of both round spermatids and mature sperm. However, under immunoelectron microscopy, gold-particles were seen over other subcellular structures of spermatocytes, spermatids, and Sertoli cells. When the labeling density in subcellular structures of spermatids was analyzed, the acrosome was found to be most heavily labeled and the Golgi apparatus was second. The complete amino acid sequence of calreticulin, deduced from the cDNA sequence, shares a high degree of identity with that of the analogous mouse protein. The cDNA encoded a protein of 416 amino acids, including a 17-residue NH2-terminal signal sequence. The mature protein contains a KDEL sequence as an ER signal at the COOH terminus. Sperm calreticulin contained no glycosyl moiety. Northern blot analysis of RNAs from purified populations of rat spermatogenic cells indicated that the calreticulin mRNA was present in both pre- and postmeiotic cells. Immunoblot analysis of calreticulin during developmental stages showed that calreticulin was detected in the testis between the ages of 5 and 50 days. Furthermore, purified rat calreticulin contained two Ca(2+)-binding sites, a low affinity/high capacity site and a high affinity/low capacity site. These results suggest that calreticulin, which is not specific to testis, is closely associated with spermatogenesis of rats. This ER protein may be incorporated into the acrosomal vesicle via the Golgi apparatus, without glycosylation, during spermiogenesis, and may play an important role in the regulation of cell functions such as sperm motility and acrosome reaction.
最近，我们从大鼠生精细胞中纯化出一种钙结合蛋白[《生物化学与生物物理研究通讯》176, 135 - 1364, 1991]。在本研究中，该蛋白被鉴定为钙网蛋白，它是内质网（ER）的驻留蛋白。免疫组织化学研究显示，钙网蛋白存在于圆形精子细胞和成熟精子的顶体中。然而，在免疫电子显微镜下，在精子细胞、精子细胞和支持细胞的其他亚细胞结构上可见金颗粒。当分析精子细胞亚细胞结构中的标记密度时，发现顶体的标记最重，高尔基体次之。从cDNA序列推导的钙网蛋白完整氨基酸序列与类似的小鼠蛋白具有高度同一性。该cDNA编码一个416个氨基酸的蛋白质，包括一个17个残基的NH2末端信号序列。成熟蛋白在COOH末端含有一个作为ER信号的KDEL序列。精子钙网蛋白不含糖基部分。对纯化的大鼠生精细胞群体的RNA进行Northern印迹分析表明，钙网蛋白mRNA存在于减数分裂前和减数分裂后的细胞中。对发育阶段钙网蛋白的免疫印迹分析表明，在5至50天龄的睾丸中可检测到钙网蛋白。此外，纯化的大鼠钙网蛋白含有两个钙结合位点，一个低亲和力/高容量位点和一个高亲和力/低容量位点。这些结果表明钙网蛋白并非睾丸所特有，它与大鼠的精子发生密切相关。这种ER蛋白可能在精子发生过程中通过高尔基体被整合到顶体小泡中，且未进行糖基化，并且可能在调节细胞功能如精子运动和顶体反应中发挥重要作用。
