Kaneko Takane, Toh Saori, Mochida Izumi, Iwamori Naoki, Inai Tetsuichiro, Iida Hiroshi
Department of Bioresource Sciences, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.
Department of Morphological Biology, Fukuoka Dental College, Fukuoka, Japan.
Mol Reprod Dev. 2020 Jul;87(7):808-818. doi: 10.1002/mrd.23396. Epub 2020 Jun 27.
We isolated the transmembrane and coiled-coil domains 2 (Tmco2) gene using a polymerase chain reaction-based subtraction technique. Tmco2 is predominantly expressed in rat testes starting from 4 weeks of age. Rat TMCO2 consists of 187 amino acids with a predicted molecular mass of 20.6 kDa. When expressed in COS7 cells, TMCO2 was found as vesicle-like structures in the cytoplasm, whereas TMCO2ΔTM lacking the transmembrane (TM) region was found diffused in the cytoplasm. These results suggest that the TM region in TMCO2 is essential for its specificity of localization. Immunocytochemical analyzes indicated that rat TMCO2 was localized as small semiluminate bodies or cap-like structures in the vicinity of round spermatid nuclei and as curved lines associated with nuclei of elongated spermatids and caput epididymal spermatozoa. However, it was detected in only a small part of cauda epididymal spermatozoa. Double immunolabeling of the spermatids and spermatozoa with the anti-TMCO2 antibody and the monoclonal anti-MN7 antibody showed that TMCO2 was predominantly associated with the inner acrosomal membrane in spermatids and caput epididymal spermatozoa. Our findings suggest that TMCO2 might be involved in the process of acrosome biogenesis, especially binding of acrosome to a nucleus, during spermiogenesis.
我们使用基于聚合酶链反应的消减技术分离出跨膜和卷曲螺旋结构域2（Tmco2）基因。Tmco2从4周龄开始在大鼠睾丸中大量表达。大鼠TMCO2由187个氨基酸组成，预测分子量为20.6 kDa。当在COS7细胞中表达时，TMCO2在细胞质中表现为囊泡样结构，而缺乏跨膜（TM）区域的TMCO2ΔTM则在细胞质中呈弥散分布。这些结果表明，TMCO2中的TM区域对其定位特异性至关重要。免疫细胞化学分析表明，大鼠TMCO2定位于圆形精子细胞核附近的小半月形小体或帽状结构，以及与延长精子细胞核和附睾头精子细胞核相关的曲线。然而，仅在一小部分附睾尾精子中检测到它。用抗TMCO2抗体和单克隆抗MN7抗体对精子细胞和精子进行双重免疫标记显示，TMCO2主要与精子细胞和附睾头精子的顶体内膜相关。我们的研究结果表明，TMCO2可能参与精子发生过程中的顶体生物发生过程，特别是顶体与细胞核的结合。
