Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.
Department of Morphological Biology, Fukuoka Dental College, Fukuoka, Japan.
Mol Reprod Dev. 2019 Mar;86(3):330-341. doi: 10.1002/mrd.23108. Epub 2019 Jan 17.
We isolated the transmembrane and coiled-coil domains 5A (Tmco5A) gene using polymerase chain reaction-based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum-nuclear membrane (ER-NM) of cells as a membrane-associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER-NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti-TMCO5A and the anti-β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.
我们使用聚合酶链反应差减技术分离跨膜和卷曲螺旋域 5A（Tmco5A）基因，并表明 Tmco5A 从出生后 4 周开始在大鼠睾丸中大量表达。当在 COS7 细胞中表达时，TMCO5A 被发现作为一种膜相关蛋白分布在细胞的内质网-核膜（ER-NM）中，而缺乏跨膜区（TM）的 TMCO5AΔC 则错误定位并扩散到细胞质中。结果表明 TM 负责将 TMCO5A 保留在 ER-NM 中。免疫细胞化学和免疫印迹分析表明，TMCO5A 沿圆形和伸长的大鼠精原细胞的核后部定位，但从附睾精子中消失。用抗 TMCO5A 和抗β微管蛋白抗体对分离的精原细胞进行双重免疫标记，结果表明 TMCO5A 始终与发育中的顶体微管密切相关，但不完全与它们共定位。另一方面，我们发现几乎所有的 TMCO5A 都与 SUN4 共定位，SUN4 是核骨架和细胞骨架复合物蛋白的连接蛋白，位于精原细胞核的后部。这些数据表明 TMCO5A 比顶体微管更靠近细胞核。TMCO5A 可能与顶体微管一起参与精子发生过程。
