College of Life Science, Hebei University, 071000 Baoding, Hebei, China.
Institute of Life Science and Green Development, Hebei University, 071000 Baoding, Hebei, China.
Front Biosci (Landmark Ed). 2023 Aug 7;28(8):160. doi: 10.31083/j.fbl2808160.
Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation.
We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of and were cloned based on the testis transcriptome of and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF).
A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane.
Our research found 7 Rab proteins based on the analysis of the sperm proteome in , and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.
Rab 蛋白是 GTP 依赖性的小蛋白，作为细胞内囊泡运输、融合和定位的调节剂发挥作用。然而，很少有研究调查 Rab 蛋白在十足目动物生殖中的功能。精子没有尾巴，核不浓缩。由于顶体形成了精子大部分的质量，它为研究顶体形成提供了一个理想的模型。
我们首先使用 LC-MS/MS 分析精子蛋白质组。为了研究与高尔基体有关的 Rab2 和 Rab6 相关基因在精子发生过程中顶体形成中的作用，根据 和 的睾丸转录组，克隆了 和 的基因，并制备了多克隆抗体。Western blot 证实了 2 种 Rab 蛋白在睾丸和精子中的存在。我们进一步通过免疫荧光（IF）观察了靶标 2 种 Rab 蛋白的特征。
在精子蛋白质组中鉴定出 1247 种蛋白质，包括 7 种 Rab 蛋白（Rab1、Rab2、Rab5、Rab6、Rab11、Rab14 和 Rab18）。IF 结果显示，Rab2 与 GM130 共定位，GM130 是顺式高尔基体基质蛋白，在精原细胞和精母细胞中存在。在早期精子细胞中，Rab2 和 Rab6 参与前顶体小泡的形成。在成熟精子细胞中，Rab2 和 Rab6 都定位于顶体膜上，但呈现出不同的特征，包绕着前顶体。在成熟精子中，Rab2 定位于核杯周围的核周质膜，而 Rab6 则保留在顶体膜上。
我们的研究基于 精子蛋白质组的分析，发现了 7 种 Rab 蛋白，并证实了 Rab2 和 Rab6 参与了顶体形成。这些发现为研究十足目动物精子发生过程中 Rab 蛋白的功能提供了基础。
