Abstract：
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 Eriocheir sinensis 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 Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis 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 E.sinensis, 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.
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 Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis 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 E.sinensis, 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.
摘要：
背景：Rab蛋白是GTP依赖性小蛋白，可作为细胞内囊泡转运的调节剂，聚变,和本地化。然而,很少有研究调查它们在十足类繁殖中的功能。中华绒螯蟹精子没有尾巴，细胞核未凝聚。顶体形成了大部分精子，它为研究顶体形成提供了一个理想的模型。
方法：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的功能，在精子发生期间的顶体形成中，基于中华绒螯蟹的睾丸转录组克隆了Rab2和Rab6基因，并制备了多克隆抗体。通过蛋白质印迹证实了2种Rab蛋白在睾丸和精子中的存在。我们使用免疫荧光（IF）进一步观察了靶2Rab蛋白的特征。
结果：总共1247种蛋白质，包括7种Rab蛋白，在精子蛋白质组中鉴定出Rab1、Rab2、Rab5、Rab6、Rab11、Rab14和Rab18。IF结果显示Rab2与顺式高尔基基质蛋白GM130共定位,在精母细胞和精母细胞中。在早期精子中，Rab2和Rab6参与顶体前囊泡的形成。在成熟的精子细胞中，Rab2和Rab6均沉积在顶体膜上，但具有包裹顶体前的不同特征。在成熟的精子中，Rab2位于核杯周围的核周卵泡膜中，而Rab6保留在顶体膜上。
结论：我们的研究发现了7个Rab蛋白基于对中华绒螯蟹精子蛋白质组的分析，并证实Rab2和Rab6参与顶体形成。这些发现为研究Rab蛋白在十足类动物精子发生过程中的功能奠定了基础。
方法：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的功能，在精子发生期间的顶体形成中，基于中华绒螯蟹的睾丸转录组克隆了Rab2和Rab6基因，并制备了多克隆抗体。通过蛋白质印迹证实了2种Rab蛋白在睾丸和精子中的存在。我们使用免疫荧光（IF）进一步观察了靶2Rab蛋白的特征。
结果：总共1247种蛋白质，包括7种Rab蛋白，在精子蛋白质组中鉴定出Rab1、Rab2、Rab5、Rab6、Rab11、Rab14和Rab18。IF结果显示Rab2与顺式高尔基基质蛋白GM130共定位,在精母细胞和精母细胞中。在早期精子中，Rab2和Rab6参与顶体前囊泡的形成。在成熟的精子细胞中，Rab2和Rab6均沉积在顶体膜上，但具有包裹顶体前的不同特征。在成熟的精子中，Rab2位于核杯周围的核周卵泡膜中，而Rab6保留在顶体膜上。
结论：我们的研究发现了7个Rab蛋白基于对中华绒螯蟹精子蛋白质组的分析，并证实Rab2和Rab6参与顶体形成。这些发现为研究Rab蛋白在十足类动物精子发生过程中的功能奠定了基础。
