The characterization and potential roles of bone morphogenetic protein 7 during spermatogenesis in Chinese mitten crab Eriocheir sinensis
The characterization and potential roles of bone morphogenetic protein 7 during spermatogenesis in Chinese mitten crab Eriocheir sinensis
复制标题
中华绒螯蟹精子发生过程中骨形态发生蛋白7的特征及潜在作用
作者：
徐亚茹;王高远;周一超;杨万喜
骨形态发生蛋白（BMP）属于转化生长因子-β超家族，参与多种生物和生理过程，特别是性腺发育。然而，很少有研究关注 BMP 在甲壳类动物生殖系统中的作用。本研究首次在中华绒螯蟹中克隆并鉴定了BMP7蛋白全基因。推导的氨基酸序列的生物信息学分析表明Es-BMP7由前结构域/潜伏相关肽和TGF-β特征结构域组成。还进行了序列保守和系统发育分析。在不同组织中进行实时定量PCR。睾丸中的最高表达表明 BMP7 对男性性腺发育的潜在作用。 Western blot结果显示，不同组织中BMP7的翻译水平不同。原位杂交显示，ofes-bmp7信号的表达呈双峰方式：在精原细胞中最高，在精母细胞和I期精子细胞中减少，在II期精子细胞中消失，在III期精子细胞和成熟精子中再次出现。为了进一步验证其在精子发生过程中的潜在作用，进行了免疫荧光，结果显示原位杂交中相似的表达趋势。精原细胞细胞质中蛋白信号最高，精母细胞及以下阶段细胞质中蛋白信号持续下降，成熟精子中信号较弱。综上所述，我们的结果表明，Es-BMP7可能在中华绒螯蟹睾丸发育中发挥作用，可能是通过维持精原细胞的自我更新并促进生殖细胞分化/减数分裂有丝分裂，或促进成功受精。
Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, have been implicated in various biological and physiological processes, especially in the gonad development. However, scarce studies were focused on the roles of BMPs in the reproductive system of crustaceans. In this study, the whole gene encoding BMP7 protein was cloned and characterized firstly in Chinese mitten crabEriocheir sinensis. The bioinformatics analysis of the deduced amino acid sequence showed that Es-BMP7 was composed of prodomain/latency-associated peptide and the TGF-β characteristic domain. The sequence conservation and phylogenetic analysis were also conducted. Quantitative real-time PCR was conducted indifferent tissues. The highest expression in testis indicated the potential role of BMP7 to male gonad development. Western blot results showed the different translational levels of BMP7 in different tissues.In-situhybridization revealed that the expression ofes-bmp7signals presented in a bimodal manner: highest in spermatogonia, decreased in spermatocytes and stage I spermatids, disappeared in stage II spermatids, and showed up again in stage III spermatids and mature sperm. To further verify the potential roles during spermatogenesis, immunofluorescence was conducted and results showed the similar expression tendency within situhybridization. The protein signal was highest in the cytoplasm of spermatogonia, continued to decline in the cytoplasm of spermatocytes and the following stages, and weak signal was found in the mature sperm. Taken together, our results revealed that Es-BMP7 might play a part in testis development inEriocheir sinensis, presumably by maintaining the self-renewal of spermatogonia and promoting the germ cell differentiation/meiotic mitosis, or facilitating the successful fertilization.