Milani Liliana, Maurizii Maria Gabriella
Department of Scienze Biologiche, Geologiche ed Ambientali, University of Bologna, Bologna, Italy.
J Exp Zool B Mol Dev Evol. 2015 Jul;324(5):424-34. doi: 10.1002/jez.b.22628. Epub 2015 May 6.
The vasa gene encodes a DEAD-box ATP-dependent RNA helicase that regulates the translation of multiple mRNAs involved in germ line differentiation. This protein has been deeply studied in many animals, but few data are available to date on reptiles. In this work, we sequenced a portion of Podarcis sicula vasa gene (Ps-vasa), developed a specific antibody and verified its specificity. Using anti-Ps-Vasa and confocal microscopy, we studied Vasa expression in male germ cells during the reproductive cycle of P. sicula: during full gonadal activity (spring), during regression of gonadal activity (summer) and during slow autumnal recrudescence. We also analyzed Vasa expression in young testes when the walls of the seminiferous tubules were forming. The aim was to verify if Vasa is involved in the process of male germ cell differentiation in all phases of the reproductive cycle. In adult testes, during full gonadal activity and during recrudescence, Vasa staining was detected from spermatogonia to spermatids. Vasa spots were also observed in the nucleus of germ cells supporting its function in different cellular compartments. No Vasa staining was observed in mature spermatozoa during the spring and mid-late November. The seminiferous epithelium analyzed in the summer appeared reduced with only spermatogonia, all Vasa-immunostained, some in division to replace germ cells. In immature testes, the seminiferous epithelium contained only spermatogonia and spermatocytes. The clear immunostaining in their cytoplasm revealed that Vasa is already expressed in juvenile male gonads, suggesting a role in the differentiation process since P. sicula early developmental stages.
瓦萨基因编码一种依赖ATP的DEAD盒RNA解旋酶，该酶可调节参与生殖细胞分化的多种mRNA的翻译。这种蛋白质在许多动物中都得到了深入研究，但迄今为止关于爬行动物的数据却很少。在这项研究中，我们对意大利壁蜥的瓦萨基因（Ps-vasa）的一部分进行了测序，制备了一种特异性抗体并验证了其特异性。我们使用抗Ps-Vasa抗体和共聚焦显微镜，研究了意大利壁蜥生殖周期中雄性生殖细胞内瓦萨蛋白的表达情况：在性腺完全活跃期（春季）、性腺活动消退期（夏季）和秋季缓慢恢复生长期。我们还分析了生精小管壁正在形成时幼龄睾丸中瓦萨蛋白的表达情况。目的是验证瓦萨蛋白是否参与了生殖周期所有阶段的雄性生殖细胞分化过程。在成年睾丸中，在性腺完全活跃期和恢复生长期，从精原细胞到精子细胞都能检测到瓦萨蛋白染色。在生殖细胞的细胞核中也观察到了瓦萨蛋白斑点，这支持了其在不同细胞区室中的功能。在春季和11月中下旬，成熟精子中未观察到瓦萨蛋白染色。夏季分析的生精上皮细胞减少，只有精原细胞，所有细胞都被瓦萨蛋白免疫染色，有些正在分裂以替代生殖细胞。在未成熟睾丸中，生精上皮细胞仅包含精原细胞和精母细胞。它们细胞质中清晰的免疫染色表明，瓦萨蛋白在幼年雄性性腺中已经表达，这表明自意大利壁蜥发育早期阶段起，它就在分化过程中发挥作用。
