中华鳖胚胎期雌雄生殖细胞的差异  

Sexual difference of embryonic germ cells in Pelodicus sinensis

作　　者：王金霓 黄安 孙伟[1,2] 钱国英[2] 肖玲[1,2] 郭银 葛楚天 WANG Jinni;HUANG An;SUN Wei;QIAN Guoying;XIAO Ling;GUO Yin;GE Chutian(Institute of Animal Sex and Development,Zhejiang Wanli University,Ningbo 315100,China;College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,China;College of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]浙江万里学院,动物性别与发育重点研究所,浙江宁波315100 [2]浙江万里学院生物与环境学院,浙江宁波315100 [3]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2025年第5期66-77,共12页Journal of Fisheries of China

基　　金：浙江省自然科学基金(LTGN23C190006);甬江人才工程(2022B-033-G);浙江省“生物工程”一流学科(ZS2022003);宁波市自然科学基金(2022S235,2022J187)。

摘　　要：【目的】确定中华鳖雄性和雌性性腺中生殖细胞的发育差异。【方法】以胚胎发育阶段14~20期的中华鳖为研究对象,利用组织学切片、免疫荧光和转录技术分析中华鳖胚胎性别决定期至性别分化时期雌雄生殖细胞的差异变化以及相关基因的表达。【结果】(1)胚胎期中华鳖的生殖细胞体积和细胞核比周围的体细胞大且核仁明显。(2)通过生殖细胞标记蛋白DDX4的免疫荧光染色显示,中华鳖14~17期的雌雄性腺内的生殖细胞分布无显著差异。(3)组织切片结果显示17~20期ZW雌性性腺内生殖细胞集中在皮质区,而ZZ雄性生殖细胞集中在髓质区。(4)分析中华鳖14~20期中华鳖性腺的转录组发现,Ddx4、Mael、Dazl、Dnd1、Stra8和Dmc1在17期后的雌性性腺中呈现高表达,而Rmb38、Hes1、Notch和Bcl2在17期后的雄性性腺中呈现高表达。(5)利用荧光定量PCR验证发现,生殖细胞相关基因Dazl、Ddx4和减数分裂相关基因Stra8、Dmc1在17~20期ZW雌性性腺中显著高于ZZ雄性。【结论】Stra8和Dmc1在17期的雌性生殖细胞中高表达,说明雌性生殖细胞在17期后可能进入减数分裂,而Notch、Hes1在17期的雄性生殖细胞开始高表达,说明雄性生殖细胞可能进入了细胞周期停滞期。本研究为中华鳖的生殖细胞发育研究提供了参考,也为中华鳖的性控育种提供了参考。The Chinese soft-shelled turtle(Pelodiscus sinensis)is a valuable aquaculture species in China that exhibits typical sexual dimorphism and is classified as GSD(genetic sex determination)species due to the presence of ZZ/ZW micro-sex chromosomes.However,the development and differentiation of germ cells in the gonads of male and female P.sinensis have been rarely studied.To characterize the profile of germ cells in both male and female developmental gonads,we utililzed embryos at developmental stages 14 to 20,which encompassed the sex determination and sex differentiation periods of P.sinensis.Germ cells from stages 14 to 20 were sampled,and their morphology was examined using tissue sections.Immunohistochemistry was employed to detect the expression of related proteins,while RNA was extracted from gonads at stages 14 to 20 for RNA-seq and qPCR analyzes to investigate the differential expression of male and female germ cell-related genes.Germ cell marker genes included Ddx4 and Dazl,while genes associated with reproductive cell growth and development included Mael,Dnd1,Stra8 and Dmc1,Rbm38,Hes1,Notch and Bcl2.The tissue sections revealed that germ cells in the embryonic stage were larger than surrounding somatic cells,with large nuclei and prominent nucleoli in both ZW female and ZZ male gonads.Immunofluorescence staining of DDX4 showed no differences in germ cell distribution between male and female gonads from stages 14 to 17.However,from stage 17 to 20,germ cells in the gonads of ZZ females were concentrated in the cortex,whereas male germ cells were concentrated in the medulla.RNA-seq analysis indicated that Ddx4,Mael,Dazl,Dnd1,Stra8 and Dmc1 were highly expressed in female gonads,while Rbm38,Hes1,Notch and Bcl2 were highly expressed in male gonads during stage 17 to 20.Quantitative Real-time PCR analysis confirmed that mRNA expression levels of the germ cell-related genes Ddx4 and Dazl were significantly higher in ZW female gonads than in ZZ male gonads.Similarly,expression levels of meiosis-related genes Stra

关 键 词：中华鳖 生殖细胞 胚胎 性别 

分 类 号：S966.5[农业科学—水产养殖]