中国科学院海洋研究所机构知识库

生殖细胞系是两性配子生成和种族延续的基础。作为生殖细胞的祖先，原始生殖细胞(PGCs)会经历特定迁移，增殖，并分化为雌雄性原细胞。在模式及少数淡水鱼类中发现，性原细胞增殖数量决定雌雄性别分化方向。然而目前鱼类性原细胞增殖相关的规律及机制却仍不清楚，并缺乏相关海水鱼类的研究。牙鲆 (Paralichthys olivaceus)是我国重要的海水养殖经济鱼类，具有XX/XY性别决定机制。养殖过程中，牙鲆雌鱼较雄鱼生长快，个体大，且其性别决定受到环境因素的影响。因此本研究采用雄激素(17α-甲基睾酮)，高温(27.5±0.5℃)，雌激素(17β-雌二醇)及对照（18°C ± 0.5 °C）处理XX牙鲆幼鱼，并分别获得4组雌雄主导群体，性别比率分别为雄93%，雄95.24%，雌99%和雌95%。我们以生殖细胞为研究主体，分析了雌雄生殖细胞增殖分化的基本规律；并进一步分析了性分化过程中，gsdf，amh和p450arom三个基因在雌雄主导群体中的表达及其对生殖细胞增殖的调控机制。

首先我们以牙鲆雌雄主导群体为研究对象，详尽系统地描述了生殖细胞迁移，增殖和分化的基本规律。孵化后，牙鲆PGCs经历特定胚后迁移，于15dph迁移至性腺原基，并于22dph由性腺体细胞包绕PGCs共同形成原始性腺；35dph，性原细胞进入第一次有丝分裂慢速增殖时期，同时基底膜形成以维持性原细胞自我更新的微环境；52dph，雄性性原细胞持续慢速增殖，呈单个独立分布，而雌性性原细胞进入第二次有丝分裂快速时期，形成2细胞生殖囊，且其生殖细胞数量是雄性的1.5倍，至66dph，雌性生殖细胞增加为雄性的2倍；73dph，雄性性原细胞持续慢速增殖，雌性性腺中传统卵巢分化的标志-卵巢腔和生殖上皮开始形成；85dph，雄性性原细胞进入第二次有丝分裂快速增殖时期，生殖细胞数量呈指数增长，2-8个雄性性原细胞形成一个由基底膜包绕界定的腺泡状细胞团，同时传统精巢分化的标志-输精管原基形成，而卵巢中卵母细胞开始进入第一次减数分裂的胞核改变期；120dph，雄性性原细胞团中精原干细胞开始分化为B型精原细胞，丢失干细胞标记基因Nanos2的表达，并发育为精小叶的结构。

然后我们克隆得到牙鲆gsdf基因，通过RT-PCR发现gsdf在性腺组织中特异表达。通过原位杂交和免疫组化发现，性腺发育过程中gsdf mRNA和蛋白均特异地表达于生殖干细胞即性原细胞，精原细胞和卵原细胞周围的性腺体细胞。进一步敲降和过表达XX牙鲆幼鱼中gsdf基因，结果显示过高的gsdf表达量显著抑制性原细胞增殖，而过低的gsdf表达量甚至凋亡生殖细胞，这些结果证实gsdf的表达与牙鲆早期性原细胞增殖具有密切的关系。我们采用荧光定量分析，性分化中牙鲆四组雌雄主导群体的gsdf，amh和p450arom基因的表达。结果显示，高温增加了gsdf和amh基因的表达，并抑制了芳香化酶p450arom的表达，从而导致雄性化，而雄激素却仅增加了amh的表达，并抑制了p450arom的表达，导致雄性化。

上述结果证实，牙鲆早期性原细胞增殖存在性别二态性，TGF-β信号通路(gsdf/amh/p450arom)基因的表达与性原细胞有丝分裂增殖存在密切关系，而其如何调控生殖细胞增殖还需进一步探索。本研究为丰富海水鱼类生殖细胞增殖发育积累必要资料，并为鱼类性别分化及生殖细胞的调控提供必要的理论知识。

Germ cell, a highly specialized cell, play an extremely important role in fish reproduction. As the precursors of gametes, primordial germ cells (PGCs) undergo a series of cellular changes including migration, proliferation and eventually differentiate into spermatogonia or oogonia. The studies on early gonium proliferation reveal that the number of germ cells determines the directions of sex differentiation in fish. However, little is known in the marine fish species. As a flat fish, the Japanese flounder (Paralichthys olivaceus) is one of the most important marine aquaculture species and widely distributes in the northwest Pacific Ocean, including China, Korea and Japan. Similar with other gonochorism fish, it has a XX (female)/XY (male) sex determination mechanism, but the sex of Japanese flounder would be influenced greatly by environmental factors. In this study, we reared XX flounder larvae with high temperature (27.5±0.5℃), androgen (17α-alphamethyltestosterone), estrogen (17β-estradiol) and normal condition (18°C ± 0.5 °C), and obtained four predominantly phenotypic male or female population. And the sex rations were male 93%, male 95.24%, female 99% and female 95% respectively. We investigated the gonadal differentiation and germ cell proliferation of Japanese flounder, and further analyzed the mechanisms of TGF-β signal pathway (gsdf/amh/cyp19a1a) regulating germ cells proliferation.

We first described the germ cells migration, proliferation and differentiation in Japanese flounder in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region, and at 15 dph, a part of PGCs arrived at the genital ridge. At 22 dph, the PGCs were totally enclosed by somatic cells and formed the gonadal primordia under the ventral kidney; 35dph, gonium began first mitosis and the basement membrane firstly appeared and gradually surrounded and separated the germ cells; from 52 to 66 dph, individual spermatogonia maintained continuous slow proliferation, and oognia began second mitosis forming 2 germ cell cysts and intensive increased in number; 73 dph, spermatogonia maintained continuous slow proliferation, and ovarian lumen and germinal epithelium formed in ovary; 85 dph, with the second mitosis of spermatogonia, 2-8 spermatogonia formed germline acinar-clusters and the spermaduct anlag also formed, and oocytes develop into first meiosis; 120 dph, type A spermatogonia in the clusters differentiated into type B, lost stem cell activity and formed seminiferous lobules.

The full-length cDNA of Japanese flounder gsdf homolog was cloned. Japanese flounder gsdf transcript was exclusively detected in gonadal tissue by RT-PCR. In situ hybridization and immunohistochemistry showed that both gsdf mRNA and protein restrictedly expressed in somatic cells around germline stem cell during ovary and testis development of Japanese flounder. Furthermore, in the gsdf overexpression and knockdown experiments, germ cell number in XX flounders significantly decreased even disappeared, showing gsdf expression was important for early gonium proliferation. The expressions of gsdf, amh and p450arom were analyzed by RT-PCR in four groups of Japanese flounder. The results showed that high temperature induced gsdf and amh expression and inhibited p450arom expression, causing masculinization; and androgen only induced amh expression, inhibiting p450arom expression.

Those results above verify the Japanese flounder early gonium proliferation exist sexual dimorphism, and the genes expression of TGF-β signal pathway (gsdf/amh/p450arom) probably participate in regulating germ cell proliferation. And the mechanism of germ cell proliferation need to be further analyzed. This study will provide the basic knowledge in regulating the sex differentiation and germ cell development in marine fish.