| 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 p450 arom were analyzed by RT-PCR in four groups of Japanese flounder.The results showed that high temperature induced gsdf and amh expression and inhibited p450 arom expression,causing masculinization;and androgen only induced amh expression,inhibiting p450 arom 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. |
