Studies On Early Life History,Gonadal Development And Expression Patterns Of Germ Cell Marker Genes In Cobia (Rachycentron Canadum)

The morphological characteristics were observed and studied in the early life stage of cobia(Rachycentron canadum),the growth and development characteristics of the embryo,larvae and juvenile were described.To gain foundament data,which was provided for cobia biology and artifical propagation investigate.Histological observation of gonadogenesis,gonalal differentiation and annual development in cobia was performed based on paraffin section technique and Haematoxylin-Eosin(H.E.)staining method.We cloned the vasa and dnd homologue gene from cobia,analyzed its tissues expression distribution and the expression profile during embryonic development and annual gonadal development.Furthermore,we also investigated its expression location in the gametogenesis stage by chromogenic in situ hybridization(CISH).This study will provide a theoretical reference for understanding reproduction laws of cobia and revealing the regulation mechanism of germ cells differentiation during gametogenesis in teleost.The main results are as follows:(1)The fertilized eggs obtained by artificial spawning were spherical in shape,translucent and buoyant,there was an oil globule in the central,the diameter of egg and oil globule were limited to(1.245±0.065)mm and(0.325±0.027)mm respectively.The fertilized eggs hatched 26 h 30 min after fertilization in the sea water at 27.0±0.5℃,salinity of 29 and p H of 8.3.The embryonic development process was divided into 7stages,including fertilized egg stage,the cleavage stage,the blastocyst stage,the gastrula stage,the neurula stage,the organogenesis stage and the hatching stage(total24 developing periods).The total length(TL)of newly hatched larvae was(3.254±0.096)mm.The larvae started to eat protozoa and exhibited blackened eyes at3 dph(days post hatching),meanwhile,the yolk-sac decreased in volume by approximately 80%.The yolk-sac and oil globule completely absorbed at 5 dph and 7dph respectively,and the larvae completely entered the exogenous nutritional stage.The larvae started to develop into juvenile at 14 dph(TL of 19.933 mm±1.118 mm)when the development of each fin completed.At 22 dph,the juvenile with a total length of(41.140±3.779)mm,there were some scales on the caudal peduncle formed.The 46 dph juvenile reached(116.667±5.916)mm in TL,the surface body covered with cycloid scales and their general appearance was similar to that of adults,except for the shape of caudal fin.(2)After hatching,the primordial germ cells(PGCs)of cobia underwent a specific migration and arrived at genital ridge at 7 dph.Then the somatic cells began to aggregate around the PGCs,and at 15 dph,the PGCs were enclosed entirely by somatic cells and formed the primary gonad.Two types of primary gonads with noticeable histological differences could be observed at 34 dph.The presence of clusters of oogonia characterized a part of primary gonads.The other primary gonads showed narrower cross section with fewer germ cells,which were presumed to be undifferentiated testis.Similar to other fishes,ovarian differentiation of cobia was anterior to testicular differentiation.The anatomical indication of ovarian differentiation was the ovarian cavity formation,which occurred at 44 dph.The cytological differentiation of testis began at about 50 dph and was characterized by the presence of spermatogonial acinar-clusters.(3)The first annual development of testes could be divided into five stages(Stage I,II,III,IV,V),while the ovaries was divided into three stages(Stage I,II,III).Testes and ovaries were both developed to early stage I at 60 dph.All of the testes developed to Stage II at 90 dph;the ovaries were still at stage I.More than half of the testes developed to Stage III and part of the ovaries developed to Stage II at 120 dph.The testes were mainly developed to Stage III at 150 dph,most of the ovaries were at Stage II.The testes were still at stage III and the ovaries were basically developed to Stage II at 185 dph.Most of the testes developed to Stage IV at 210 dph,but the ovaries were still at Stage II.At 360 dph,the testes entered Stage V and most of the ovaries developed to Stage III.(4)The full-length c DNA sequence of cobia vasa and dnd homologue were cloned through RACE technology.The results of q RT-PCR revealed that Rcvasa and Rcdnd specifically expressed in gonad.The expressions of Rcvasa and Rcdnd were detected in all stages examined during embryogenesis.The expression levels of Rcvasa during the early embryonic development stage were significantly higher than those after the gastrula stage,and Rcdnd first increased and then decreased as the embryo developed,the highest expression was found at multi-cell stage.During the development of testis,the expression of Rcvasa first increased significantly then stabilized,while Rcdnd tended to gradually increase.During the development of ovary,the expression of Rcvasa first increased to the maximum at 210 dph and then decreased.Rcdnd expression first increased significantly then stabilized as the ovary developed,and the expression level reached the maximum at 150 dph.(5)The results of CISH revealed that Rcvasa and Rcdnd m RNA was exclusively expressed in germ cells but barely detected in somatic cells.In the testis,Rcvasa and Rcdnd m RNA signals were concentrated in the periphery of spermatogonia and primary spermatocytes,weakly in secondary spermatocyte,barely detected in spermatid and spermatozoa.In the ovary,the hybridization signals of Rcvasa and Rcdnd m RNA were uniformly distributed in oocyte cytoplasm.Rcvasa m RNA was expressed highly in early primary oocytes(phase I and II),while the oocytes developed to phase III,the Rcvasa m RNA signals were significantly weakened.There were no significant difference of Rcdnd m RNA signals detected in phase Ⅰ,Ⅱ and Ⅲ oocytes.