Establishment And Characterization Of Three Cell Lines And Expression Patterns Of Dnmt3 In Olive Flounder Paralichthys Olivaceus

Since the first permanent cell line(RTG-2)of rainbow trout(Oncorhynchus mykiss)established by Wolf and Quimby in 1962,more than 275 fish cell lines have been reported by 2011 in the world.However gonad or muscle-derived cell lines are rarely established.Fish gonadal somatic cells,such as ovarian stromal cells,granulosa cells and sertoli cells,leydig cells of the testes etc.,play crucial roles in the process of normal division and differentiation of germ cells,and in the gonadal structure.Then,the research of gonadal somatic cells become inevitable.As is known,the delicious fish food was mainly derived from the tissue of fish muscle,and the cell line from fish muscle will become a platform for the further study in growth,differentiation and development of fish muscle,and which will be helpful for the study and application of fast growth mechanism in the cultured fish.Furthermore,fish cell lines have been widely used in the theory and applied research of virology,genetics,gene regulation and functional analysis,fish physiology,endocrinology,fish resources protection and so on.Therefore,the research on fish cell culture and the establishment of fish cell lines will be of profound significance.The research on fish sex determination and gonad differentiation has been a hot spot,while study on DNA methylation of fish sex determination and gonadal differentiation is currently only in its infancy.The study on DNA methyltransferase such as dnmt3 is very scarce and the influence of environmental factors such as temperature,exogenous hormones on the methylation patterns is poorly understood.Furthermore,dmrt1 is considered to be the only conservative sex related gene,while research about it is more focused on freshwater fish.Research reports on methylation epigenetic modifications of dmr1 in gonad is still very weak,so it is urgent to carry out relevant research.Olive flounder(Paralichthys olivaceus),is an important marine aquaculture species in Japan,Korea and China.The females grow faster and larger than males when cultured in the same environment,so there has been considerable attention given to all-female production and the mechanisms of flounder sex determination and gonadal differentiation.However,little was known in particular at the cellar level due to the lack of appropriate gonadal cell lines to study the mechanism.Studies have shown that triploid ones of flounder grow faster than the diploid obviously.Furthermore triploid induction of flounder was succeed in 2010 and mass production has been achieved.So far,however,there is no research about the mechanism of growth and development of triploid muscle,neither report about in vitro culture of satellite cells from triploid muscle.Therefore,this study established and characterized three new fish cell lines from the testis,ovary and triploid muscle of P.olivaceus,and transfected exogenous plasmid to these three cell lines as in vitro systems for genetic operation.In addition,we cloned and got the full cDNA length of methyltransferase gene dnmt3 a and dnmt3 b.The gynogenetic flounder fry were cultured at 28 oC or were treated by estradiol(E2)in their critical transition period and we got the pseudo-male juveniles and all female juveniles.Expression levels of dnmt3 a,dnmt3b and dmrt1 during differentiation developmental stage of gonads of gynogenetic and its pseudo-male juveniles were detected by real-time PCR to analyze the regulation of methylation on sex-related dmrt1 gene.The main details and results were as follows:The cell lines POSC and POOC respectively derived from testis and ovary tissues of flounder have been subcultured 55 and 34 times.The two cell lines were optimally maintained at 25 oC in DMEM/F12 supplemented with 20% fetal bovine serum,and 15 ng/ml EGF for POSC and 20 ng/ml for POOC.POSC and POOC are mainly composed of fibroblast-like cells.COI were amplified by using RT-PCR method and subsequent comparative analysis demonstrated a 99% match with the known mitochondrial COI sequence of P.olivaceus,the results showed that POSC and POOC cells indeed originated from P.olivaceus.Chromosome analysis revealed that most POSC and POOC cells maintained the normal diploid karyotype with chromosome number of 48 respectively,which demonstrated that the two cell lines are normal flounder diploid cells.POSC cells were strongly positive for Fas-L and POOC cells were positive for P450 SCC and 3β-HSD,which suggested that POSC were mainly testicular sertoli cells and POOC were ovarian stromal cells.Both POSC and POOC cells were transfected with pEGFP-N3 plasmid by lipofectamineTM 2000 and the transfection efficiency was approximately 10% and 15% respectively.The skeletal muscle satellite cell line POMSCS(3n)from triploid flounder has been subcultured 66 times for about a year.POMSCS(3n)cells were optimally maintained at 25 oC in MEM supplemented with 20% FBS.POMSCS(3n)were mainly composed of fusiform or spindle-like cells.Chromosome analysis revealed that most POMSCS(3n)cells maintained the normal triploid karyotype with chromosome number of 72,which demonstrated that the cell line are triploid cells.POMSCS(3n)cells expressed the muscle satellite cell gene marker pax7 b by RT-PCR detecting and were strongly positive for Desmin,which both suggested that POMSCS(3n)were skeletal muscle satellite cells.The differentiation experiment showed that at 48 h after changed to DM,long fibrous cells could be observed in POMSCS(3n)compared with the control group all along with GM.What’s more,certain number of multinucleated myotubes with about 2 or 3-4 nuclei per cell after DAPI staining were found in the DM group.The results revealed that POMSCS(3n)cells have the differentiation ability to differentiate into small multinucleated myosatellite fibres.Similarly,POMSCS(3n)cells were transfected with pEGFP-N3 plasmid by lipofectamineTM 2000 and the transfection efficiency was approximately 20%.The full lengths of dnmt3 a and dnmt3 b cDNA sequences were cloned by using RT-PCR and RACE techniques.Four transcripts of dnmt3 a and three transcripts of dnmt3 b were obtained with high similarity,respectively.Among them,there all have the conservative PWWP domain.In order to compare the expression patterns between dnmt3 and dmrt1,the gynogenetic flounder fry were separately treated by E2 and cultured at 28 oC in their critical transition period and finally we got the expected 100%(genetic and phenotypic)female flounders and 100% pseudo-male juveniles.Expression patterns of dnmt3 a,dnmt3b and dmrt1 during gonadal differentiation developmental stage of gynogenetic respectively treated by E2 and 28 oC were detected by real-time PCR to analyze the regulation of methylation on sex-related dmrt1 gene.The results revealed that the expression of dmrt1 was regulated by both dnmt3 a and dnmt3 b,and dnmt3 a meight play more important role than dnmt3 b during the process.Then we implied that the sex determination and gonad differentiation of P.olivaceus could also be regulated by DNA methylation.