| Many research field in life science,animal sex determination and differentiation as a hotspot of biology can implement specific gender in the actual production requirement,which for some economic animal breeding is of great significance.Among them,for the research results show that the fish fish sex determination as well as the comprehensive effect of the factors such as the endocrine and exogenous hormones.Through sex determination of fish genes and affected by exogenous hormones,is not only beneficial to the development of the vertebrate research,at the same time will promote the development of economic fish breeding.M.anguillicaudatus is an important fresh water fish economy in China,due to its huge commercial value,in China,more and more people choose in the agriculture field collection loach is to implement a kind of rice and the breeding of loach industry a win-win situation.Due to P4 is often used to control pregnancy time of animals in the livestock industry,environmental residues of P4 may pose potential risks to M.anguillicaudatus in the receiving aquatic environment.Hence,the aim of this study was to investigate the effects of P4 on the sex differentiation by quantitative determination of transcriptional changes of target gene(Sox4 and Dax1)induced by P4 in M.anguillicaudatusIn this paper,we firstly introduce and analyze the M.anguillicaudatus Sox4 and Daxl cDNA sequence.The full length cDNA of Sox4(designated as Ma-Sox4)was 2122 bp,encoding a putative protein of 378 amino acids.The amino acids contained the conservative sequences in HMG-box.The full length cDNA of Daxl(designated as Ma-Daxl)in was 1208 bp,encoding a putative protein of 264 amino acids.The signal peptide analysis predicted that Ma-Daxl and Ma-Sox4 protein are two non-secretory protein.The hydropathy profile of Ma-Daxl and Ma-Sox4 protein revealed that all two protein are hydrophilic in nature.Then we evaluated few of such genes to identify suitable housekeeping genes as references.qRT-PCR detection of mRNA transcription was applied to four putative reference genes,?-actin(ACTB),elongation factor 1 alpha(EF-1a),glyceraldehyde-3-phosphate(GAPDH),and 18S ribosomal RNA(18S rRNA)in different groups based on gender,tissue type,developmental stages and chemical treatment.After analyzed by three independent common statistical algorithms,,?-actin(ACTS)showed highest stability in all the experimental conditions.qRT-PCR and in situ hybridization were conducted to determine the expression of Ma-Sox4 and Ma-Daxl gene in different stages during embryonic development and adult tissues.The Ma-Sox4 and Ma-Daxl were highly expressed in testis,while the other tissues taken for basal expression pattern studies showed low expression.In situ hybridization results demonstrated that Ma-Sox4 and Ma-Daxl gene expressed in developing germ cells.In different embryonic developement stages,Ma-Sox4 and Ma-Daxl transcript was expressed in all examined developement stages.Whole mount in situ hybridization results showed that Ma-Sox4 and Ma-Daxl mRNA expressed in all examined developement stages,and broadly in the CNS and strong expression was observed in the optic vesicle and otic vesicle.Those results showed that Ma-Sox4 and Ma-Daxl gene highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis,central nervous system development and gonad development.For the P4 administration assay,groups of mature fish were exposed for 1,7,14,21 and 28d to nominal concentrations of 10,100,and 1,000 ng/L P4 in a flow-through system.Quantification of Ma-Sox4 and Ma-Daxl transcripts revealed the expression of Ma-Sox4 and Ma-Daxl mRNA is altered after P4 treatment in mature gonads at different time points.The overall results showed that P4 could influence the sexual development and sex differentiation in M.anguillicaudatus by disturbing sex-differentiation associated gene expression,and Ma-Sox4 and Ma-Daxl can be used as sensitive molecular biomarkers for early warning to monitor the environmental progestins chemicals in fresh water environment.In conclusion,we have cloned the full length cDNA of Ma-Sox4 and Ma-Daxl and characterized the mRNA spatio-temporal expression patterns.The results showed that Ma-Sox4 and Ma-Daxl gene highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis,central nervous system development and gonad development.In addition,the results of P4 exposure suggest that P4 could cause a disruption of sexual development and sex differentiation at environmentally relevant concentrations based on the regulation of the transcriptional levels of Ma-Sox4 and Ma-Daxl,The study is to explore the Sox4 and Daxl gene expression and regulation pattern in loach,the gender of the molecular mechanism for further in-depth study of M.anguillicaudatus a strong foundation,but also for the research in the field of expanded the broader thinking,then it is necessary to delve into P4 impact on fish and regulation mechanism. |
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