Transcriptome Analysis Of Male And Female Gonads And Study On Sex Steroid Hormone In Spotted Scat(Scatophagus Argus)

Scatophagus argus is an important farmed fish with both edible and ornamental value,has the typical sexual dimorphism between the male and female.They have different growths feature obviously,the female fish grows is fast and the body is large,has a good character on aquaculture.While the male fish grows is slow and the body is small,which is valuable in ornament.The female and male fish do not have synchronized development,the ovary attains maturity later than the testis,which is difficult to ensure the synchronous aging of male and female fish in artificial breeding that resulting in a low production efficiency.Sexual dimorphism is a physiological activity that is prevalent in the kingdom of animals.Part of the reason for its formation probably are many related genes differential expression,and the difference level of sex steroid hormones or various eating habit.It is a complex growth and development process in which various factors coordinate in this system.The male and female gonads of fish have different developmental feature.After sex determination,they begin to differentiate into testis or ovary,which make the internal gonad structure and external morphology show obvious sexual dimorphism,and some gene expression are also significant differences.Many researches have focused on the discovery of mRNA expression in fish gonads.In this study,the S.argus gonadal transcriptome sequencing was carried out.Discovered differentially expressed genes and enriched signaling pathways in testes and ovaries from mRNA levels.The levels of 17?-estradiol,11-ketotestosterone and testosterone in serum were determined.Analyzed gonads mRNA expression levels of sex steroid synthesis related genes and their receptors at different developmental stages.Initially answered the question for the difference in serum steroid hormone levels,providing basic data for the deep analysis of the possible molecular mechanism between male and female dimorphism.The main results were as follows:1.Transcriptome analysis of S.argus gonads,discovery of differentially expressed genes and signaling pathwaysHigh-throughput sequencing S.argus testes?n=3?and ovaries?n=3?of mature stage III using RNA-seq technology.cDNA libraries were constructed in the male and female gonads.After de novo assembly,136,561 unigenes were obtained,which acquired many annotation information by blast in various database.32,122 differentially expressed genes were obtained by differential discovery conditions?|log₂FoldChange|?2,P-values<0.01?.Among them,11,156?34.73%?were highly expressed in testes,and 20,966?65.27%?were highly expressed in ovaries.20 differentially expressed gene were verified the expression levels using quantitative real-time PCR,which were consistent with the RNA-seq that confirmed the reliably.According to the database annotation and others research,further discovered 73 known functional differentially expressed genes.Among them,53genes were highly expressed in males,such as dmrt1,amh,gsdf,wt1a,sox9b and nanos2.20 genes were highly expressed in females,such as foxl2,sox3,gdf9,bmp15,zar1 and figla.In addition,we found some important sex-related functional classification in differentially expressed genes by blast in different database,including sex differentiation,sexual reproduction,reproductive process,gamete generation and gonad development.Also enrichment some important signaling pathway,including Steroid hormone biosynthesis,Ovarian steroidogenesis,PI3K-Akt signaling pathway,Insulin signaling pathway and FoxO signaling pathway.2.Serum steroid hormone levels in different developmental stages of male and female S.argusThe levels of 17?-estradiol?E2?,11-ketotestosterone?11-KT?,testosterone?T?in serum of female II?III?IV stages and III?IV?V male stages were determined.The results showed that the E2 level was significantly higher in female than that of male at different developmental stages,and its level increased with the developmental stages,the female reached the highest level?0.80 ng/mL?in stage IV.The 11-KT level was higher in males than in females at different developmental stages,and gradually increased with developmental stages.The male reached the highest level?0.47 ng/mL?in stage V.The T level is higher in females than in males,and the females level at each developmental stage is significantly higher than that of males,the female reached the highest level?0.41 ng/mL?in stage IV.T is a precursor in the synthesis of E2 and 11-KT,maybe balancing the regulation during the process of synthesis of E2 and 11-KT.The high levels of E2 in females and the high levels of 11-KT in males may play an important role in maintaining male and female sexual feature.It is speculated that the S.argus may be affected by the relative levels of estrogen and androgen to regulate the growth.3.Expression of sex steroidogenesis related-genes and receptors genes in different developmental stagesNineteen important genes related to sex steroidogenesis synthesis pathways and their receptor genes were discovered from the RNA-seq.The mRNAs were quantitative verified in ovaries of II?III?IV stages and testes of III?IV?V stages,which showed sexual dimorphism expression level in different developmental stages of ovaries and testes.The expression levels of StAR1,StAR2,cyp11a1,cyp11b2,cyp19a1b,hsd11b2,hsd3b1,hsd3b7,ar,er?,er?1 and er?2 were higher in the testes than in the ovaries.The expression level of cyp19a1a,hsd17b1,hsd17b8,hsd17b12 and hsd17b14 were higher in the ovaries than in the testes.Cyp11b2 acts as a key gene for the conversion of T to 11-KT,its high expression in males probably contribute to the high level of 11-KT in males.The high expression of the aromatase gene cyp19a1a in different development stages of the female is promote the conversion of T to E2,which maintain a higher E2 level at the females S.argus.The different expression levels of putative sex steroidogenesis synthesis related-genes and their receptors probably is one of the reasons for the difference in growth between male and female fish.Sex steroidogenesis synthesis related-genes and their receptor genes probably involved in regulating estrogen and androgen levels to affecting the growth and reproductive processes of female and male S.argus.This is the first research on sequenced and analyzed gonad transcriptome of the S.argus,which provides a basic reference for further study of the sex control mechanism and molecular characteristics,and provides some basic data support for the sex control and reproductive development in S.argus aquaculture.