| Pelodiscus sinensis (P. Sinensis), belonging Reptilia, Testudines, turtle family, isan important aquaculture species in southern China. During breeding process, the maleturtle grow faster than female turtle by25.0%to30.0%, and the male turtle has less fat,therefore, males are more welcomed by the market. Given this context, the Soft-shelledturtle farming enterprises have been carried out a series of technical explorationregarding the sex control, but still cannot make the male offspring ratio exceed65%.This is due to that the molecular mechanism of sex determination is unclear in P.Sinensis.To illustrate the molecular mechanism of sex determination, the DMRT1(doublesex and relatated mab3transcription factor1) was chose as a sex determinationcandidate gene in this study. At first, full-length cDNA sequence of DMRT1gene wascloned. Subsequently, we study the mRNA and protein expression of DMRT1gene andits cellular localization in different tissues and gonads of different developmental stagesof both sexes. Furthermore, the mRNA expression changes of DMRT1in estrogen oraromatase inhibitor-induced sex reversal were investigated. This study aims topreliminarily clarify the effect of DMRT1in sexual differentiation, which supplies thebasis for the subsequent functional research of DMRT1and the elucidation of molecularmechanisms of sex determination in P. Sinensis. Detailed results are as follow:(1) By RACE technique, we obtained full-length cDNA sequence of DMRT1gene (2409bp), which has1107bp of the open reading frame (ORF) encoding368amino acids. Besides, the full-length cDNA also contains a67bp of5’ untranslatedregion, a1058bp of3’ untranslated region and the TAA stop codon and poly-Atail. The phylogenetic tree analysis showed that P. sinensis has the closest geneticrelationship with Chrysemys picta bellii. Additionally, the mRNA expression ofDMRT1in heart, liver, spleen, lung, kidney, muscle, ovary and testes tissues wereexamined by qPCR, Results showed that DMRT1gene is highly expressed in the testis,however, no DMRT1mRNA was detected in other seven tissues, indicating thatDMRT1is a specific gene in male gonad.(2) The mRNA and protein expression of DMRT1gene and its cellular localizationin gonads of different developmental stages of both sexes were examined to investigate the involvement of DMRT1in sexual differentiation in P. Sinensis. Immunofluorescenceanalysis showed that DMRT1protein expression initiated at the stage16of male gonadprior to gonadal differentiation, localizated in nuclei of somatic cell, with no detectionin germ cells. The protein expression of DMRT1was increased by the stage17of malegonad, whereas female gonads showed no DMRT1expression. By the stage19, gonadaldifferentiation began, DMRT1protein was localized in nuclei of sertoli cell-precursor,around the primordial germ cells. As the embryo grew, the male gonad cortex wasfurther degraded to form a single cell layer, whereas the medulla region was continuedto develop as sex cord, and DMRT1protein were abundantly expressed in the precursorof sertoli cells. By this time point, female gonads appeared the further development ofthe cortex, medullar degeneration and vacuolar structure, without DMRT1proteinlocalization. After birth, the male gonads differentiated into the testes, primary sex corddeveloped into seminiferous tubules with sertoli cells surrounding germ cells. In the1-3year of testes, DMRT1protein was continued to position on the sertoli cells, no DMRT1protein was detected in germ cells. Moreover, adult ovaries did not express DMRT1protein. Given that stage16-19is the critical period in turtle sex determination andgonadal differentiation, these data suggest that DMRT1is likely to play a role in malesex determination differentiation in P. Sinensis.(3) In many vertebrate species, estrogens play a key role in sex determination andgondal differentiation. To investigate the effect of estrogens in the gonadaldifferentiation of P. Sinensis and the underlying molecular mechanism, we treated theundifferentiated embryos with17β-estradiol (E2) and aromatase inhibitor (AI). Resultsshowed that the female ratio increased by92.3%after E2treatment, whereas femaleratio in AI treatment group decreased dramatically to13.1%. Meanwhile, the obviousmasculinization of genetically female embryos and feminization of genetically maleembryos were observed after AI and E2treatment, respectively, indicating the importantrole of estrogens in the female sexual differentiation in P. Sinensis. Based on theestablishment of the hormone-induced sex reversal model, this paper also analyzes theexpression change of DMRT1gene in the sex reversal. RT-PCR andimmunofluorescence analysis showed that mRNA and protein expressions of DMRT1were significantly down regulated in E2-induced male to female sex reversal. However,AI exerted the opposite effect. These results indicated that DMRT1participate in themale gonadal differentiation process in P. Sinensis.In conclusion, these above data suggest that the DMRT1gene is a male specificgene and participates in the male gonadal differentiation, which supply the basis for thesubsequent function research of DMRT1in P. Sinensis. |
PDF Full Text Request