| Animal sex determination mode who has a profound impact to ontogeny and naturalsex ratio composition is a hot research issue of the field of biology. The sex determinationmechanisms can be divided into two categories: genotypic sex determination (GSD),temperature-dependent sex determination (TSD). However, fish sex determination isinfluenced by a combination of genotype and temperature. In other words, sexdetermination in some fish can be affected by temperature changes when the gonad isundifferentiated. But it is still unknown that how temperature can override the role ofgenetic factors and switch the mechanism to affect the sex determination and differentiationof fish. Nile tilapia (Oreochromis niloticus) is a global farmed fish, whose whole-genomesequencing had been completed in2012, and many sex determination and differentiationgene sequences also had been cloned. It has a good basis for further study in sexdetermination and differentiation. In this study, with temperature treatment during sexdifferentiation, the expression of18genes include12sex differentiation related genes,4heat shock protein genes and2DNA methylation genes were systematically analyzed byquantitative real-time RT-PCR techniques.1. Genes selected and sequencingThe18genes selected in the present study included12sex differentiation genes(Cyp19a1a, Cyp19a1b, Cyp11b2, Dmrt1, Foxl2, Sox9a, ERα, ERβ, GTHα, FSHβ, LHβ andARα),4heat shock protein genes (Hsp27, DNAJB1or Hsp40, Hsp70and Hsp90), and2DNA methyltransferase genes (Dnmt1, Dnmt3a). In addition, elongation factor1α wasselected as internal quantitative control. For7predicted genes (Hsp27, DNAJB1, Hsp70,Hsp90, Dnmt1, Dnmt3a, and EF-1α), the PCR products were purified, cloned andsequenced. Aligning the sequenced result of7genes with predicted sequences, it showedthat the homology between the deduced Hsp27, DNAJB1, Hsp70, Hsp90, Dnmt1, Dnmt3a,EF-1α sequences and the predicted sequences was99%. The deduced Hsp27, DNAJB1, Hsp70, Hsp90, Dnmt1, Dnmt3a, and EF-1α partial amino acid sequences shared a higheridentity (50%~90%) with that of zebrafish.2. Genetic sex determination of Nile tilapia larvaeCyp19a1a and ERβ have the characteristics of sexual dimorphic expression. The realtime PCR, the Cyp19a1a as primer and the gonad cDNA as template, was utilized toidentify the genetic sex of Nile tilapia larvae. Similarly, qRT-PCR of ERβ was done tofurther verify the genetic sex result obtained from the Cyp19a1a. The result showed that theamplification curve of Cyp19a1a was significantly divided into two clusters, and the twoclusters respectively represented genetic female larvae and male larvae.3. Differential expression analysis of sex differentiation related genes after hightemperature inductionThe relative expression level of Cyp19a1a and Foxl2who is the transcriptionalregulating factor of Cyp19a1a was significantly decreased after high temperature induction(P<0.05). Expressions of GTHα, LHβ and ERα, which are known to be implicated inovarian development, were significantly down-regulated in the brain of females exposed tohigh temperature (P<0.05). These results showed that the expression of ovary developmentrelated genes can be suppressed by high temperature. The result of spearman rankcorrelation indicated that the expression of Cyp19a1a, was significant positively correlatewith the expression of GTHα and LHβ (P<0.05).The results showed that the expression levels of Cyp11b2and Dmrt1, which were thetestis development related genes, were significantly elevated after high temperatureinduction (P<0.05). Expression of ARα in the brain was also significantly increased afterhigh temperature induction (P<0.05). These results showed that high temperature inductionprovided strong masculinizing signal by increasing the expression of testis developmentrelated genes. The result of spearman rank correlation indicated that the expression ofCyp11b2, ARα and the expression of Cyp19a1a had significant negative correlation.4. Heat shock protein mRNA expression changes after high temperature inductionThe results of qRT-PCR showed that the expression levels of Hsp27, Hsp70and Hsp90in the gonad of females and males were significantly increased after high temperatureinduction (P<0.05) and then decreased after recovery. The result is consistent with the expectation. The expression levels of Hsp27, DNAJB1and Hsp70were significantlyelevated (P<0.05) in the brain of females in induction and recovery group when comparedto the control group. However, the expression levels of Hsp27, DNAJB1and Hsp90in thebrain of males had no significant change after high temperature induction. The Hsp70relative expression level in the brain of males was significantly increased after hightemperature induction and the expression continued to increase after recovery (P<0.05).5. Differential expression analysis of methyltransferase genes after high temperatureinductionThe expression levels of Dnmt1and Dnmt3a in the gonad of females and males wereboth increased after high temperature induction, and existed sexual dimorphic expression.In female tilapias, the expression changes of Dnmt1and Dnmt3a in the brain wereconsistent with that in the gonad. However, the expression changes of Dnmt1and Dnmt3ain the brain of males were opposite to that in the gonad. These results showed that hightemperature induction could up-regulate the expression of Methyltransferase in female andmale tilapias, especially the Dnmt1in the gonad of males. High temperature inductioncould probably regulate the expression of other genes, especially the sex differentiationrelated genes by affecting the level of methylation of tilapia.In conclusion, the results of the present study showed that expression of ovarydevelopment related genes could be down-regulated but expression of testis developmentrelated genes could be up-regulated in genetic females Nile tilapia exposed to hightemperature. It preliminarily proved that high temperature induction could probably affectthe synthesis of gonadal hormone and induce the sex-reversal of genetic Nile tilapia byregulating the expression of gonad differentiation genes. The expression changes of Hspsand DNA methylation genes in Nile tilapia under high temperature induction showed thatthey may play an important role in the process of sex-reversal induced by high temperature.The results of the present study provide an important foundation for further studying themolecular mechanism of high temperature effects on Nile tilapia sex differentiation. |
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