| The sex determination of fish divided into genetic sex determination(GSD)and environmental sex determination(ESD),and the genetic sex of a variety of fish can be affected by the environment,which temperature is one of the most important environmental factors.When the environmental temperature changes,fish can rely on temperature-sensitive response genes such as heat shock proteins(hsp),heat shock cognate proteins(hsc)and eukaryotic translation elongation factor(e EF1A)to transmit signals to the body.However,the role of these temperature-sensitive response genes or proteins responding to external high temperature and affecting sex determination and differentiation in vivo remains unclear.Chinese tongue sole(Cynoglossus semilaevis)is broadly distributed in the Yellow Sea and Bohai Sea of China,and it is a vital valuable marine fish for aquaculture.C.semilaevis is a typical female preference sexual dimorphism species,and the growth rate of the female is 2–4 times faster than that of male individuals,and the full length and weight of mature females can reach several times that of males.C.semilaevis has a female heterotypic sex determination system(ZW ♀/ZZ ♂),which is determined by GSD and ESD.Its original sex can be overturned by environmental factors.Under natural conditions,about 14% of the females in the C.semilaevis population will undergo sex reversal and change into ZW phenotypic males,namely pseudomales.If the high temperature treatment at 28 °C is carried out during the key period of sex determination of C.semilaevis,the proportion of pseudomales of ZW genotype will rose to 73%,and these pseudomales are fertile,and most of their offspring will continue to have sex reversal even under natural conditions.Thus,C.semilaevis can be used as an appropriate model for studying the response to temperature changes in teleost.In this study,the effects of short-term high temperature heat shock on C.semilaevis with different sex were analyzed.Transcriptome sequencing and analysis of the gonads of female and male of C.semilaevis were performed under high temperature(HS,28 °C)short-term time series(1 h,2 h,3 h,5 h)and normal temperature treatment(CT,22 °C).A total of 217 Gb of Clean data were obtained,and 87.71% of Clean Reads could be well compared to the reference genome of C.semilaevis.PCA analysis also proved that there were significant differences between the sexes of C.semilaevis,and there were also differences at different time of high temperature treatment.A large number of differential expressed genes(DEGs)were analyzed among different treatment groups.The normal temperature treatment group and the heat shock treatment groups for 1,2 and 3 hours respectively,and the number of DEGs was more than 10000,and the number of down-regulated DEGs was more than that of up-regulated DEGs.However,the female and male groups treated with heat shock for 5 hours were special,with only 134 DEGs,of which 104 were up-regulated and 30 were down-regulated.There were 6531 DEGs in females in different treatment groups of the same sex,and151 DEGs in each treatment group.There were 6743 DEGs in males and 488 DEGs in each treatment group.Functional analysis of differential genes compared between different dimensions showed that up-regulated genes were mainly concentrated in metabolic pathways,RNA transport,apoptosis,neuroactive ligand-receptor interaction,calcium signaling pathway,cytokine-cytokine receptor interaction,etc.Downregulated genes were mainly concentrated in neuroactive ligand-receptor interaction,MAPK signaling pathway and calcium signaling pathway.In addition,heat shock protein gene family and translation extension factor related genes in gonads were found to be involved in the process of high temperature heat shock and showed sex-specific expression characteristics.The above research results can help us screen candidate genes for high temperature response,and lay a foundation for revealing that fish respond to external temperature changes and thus affect their own sex determination and differentiation.In this paper,the full-length sequences of hsc70,hsc70-like and eef1a1 genes were cloned from C.semilaevis and their expression patterns were analyzed.The results showed that the expression of hsc70 and hsc70-like in the testis was much higher than that in the ovary,and the hsc70 was widely distributed in other tissues except muscle,the hsc70-like was almost not expressed in other tissues.eef1a1 was highly expressed in gonads,and its expression in ovary was significantly higher than that in testis.It also had high expression in liver and low expression in other tissues.At different stages of gonadal development,hsc70 was abundant in the gonads with a testicular-higher expression at all gonadal development stages except for 6 months post fertilization(mpf).Intriguingly,hsc70-like showed higher expression in testes from 6 mpf on,and eef1a1 showed higher expression in ovaries from 6 mpf on.Both long-term heat treatment during the temperature-sensitive sex-determining period and short-term heat stress at the end of this period caused different expression of hsc70,hsc70-like and eef1a1 between sexes.The dual-luciferase assay results also suggested that these genes can respond to high temperature rapidly in vitro.Heat treatment of C.semilaevis testis cells overexpressed with hsc70,hsc70-like and eef1a1 could affect the expression of sex-related genes sox9 a and cyp19a1 a.Our results indicated that hsc70,hsc70-like and eef1a1 were key regulators linking external high-temperature signals with sex differentiation in vivo and provide a new idea for understanding the mechanism by which high temperature affects sex determination/differentiation in teleost. |
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