| Chinese soft-shelled turtle(Pelodiscus sinensis)is an important aquaculture species in China with delicious meat and high nutrition.P.sinensis has obvious sex dimorphism,the growth rate of males was significantly faster than that of females,and the market value of males was higher than that of females,which was deeply popular to consumers.Estrogen was used to artificially induce P.sinensis,and a pseudo female population(△ZZ)with male genotype(ZZ)and female gonads was obtained.Then,the pseudo female population(△ZZ)was mated with a normal male population(ZZ)to obtain all-male offspring,which could significantly improve the market seedling value of P.sinensis.The sex differentiation and sexual reversal induced by estrogen receptor in P.sinensis were analyzed in this study.As an important steroid hormone,estrogen plays an important role in the development of gonads and the maintenance of secondary sex characteristics.Estrogen exerts its corresponding effect mainly through estrogen receptor(ERs).ERs are classified into two classes,which are the estrogen nuclear receptors,including estrogen receptor α(ERα),and estrogen receptor β(ERβ).One is the G protein coupled estrogen receptor(GPER).The purpose of this study aims to investigate the function of estrogen receptor during sex differentiation and sexual reversal of P.sinensis in different tissues,embryonic development stage and different treatments.The main results of the research are as follows:(1)Clone and expression analysis of estrogen receptor gene in P.sinensisThe full-length cDNA sequence of estrogen receptor was obtained by rapidamplification of cDNA ends(RACE)technique using the gonads of P.s.The full-length of ERα was 3014 bp,5’ UTR 900 bp,3’ UTR 260bp,open reading frame(ORF)1854 bp,encoding 617 amino acids;The full-length of ERβ was 2950 bp,5’ UTR 483bp,3’ UTR 796bp,ORF 1671 bp,encoding 556 amino acids;The full-length of GPER is 2023 bp,5’UTR 705 bp,3’ UTR 241 bp,ORF 1077 bp,encoding 358 amino acids.The cloned sequences of estrogen nuclear receptors contain the classical DBD and LBD domains of the nuclear receptor superfamily.The DBD and LBD domains of ERα and ERβ shared 66%and 59%identity respectively.The GPER sequence contains seven transmembraneα-double helix classical structures.The expression of different tissues showed that estrogen receptor was ubiquitously expressed in many tissues,and estrogen nuclear receptors were mainly expressed in gonads.GPER was widely expressed in multiple organizations.During embryonic development,the expression level of ERα gradually increased,and in both female and male individuals,the expression level of ERα was much higher than those of the other two receptors.In male individuals,the expression levels of ERβ and GPER were basically the same.In female individuals,the expression level of GPER gradually increased over time and was higher than that of ERβin the late developmental stage.The fertilized eggs of P.sinensis during embryonic development were induced by exogenous estradiol(E2).The expression level of estrogen receptor increased significantly,the peak stage of estrogen receptor expression was postponed with the increase of E2 concentration,indicating that estrogen can prolong the time of estrogen receptors action.After 1 mg/mL and 5 mg/mL E2 treatment,the expression level of ERαwas significantly increased,reaching the peak at St.17,and extended to St.19 after 10 mg/mL E2 treatment.The expression level of ERβincreased most obviously after 1 mg/mL and 10 mg/mL E2 treatment,and the peak expression stage was extended from St.15 of 1 mg/mL to St.17.E2 has the most obvious promoting effect on GPER.With the increase of concentration,the expression level of GPER gradually increased,and the peak stage of expression extended from St.18 to St.19.The expression level of genes related to sex differentiation was detected in St.17 with high expression of estrogen receptor.The results showed that Rspol,Wnt4/βcatenin,Cyp19a1,Foxl2 and Sox3 were significantly promoted by E2.At the same time,it could significantly inhibit the expression of Dmrt1,Amh and Sox9,which were important factors in the early morphological stage of male gonad.(2)Receptor selectivity of estrogen during sex reversal in P.sinensisIn order to study the role of estrogen receptor in the sexual reversal in P.sinensis,estrogen receptor agonists(EB,DPN,G-1)and estrogen receptor inhibitors(AS,PHTPP,G-15)were used to induce the fertilized eggs of P.sinensis during embryonic development stage.EB significantly promoted the expression level of female pathway genes Rspol and Wnt4/β-catenin,and significantly inhibited the male pathway genes in late embryonic development stage.The expression levels of Rspol and Wnt4/β-catenin in the AS experimental group were decreased significantly,and the expression levels of Rspol and Wnt4 were decreased gradually.After DPN treatment,the expression level of Rspol was significantly inhibited,but the expression level of male related genes was not significantly promoted,and the expression level of male related genes was significantly decreased in PHTPP treatment.G-1 inhibited the expression of Rspol and Wnt4 in the middle and late stages of embryonic development,while G-15 promoted the expression of Dmrt1 and Amh in the late stages.The results of development vitro experiments showed that ERα agonist EB can simulate the promoting effect of E2 on the female sex differentiation gene Rspol and the inhibiting effect on Dmrt1.The combination of membrane agonists G-1 and E2 or inhibitors G-15 and E2 failed to significantly change the expression level of Rspol,indicating that membrane receptors had no effect on the expression of Rspol.In addition,in order to avoid competitive inhibition between receptors,the cells were incubated with three agonists in pairs.The results showed that the mixed treatment with EB,DPN and E2 had a significant promoting effect on the expression of Rspol and Dmrt1,which further indicated that nuclear receptors played a major role in the regulation of gene expression.In DPN+G-1 and E2,PHTPP+G-15 and E2,the expression of Rspol was not significantly different from that of the control group,which indicated that ERα had a direct regulatory effect on Rspol expression.In the mixed treatment group,the expression level of Dmrt1 was significantly increased,indicated the diversity of receptors regulated by ERs on Dmrt1 expression.The expression of Dmrt1 in AS+G-15 and E2 treatment groups was significantly higher than that in the control group,which suggested that the regulatory effect of Dmrt1 may be more closely related to ERβ.In conclusion,estrogen receptors in P.s transmit estrogen signals through different action pathways.Differences of the estrogen receptors ERα,ERβ,and GPER lead to differences in their functions.ERα showed high expression in ovary,while ERβ was slightly higher than ERα in testis,which may suggest that estrogen nuclear receptors played different roles in gonad development.Combined with the results of estrogen receptor agonists and inhibitors,it is further speculated that ERα plays a major role in ovarian development.ERβ may promote the development of testis.The widespread expression of GPER in tissues and its persistent expression during embryonic development suggested that its role is widespread. |
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