Molecular Cloning Of ESR1 And ESR2 Genes And The Effect Analysis Of The Gonadal Development In Pelodiscus Sinensis

Estrogen is one of the most important steroid hormones which is closely related to sex differentiation and gonadal development in vertebrates.The estrogen response is mediated through estrogen receptors（ER）.At present,two types of classical estrogen receptors（n ER）are known which namely estrogen receptor 1/alpha（ESR1,ERα）and estrogen receptor 2/beta（ESR2,ERβ）.G protein coupled estrogen receptor（GPER）is membrane estrogen receptors.n ER are the first discovered estrogen receptors and involved in a variety of physiological activities in animals.Knockout experiments showed that n ER deficiency could affect gonadal development and even leaded to sterility in animals.The Chinese soft-shelled turtle（Pelodiscus sinensis）is one of the valuable freshwater species that has a long history of breeding in China.Their whole body are very valuable that have medicinal value in their meat,blood,back armour and so on.The turtle is more popular in consumers and more higher economic value than common aquatic products.The turtle is also an ideal material for studying the molecular mechanisms of vertebrate sex differentiation and reproduction.In this study,we first cloned ESR1 and ESR2 cDNA full-length squences by RACE technology.Secondly,we explored ESR1 and ESR2 m RNA expression patterns in different tissues and embryonic development stages by q-RT PCR.Finally,The effects of n ER on gonads of turtle were studied by intraperitoneal injection agonists and antagonists.The results were as follows:The aims of this study were to identify the sequence information and function of ESR1 and ESR2 in the turtle.The full-length P.sinensis ESR1 and ESR2 cDNA sequences were cloned.The reverse transcription template was the total RNA of the gonad of P.sinensis.The results showed that the full-length cDNA sequence of ESR1 cDNA was 6061 bp long and encoded 588 amino acids.The total length of ESR2 cDNA was 6396 bp and encoded 556 amino acids.They were both contain the typical DBD and LBD domains of the nuclear receptor superfamily.The phylogenetic tree showed that the cDNA sequences ESR1 and ESR2 which we cloned by the RACE were right.q-RTPCR results showed that ESR1 was expressed in all tissues of P.sinensis,the highest expression in testis,followed by ovary.But ESR2 was only expressed in gonad and brain.The expression of ESR1 gene increased gradually with the development of male embryo at the key stages in gonad development,reached the highest level at 18 stage and then decreased.The expression of ESR1 gene in female embryo gradually decreased with the development of female embryo,the expression level in 20 stage increased and then decreased significantly（P < 0.05）.ESR2 was no expression at m RNA level during the same periods in male and female embryo.To investigated the protein localization of ESR1 and ESR2 in the gonad of P.sinensis,we used p ET-32 a as expression vector to constructed the recombinant plasmids p ET32a-ESR1 and p ET32a-ESR2.Recombinant ESR1 protein polypeptide was 34.6 k D,ESR2 was 33.4 k D.Their titers of antibody were 2.7×105 and 2.43×106,respectively.Immunohistochemical results showed that ESR1 and ESR2 were located at leydig cells and spermatogenic cells in testis,located at membrane cells in ovary.These results suggest that ESR1 and ESR2 may play important role in gonadal development and maintenance.In order to reseach the role of ESR1 and ESR2 in gonadal development,4 month turtles were injected by specific agonists PPT and DPN and adult male turtles were treated by the n ER antagonist ICI 182,780.The results showed that PPT and DPN both increased the ovary size,the number of oocyte also increased.we did not found morphological changes in the testis after treated by ICI 182,780.The gene expression of tsc2,Gn RH,eif4 b,fshb,rps6 and mtor were significantly increased,but the expression of rps6 and mtor were decreased in ovary after treated by PPT and DPN.In the testis after treated by DPN,the expression of sycp3,spata6,picalm,evl and stra8,cyclin D were significantly increased.While in the testis after treated by PPT,the expression of stra8 and picalm were increased.the expression of sycp3,dmc1 and spata6 were decreased.After ICI 182,780 was injected into adult male turtles,the E2 level was significantly increased,the number of spermatocytes were increased and the number of sperm were decreased.The relative expression of stra8,sycp3,dmc1,picalm,evl,boule and cdk1 were significantly increased in testis by q-RT PCR.To sum up,ESR1 and ESR2 were expressed in the gonads of P.sinensis both at m RNA level and protein level,q-RT PCR results showed that in the critical stage of gonadal differentiation,ESR1 was differentially expressed in female and male embryos,while ESR2 was not expressed at the same stages.The research results of n ER antagonists and specific agonists suggest that n ER plays an important role in the sex differentiation and development of P.sinensis.