| Steroid hormones exert a significant influence on the teleost,for growth,reproduction,development,metabolism as well as maintaining the stability of the internal environment,all the above-mentioned biological activities need the participation of hormones.Therefore,the synthesis of steroid hormone is particularly important,which needs a series of steroid synthetases.Among them,hydroxysteroid hormone synthase 3β-HSD2 is a key enzyme participating in several essential steps.Until now,there is less report about the role of 3β-HSD2 in the female sex differentiation in teleostsQi river crucian carp（Carassius auratus）is a local population of gibel carp,formed by the long-term evolution and is named after being produced in the Qi river,Henan Province,it can reproduce all female through gynogenesis,therefore,it is of great significance in studying the female sex differentiation.In this paper,Qi river crucian carp was used as the experimental model to study the role of 3β-HSD2 in female sex differentiation,the major results were as follows:1.The full length c DNA of 3β-HSD2 gene was cloned,which includes an open reading frame of1104 bp and encodes 367 amino acid residues.The relative molecular weight of the protein was predicted to be 41.95 k Da and the corresponding protein isoelectric point was 6.50.In addition,it also contains 218 bp 5’UTR,401 bp 3’UTR.The phylogenetic tree shows that the 3β-HSD2 of Qi river crucian carp and other fishes form a branch.3β-HSD2 of Qi river crucian carp has the typical steroid binding domains and cofactor binding domains of the hydroxysteroid family（Romans fold,GXGXXG）and enzyme activation site（YXXXK）,in Qi river crucian carp they were GCGFLG and YPKTK,respectively.2.Real-time PCR analysis shows that,the expression level of 3β-HSD2 showed the highest in the spleen and medium in the ovary of adult fish.During embryonic development,the expression of3β-HSD2 was always at a medium level and reached the highest level in the hatching stage.During 30-40 days after hatching（the critical period of ovarian differentiation in Qi river crucian carp）,the level of3β-HSD2 increased significantly.And continue to increase from 120 dah,and then peaking at 720 dah.The above research results indicated that 3β-HSD2 might have an influence on the process of embryonic development and sex determination as well as maintaining of ovarian development process.3.We prepared 3β-HSD2 polyclonal antibodies to verify its expression in different tissues through Western Blot.Then we determined the cellular localization of 3β-HSD2 in the gonads of Qi river crucian carp through immunohistochemistry（IHC）.The results suggested that the prepared polyclonal antibody can specifically recognize a single band around 42 k Da in the total protein of Qi river crucian carp ovary,which revealed the prepared 3β-HSD2 antibody had high specificity.Western Blot results indicate that 3β-HSD2 showed the highest level in the spleen and moderately in the ovary and other tissues,which is in accordance with the real-time PCR result.4.The results of IHC show that 3β-HSD2 was specifically expressed in somatic cells at 15 dah.At 35-80 dah it was expressed in oogonia,oocytes and somatic cells.Furthermore,at 100-120 dah and was,3β-HSD2 was expressed in oogonia and somatic cells.From 150 dah it was specifically expressed in somatic cells.Its widespread expression in germ cells and somatic cells indicates that it might have multiple roles in the ovary and have an influence on the ovarian development and subsequent maintenance.5.In order to further explore the role of 3β-HSD2 in female sex determination,we use the inhibitor Trilostane to treat 5 dah Qi river crucian carp for 75 days.Results showed that Trilostane can induce sexual reversal.Real-time PCR results showed that after Trilostane treatment,Qi river crucian carp 3β-HSD2 expression was significantly down-regulated.IHC showed that,in the sex reversed testis resulted by Trilostane treatment,3β-HSD2 was expressed in interstitial cells.At the same time,the expression levels of some sex-biased genes we also detected.The results showed that the expression of female sex determining genes（cyp19a1a,β-catenin1 and foxl2）was significantly decreased,and the expression of male sex determination genes（dmrt1 and cyp11b2）significantly increased.In summary,this study proves that the role of 3β-HSD2 is inseparable in the process of estrogen synthesis.By inhibiting 3β-HSD2,Qi river crucian carp was sexually reversed.Therefore,this study supports the "deficiency hypothesis" that in the early stage of fish gonadal differentiation,estrogen does not appear and develops into male fish. |
PDF Full Text Request