Molecular Characterization And Expression Patterns Of Glucocorticoid Receptor Genes In Yellowtail Clownfish,Amphiprion Clarkii

The yellowtail clownfish(Amphiprion clarkii)is a protandrous hermaphrodite teleost,and their gonadal development is closely associated with their social status in the group.There is only one dominant female and one male with reproductive capacity in a group.When the female is absent,the male undergoes the sex change to become a dominant female.In vertebrates,glucocorticoid,as an important stress hormone,can directly act on the brain by glucocorticoid receptor(GR)to induce a wide range of neuroendocrine responses.Some studies have revealed that social stress response may directly act on the reproductive axis through glucocorticoids and GR to participate in the regulation of gonadal differentiation and sex change.In this paper,the GR system of yellowtail clownfish with different social status was studied.The main contents and results are as follows:1.The c DNA sequences of yellowtail clownfish gr1 and gr2 genes were cloned by RACE PCR,encoding 812 amino acids and 782 amino acids respectively.It can be found that GR1 had a nine-amino-acid insert unique to teleost,WRARQNTDG,into the DNA binding domain.Phylogenetic tree analysis showed that the amino acid sequences of GR1 and GR2 were clustered to teleost GR1 and GR2 respectively.Through ligand receptor binding assay,it was found that dexamethasone,exogenous glucocorticoid,can bind with GR1 and GR2,and activate the GRE(glucocorticoid response elements)signalling pathway.2.After five months breeding yellowtail clownfish in groups to form the stability of social hierarchy in the group,and their gonadal development status of each group was observed via histology.The results showed that all groups formed female,male and nonbreeder three social status respectively.Meanwhile,plasma cortisol levels in different social status individuals were measured by ELISA(enzyme linked immunosorbent assay).The results showed that the plasma cortisol level of nonbreeders was significantly higher than that of dominant individuals.It suggested that the increased glucocorticoid level caused by social stress was closely related to the mechanism of sex differentiation in yellowtail clownfish.3.The yellowtail clownfish gr1 and gr2 genes tissue distribution were studied by real-time PCR,indicating that gr1 gene was highly expressed in the cerebellum,hypothalamus and heart.The gr2 was highly expressed in the pituitary and liver of females and nonbreeders.And gr2 was also highly expressed in the medulla oblongata and middle kidney of males.The gr1 and gr2 genes expression patterns among different social status yellowtail clownfish were detected.The results showed that in the hypothalamus,the gr2 expression was significantly higher in nonbreeders than in males;in the pituitary,the expression of gr1 and gr2 genes had no significant difference among different social status;in the head kidney,gr1 transcripts of nonbreeders was significantly higher than that of females and males;in the gonad,gr1 expression level of females was significantly higher than that of males.It is suggested that the glucocorticoid level change caused by social stress may regulate the gonadal development and other reproductive functions through glucocorticoid receptor in the brain.