Expression Of EAP-1 And CUX-1 In The Hypothalamus Of Female Rats And Relationship With KISS-1 And GnRH

[Objective]The activation of the hypothalamus-pituitary-gonad axis represents the onset of puberty and is closely related with central precocious puberty of childhood.However,the neurobiological mechanisms are poorly understood.In 2007,using a combination of high throughput and bioinformatics strategies,a group of related genes including EAP-1 and CUX-1 were singled out from the hypothalamus of female nonhuman primates and rats.Increasing studies have demonstrated that no isolated gene or pathway is solely responsible for the pubertal regulation and that puberty is controlled by a gene network consisted of multiple functional genes.In this study,immunofluorescence staining,real-time PCR as well as western blotting were carried out to explore the expression and localization of EAP-1 and CUX-1,along with their relationship with KISS-1 and GnRH.[Method]Female SD rats were used in these studies.According to three different stages,juvenile(25 d of age),early puberty(35 d of age)and adult(42 d of age),animals were divided into 3 groups.10 rats in each group were killed,and the hypothalamus tissues were collected immediately for quantitative analysis of genes.The expression of EAP-1?CUX-1?KISS-1 and GnRN genes mRNA were measured with real-time PCR.?-actin was used as the internal standard.Rats of three groups with 6 in each group were killed,and brain tissues were collected for immunofluorescence staining.Medical imaging analysis software was used to count the numbers of EAP-1 or CUX-1 positive cells.The protein expression changes of EAP-1 were measured by western blotting.[Result]The KISS-1 mRNA level in the early-puberty group was significantly increased compared with the juvenile group(P<0.01),while the GnRH mRNA level reached a peak at the adult age(P<0.01 versus the juvenile group,P<0.05 versus the early-puberty group).However,EAP-1 and CUX-1 mRNA expression in the hypothalamus did not significantly changed during the different sexual development stages,as determined by real-time PCR(P>0.05),a finding inconsistent with previous results.Immunofluorescence staining results showed that EAP-1 and CUX-1 genes were localized to neuronal nuclei.Both were prominent in cells of the the arcuate nucleus(ARC)of the rat hypothalamus.Especially,CUX-1 gene was coexpressed in the Kisspeptin neurons.Moreover,the number and percentage of EAP-1 positive cells in the early-puberty group were both significantly more than the juvenile group(P<0.01),while the difference in the amounts of CUX-1 positive cells among the three groups was not statistically significant(P>0.05).The protein expression of EAP-1 in the early-puberty group was significantly higher than that in the juvenile group(P<0.05).[Conclusion]EAP1 expression increases at the early puberty in accordance with KISS1 gene and its localization is close to the kisspeptin neurons,which indicates that EAP-1 gene may be involved in the neuroendocrine control of female puberty in correlation with the Kisspeptin/GPR54 signal pathway.