The Regulatory Mechanism Of Hypothalamic Transcription Factor EAP1 In Pubety

Objective: Explore the puberty-regulatory effect of hypothalamic transcription factor EAP1 in vivo and the relationship between EAP1 and Kiss1/GPR54 signaling pathway and its function in pubety process.Methods: Four shRNAs and one negative control sequence targeting rat EAP1 mRNA were designed and constructed into lentiviral vector.And then choose the most efficient LV-EAP1-shRNA to the next experiment.21-day-old femal rats were randomly divided into three groups(LV-EAP1-shRNA group,LV-EAP1-shRNA(-)group and saline group).Rats of each group were intracerebroventricular injected with 40?l of LV-Eap1-shRNA,LV-Eap1-shRNA(-)and saline respectively.And then rats were raise in an appropriate condition to observed the age of vaginal opening at 4:00-5:00 every afternoon,and the development of ovary at three age stages(Juvenile(PND25);Early puberty(PND35);Adult(PND42))and the change of hypothalamic EAP1,GnRH and Kiss1 mRNA as well as protein level.Results: 1.The EAP1 mRNA level of hypothalamus was significant lower in the LV-EAP1-shRNA group compared to LV-EAP1-shRNA(-)group and saline group(p<0.05),at all three developmental stages.Moreover,the tendency of EAP1 protein expression was consistent with the mRNA expression,shown significant lower in LV-EAP1-shRNA group.2.At PND25 and PND35,hypothalamic GnRH mRNA level was significant lower in the LV-EAP1-shRNA group than LV-EAP1-shRNA(-)groups and saline group(p<0.05).However,the GnRH mRNA is no significant difference among groups at PND42.It suggests that GnRH mRNA level is decresed after EAP1 blocking,however,the inhibitory effect is a short-term patern,its expression can be recovered eventually.3.There is no significant difference among the three treated groups in all the development stages in both Kiss1 mRNA and protein level.4.The average age at vaginal opening of the LV-EAP1-shRNA group(35.50±1.08 d,p<0.05)was significantly delayed,while the age of vaginal opening of saline and LV-EAP1-shRNA(-)group is 30.30±0.95 d and 30.00±0.94 d respectively,and there is no significant difference between these two groups.It suggested that puberty delayed after blocking the EAP1 expression.5.Since femal rats were on their juvenile stage at PND25,the ovarian indexs(ovary weight /body weight)show no different among groups;At PND 35,the ovarian index was significant lower in LV-EAP1-shRNA group than saline and LV-EAP1-shRNA(-)group;while,the ovarian index of LV-EAP1-shRNA group shows no significant difference in PND42.It is suggest that the ovarian fucation is decresed after EAP1 blocking,while recovered in PND42.6.H&E staining shown: The ovary of femal rats has no corpus luteum producing at PND25.And the number of corpus luteum was significant lower in the LV-EAP1-shRNA group at PND35 compared to LV-EAP1-shRNA(-)group and saline group.However,it shows no significant difference among these groups at PND42.Conclusion: 1.After blocking the expression of EAP1 mRNA,the GnRH mRNA level is decressed but a short term,and it would recover eventually.2.The puberty-regulatory effect of EAP1 may not through the Kiss1/GPR54 signaling pathway.3.Knocking down the expression of EAP1 in hypothalamus leads to puberty delayed and the decline of the function of ovary in a short term,it would recovery eventurally.