eGFP (LV-eGFP). Immunohistofluorescent analysis of the sites of injection using antibodies against eGFP was used to identify the transduced cells (Fig. 6a). Kisspeptin neurons, also identified by immunohistofluorescence, were one of the cell populations transduced by the virus (Fig. 6b,c). ChIP analysis of DNA extracted from microdissected ARC tissue containing the transduced cells revealed that the LV-produced EED-HA protein had been recruited to the Kiss1 promoter (Fig. 6d). The number of detectable immunopositive kisspeptin cells per section decreased 25% in LV-EED injected animals (LV-GFP= 21.4 ± 1.98, n=31 vs LV-EED= 15.9 ± 1.22, n=6; t=3.65, p<0.001, Student t Test), and the abundance of kisspeptin immunoreactive material per cell was reduced by 30% in LV-EED-injected animals as compared to control rats injected with LV-GFP (LV-GFP= 141.4 ± 1.89, n=102 vs LV-EED= 95.2 ± 2.7, n=94; t=14.35, p<0.001, Student t Test) (Fig. 6e), indicating that EED overexpression compromises kisspeptin production in about 50% of the ARC population of kisspeptin neurons. This inhibition is consistent with our in vitro results showing a repressive effect of EED on Kiss1 promoter activity (Supplementary Fig. 5b).
