To determine if overexpression of EED alters pulsatile GnRH release from the hypothalamus we delivered LV-EED or LV-GFP to the ARC of a group of 22-day-old female rats, dissected the ARC-ME region seven days later, and incubated the tissues for 3h in Krebs-Ringer bicarbonate buffer, sampling the medium every 7.5 min, to measure GnRH output 44. The ARC-ME of animals injected with LV-GFP showed a robust pattern of pulsatile GnRH release with episodes of secretion occurring every 41 ± 2.7 min (Fig. 7a,b). In contrast, GnRH pulse frequency was reduced (p<0.05; Mann-Whitney Rank Sum test) to one pulse every 98 ± 30.7min in LV-EED injected rats (Fig. 7a,b). GnRH pulse amplitude was not affected (Fig. 7b), but total GnRH output was reduced in the LV-EED treated group (Fig. 7b). Measurement of Eed and Kiss1 mRNAs at the end of the incubation demonstrated that the ARC-ME of animals receiving LV-EED had 4 times more Eed mRNA than the ARC-ME of animals injected with LV-GFP (Fig. 7c), and that – consistent with the immunohistochemistry data – Kiss1 mRNA was reduced by 50% (Fig. 7c).
