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In the ewe, seasonal anestrus results from a change in the hypothalamic responsiveness to estradiol (E2) negative feedback. Various studies have demonstrated a role of hypothalamic dopaminergic cell groups in this seasonally dependent E2 effect. However, these neurons do not possess α-estrogen receptors (ER), therefore suggesting existence of another neural system mediating E2 inhibition. In the first study, we attempted to identify these E2-responsive neurons, by screening hypothalamic areas known to contain ER to determine if E2 induced Fos/FRAs expression in any ER-containing neurons. Based on the results of this analysis, we next tested whether the activation of a specific group of ER-containing neurons depends on the seasonal state of the animal. Seven days of E2 treatment significantly increased the percentage of ER-containing neurons expressing the immediate early gene product, Fos (1.7 vs. 17.8%) in the preoptic area (POA) near the OVLT, but not in other hypothalamic areas. To test the hypothesis that activation of these ER-containing neurons occurs only in anestrus, we compared the effects of E2 treatment on Fos/ER colocalization, during the breeding season and anestrus, utilizing the newly available mouse anti-human ER antibody (Dako Corporation). Ovariectomized (OVX), breeding season ewes were bled for 4 hours at 12 min intervals, and implanted with either a blank (n = 5) or E2 filled (n = 5), 0.5cm Silastic capsule. Seven days later, animals were bled again for 4 hours at 12 min intervals, their brains fixed, removed, sectioned and stained for ER and Fos using a dual immunofluorescence procedure. E2 treatment failed to significantly increase the percentage of ER-containing neurons expressing Fos (OVX: 3.47 ± 1.92% vs. OVX + E2: 1.81 ± 0.85%). The same procedure was repeated in another group of 9 animals during anestrus; E2 treatment resulted in 15.09 ± 1.95% ER/Fos colocalization, a significant increase compared to control animals that averaged only 4.52 ± 1.18% of ER-containing neurons that expressed Fos. These data suggest that a subset of ER-containing neurons in the POA near the OVLT plays a role in the seasonal change in response to E2 negative feedback.