PARENT SESSION

(T659) GnRH NEURONS CONTACTING CEREBROSPINAL FLUID IN ESTROUS EWES.

Billings, Heather¹, Lehman, Michael¹, Goodman, Robert², Amstalden, Marcel¹, McManus, Christina², Hileman, Stanley², ¹ University of Cincinnati, Cincinnati, OH² West Virginia University, Morgantown, WV

ABSTRACT- GnRH is released into cerebrospinal fluid (CSF) of the ewe in a secretory pattern similar to that in pituitary portal blood, and CSF GnRH may be important for facilitating behavioral estrus at the time of the GnRH surge. In mid-luteal ewes, approx. 40% of GnRH neurons are labeled with the retrograde tracer beta subunit of cholera toxin (CTb) following injection of CTb into the third ventricle (3V). The aim of the present study was to determine which GnRH neurons contact CSF in estrous ewes. Ewes (n=4) were estrous-synchronized, placed with rams, and every 4 h, estrous behavior was monitored and jugular blood collected for determining LH concentrations. When ewes were detected in standing estrus, CTb was injected into the 3V (3 x 5 l of 0.5% solution, 15 min apart). The ewes were euthanized and perfused via the carotid arteries with 4% paraformaldehyde 4 h later. Frozen coronal sections through the preoptic area (POA) and hypothalamus were processed for dual-immunofluorescent detection of GnRH and CTb. In two ewes, CTb diffused beyond the ependymal cell layer and into surrounding neuropil, preventing further determination of GnRH neurons contacting only CSF. In the two remaining ewes with CTb diffusion restricted to the ependymal cell layer, the percentage of GnRH cells labeled with CTb was 37.5% and 58.3% in diagonal band, 40.0% and 93.3% in medial septum, 38.5% and 83.5% in POA, 44.4% and 87.1% in anterior hypothalamus, and 70% and 85.7% in mediobasal hypothalamus. LH increased modestly in the final blood sample of the ewe with the higher percentage of GnRH neurons with CTb uptake, but none of the ewes exhibited LH surges prior to euthanasia. While the study needs to be repeated with additional ewes, these preliminary findings suggest the majority of GnRH neurons contact CSF in estrous ewes and are not likely a distinct population from those secreting GnRH into pituitary portal blood. Thus, in periovulatory ewes, it is possible for GnRH to be released directly into CSF, where it may contribute to facilitation of estrous behavior. (Funded by NSF #0345752 to HJB)

KEY WORDS: GnRH, cerebrospinal fluid, estrous behavior, estrus