SAT-421 Cell-Specific Ablation of GnRH Neurons Using Kisspeptin-Saporin in the Preoptic Area of Sheep, but Not Mice

Sheep have been a useful preclinical model for the exploration of the role of kisspeptin and its receptor, KISS1R, in the control of pulsatile section of gonadotropin releasing hormone (GnRH). However, lack of reagents to interrogate KISS1R neurons in sheep has limited progress in this species. To address this limitation, we tested the feasibility of using fluorescent in situ hybridization to monitor kiss1R mRNA and a kisspeptin-saporin conjugate to ablate KISS1R-containing neurons in a cell specific manner. Using custom-designed probes for RNAscope to detect ovine kiss1R and GnRH, a high degree of colocalization was observed in the preoptic area. In future work, we can use this technique to identify mRNAs for several different possible neurotransmitters in other kiss1R-containing neurons. The effects of kiss-saporin injections was tested in mice to determine optimal concentrations and survival times. Male and female mice of two strains received infusions of 200nL of 100ng/uL, 200ng/uL and 400ng/uL concentrations of kiss-sap and blank-sap into the preoptic area, which contains the greatest density of GnRH neuron cell bodies, and, at the level of the GnRH neuron dendron prior to termination in the median eminence. Mice were perfused two or three weeks after infusion. Two adult Suffolk ewes received unilateral kiss-saporin injections (1 ul of 700 ng/ul concentration) into the preoptic area, and one ewe received kiss-sap on one side and blank-sap on the other; brains were perfused three weeks later. Mouse and sheep brains were sectioned and immunostained for GnRH. Analysis of tissue from ewes revealed that GnRH neurons were severely reduced on the side ipsilateral to the kiss-sap injection compared to the contralateral side. In contrast, GnRH neurons were found to be intact in mice, independent of injection site, dosage, survival time, strain and sex of the mice. These results indicate that kiss-sap can be used in sheep to test the physiological role of different KISS1R-containing neuronal populations. They also demonstrate a striking difference between mice and sheep in the ability to lesion GnRH cells using kiss-saporin. This raises the possibility of differences in cell surface localization and/or internalization of KISS1R between these two species, as both are needed for saporin lesions. In conclusion, these results demonstrate the feasibility of detecting kiss1R expression in sheep GnRH neurons and using GnRH cell-specific lesions, while indicating potential differences in KISS1R cellular localization between sheep and mice.