Supplementary Material for: Neuroanatomical Relationship of Neuronal Nitric Oxide Synthase to Gonadotropin-Releasing Hormone and Kisspeptin Neurons in Adult Female Sheep and Primates

Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity. Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships. Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates. Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep.