Kisspeptin and GnRH Neurons in Prepubertal Female Sheep Express Neuronal Nitric Oxide Synthase

Presentation Number: SUN 475
Date of Presentation: April 2nd, 2017

Michelle N. Bedenbaugh*1, Ryan C. O'Connell2, Justin A. Lopez1, Richard B. McCosh1, Robert L. Goodman1 and Stanley M. Hileman1
1West Virginia University School of Medicine, Morgantown, WV, 2Davis and Elkins College, Elkins, WV


Although increased gonadotropin-releasing hormone (GnRH) secretion is critical for puberty onset, the neural mechanisms underlying this increase are not completely known. While kisspeptin is necessary for puberty onset, other neural inputs likely play a role as well. Nitric oxide (NO) stimulates GnRH/LH release in rats and deletion of neuronal nitric oxide synthase (nNOS) causes infertility in mice. In addition, kisspeptin neurons contact nNOS neurons in mice. This study examined the anatomical relationship between kisspeptin and nNOS neurons in prepubertal female sheep. Hypothalamic tissue from prepubertal ewes that were either ovariectomized (OVX; n=6) or ovariectomized and implanted with a single 1-cm long estradiol implant (OVX+E; n=6) was used. After specificity and lack of cross-reactivity for each antibody was confirmed, dual-label immunofluorescence for kisspeptin and nNOS was conducted and confocal microscopy was used to evaluate 10 nNOS and 10 kisspeptin cells/animal in each the arcuate nucleus (ARC) and preoptic area (POA). Due to the absence of estradiol, POA kisspeptin cells were not observed in OVX ewes. In the POA of OVX+E ewes, a high percentage of kisspeptin cells were found to contain nNOS (73.3±2.0%) and exhibited nNOS-positive contacts (2.0±0.9). In contrast, few nNOS cells contained kisspeptin (2.0±0.8%). The number of kisspeptin contacts per nNOS cell was also low (0.10±0.03). In the ARC, the percentage of nNOS cells containing kisspeptin was not different between OVX (43±5%) and OVX+E (30±8%) ewes. However, nNOS cells in OVX animals tended (p=0.067) to have more kisspeptin contacts (3.2±0.6) than in OVX+E animals (0.5±0.1). All ARC kisspeptin cells contained nNOS regardless of treatment and also exhibited nNOS contacts. However, there was no difference in the number of nNOS contacts on kisspeptin cells between OVX (4.1±0.7) and OVX+E (4.4±0.8) ewes. In addition to examining the kisspeptin-nNOS relationship, we are also examining the relationship between GnRH and nNOS using dual-label immunofluorescence. To date, we have evaluated 10 nNOS and 10 GnRH cells in the POA from 1 OVX and 1 OVX+E ewe. Preliminary analysis indicates 90% of GnRH cells contain nNOS and also exhibit nNOS contacts. In summary, these data establish a neuroanatomical relationship between nNOS and kisspeptin that particularly in the ARC, may be important for controlling GnRH secretion. Estradiol has little effect on kisspeptin colocalization with nNOS but may influence the amount of kisspeptin input to these cells. These results in combination with the finding of coexpression of GnRH and nNOS leads us to suggest that NO may influence GnRH secretion both directly and indirectly, via kisspeptin, in prepubertal female sheep.


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