Kisspeptin, GnRH, and ERα colocalise with nNOS neurones in prepubertal female sheep

Abstract

Puberty is a process which integrates multiple inputs that ultimately cause an increase in gonadotrophin-releasing hormone (GnRH) secretion. While kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesized by nitric oxide synthase (NOS). In this study, we sought to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine if oestradiol would exert effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined if nNOS neurones contained oestrogen receptor α (ERα) and could potentially communicate oestradiol (E₂) feedback to GnRH neurones. Neuronal nitric oxide synthase neurones contained ERα with the percentage of coexpression (12 to 40%) depending upon the area analyzed. We next investigated if a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (99%) colocalised with nNOS. Kisspeptin close-contacts were also associated with nNOS neurones. A greater number of close-contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, but no GnRH close-contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep.

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