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Neuroendocrine pathways of nerve growth factor during ovulation in llamas



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The overall objective of this thesis was to investigate the pathways of NGF-induced ovulation in llamas. We hypothesize that NGF influences GnRH secretion directly or indirectly at the median eminence. For this purpose, we used a battery of ultrasonic, histological, endocrine, proteomics, imaging and bioconjugation tools within in vitro and in vivo models to understand the mechanism of action of NGF. In Chapter 2 we examined the neuroanatomy of the neuroendocrine system of llamas and its relationship to NGF receptors. Using immunohistochemistry, we found that the hypothalamus of llamas contained neuronal groups expressing both NGF receptors (P75 and TrkA) and NGF itself. Although NGF receptors were colocalized in the medial septum and the diagonal band of Broca, both receptors were differentially expressed either in the periventricular area (for TrkA) or, more surprisingly, in circumventricular organs (for P75). Tanycytes in the median eminence had immunoreactivity to P75 and were in close apposition with GnRH and kisspeptin axons. In Chapter 3 we developed a radioactive NGF (conjugated to 89Zr) for positron emission tomography/computed tomography (PET/CT) imaging to determine the biodistribution and brain uptake of NGF. We validated the bioconjugation, and the stability and bioactivity of the radioactive conjugate using the PC12 cell bioassay. We investigated the biodistribution of radioactive NGF in mice and llamas by gamma counter in the former and PET/CT imaging in the latter species. The majority of NGF radioactivity was present in the kidney (mice and llama) and the liver (llama) 1 hour after administration, whereas the rest of the organs had low radioactivity. In llamas, serial brain scans revealed no substantial uptake in hypothalamic areas of llama, suggesting poor brain penetration of the 89Zr-labelled NGF. In Chapter 4, we evaluated the role of kisspeptin in inducing LH secretion in llamas. Administration of kisspeptin in llamas induced an increase in LH concentrations and ovulation (5/5), similar to NGF (5/5) but higher than saline administration (0/5). Kisspeptin-induced ovulation in llamas was ablated by pretreatment with a GnRH receptor antagonist, suggesting that kisspeptin is acting at the GnRH neuron level in the hypothalamus of llamas. Kisspeptin neurons were located in the preoptic area and arcuate nucleus, establishing synaptic contacts with GnRH neurons. Interestingly, kisspeptin neurons were devoid of NGF receptors. In Chapter 5, we tested the hypothesis that noradrenaline mediates the LH surge induced by NGF. Neuroanatomical studies revealed that tyrosine hydroxylase neurons and fibers were present in the hypothalamus establishing appositions with GnRH neurons in llamas. Intravenous administration of alpha-adrenergic receptor antagonist (phenoxybenzamine) failed to prevent or reduce the NGF-induced LH surge, whereas central administration of an alpha-1-adrenergic receptor reduced the magnitude of the LH surge induced by NGF. In Chapter 6, we evaluated the hypothalamic response to NGF in llamas and the involvement of GnRH neurons and progesterone. Administration of two doses of NGF induced an impaired LH response but not to GnRH administration, suggesting NGF receptor desensitization. No differences were found in FOS expression in GnRH neurons of llamas treated with NGF or saline, and the LH response to NGF in llamas was independent and progesterone concentrations. In Chapter 7, we evaluated NGF central actions in llamas and the role of kisspeptin receptor in GnRH neurons and TrkA receptor-expressing neurons during the LH surge. Llamas were either treated with saline, high and low doses of NGF intracerebroventricularly or intravenously. Only llamas treated intravenously with NGF displayed an LH surge and ovulation, whereas llamas treated with NGF intracerebroventricularly did not. Infusion of a kisspeptin receptor blocker and a TrkA blocker failed to impair the LH surge induced by NGF. In Chapter 8, we tested the hypothesis that an LH releasing effect of NGF is present in male camelids. Intravenous administration of NGF induced an immediate elevation of LH concentrations, larger than the response to GnRH. Testosterone concentrations were greater in males that received NGF than males that received saline, suggesting that NGF administration, via LH stimulation, supported blood testosterone concentrations in male south American camelids. In conclusion, results from the present thesis support the hypothesis that NGF triggers ovulation through a central mechanism of action in llamas. Our results provide evidence that a novel mechanism of action is followed by NGF that may be mediated by noradrenaline, perhaps at the llama median eminence. Additionally, we report that the LH-releasing effect of NGF may be independent of progesterone and kisspeptin and may be operating in male camelids as well.



Llama, NGF, Ovulation, LH, Hypothalamus



Doctor of Philosophy (Ph.D.)


Veterinary Biomedical Sciences


Veterinary Biomedical Sciences


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