STUDY OF THE ROLE OF THE ENDOGENOUS KISSPEPTIN TONE IN THE REGULATION OF HYPOTHALAMIC PITUITARY GONADAL AXIS

Abstract

Reproductive activity in mammals is progressed by the neuroendocrine coordination between the hypothalamus pituitary and gonads constituting hypothalamic-pituitary-gonadal axis (HPG-axis). The discovery of kisspeptin and its receptor and fundamental role of kisspeptin in regulating reproduction diverted the scientific attention toward investigating its role in reproduction. Present series of studies was designed to investigate the role of endogenous kisspeptin in the regulation of HPG-axis by applying a variety of paradigms using adult male rhesus monkey as a model: 1. for checking the role of endogenous kisspeptin tone in the regulation of HPG- axis, we peripherally antagonized kisspeptin by using peptide 234 (kisspeptin receptor antagonist) 2. to assess if seasonal modulation of the HPG-axis is mediated by the kisspeptin signaling. We used assay, immunocytochemical and RT-qPCR approaches to report the effect of seasons on gene and protein correlation of kisspeptin signaling in the mediobasal hypothalamus (MBH) of adult male rhesus monkeys and finally 3. to determine whether kisspeptin can bring any change in the pluripotent stem cells, we determined the effect of kisspeptin-10 on rhesus monkey derived Lyon -ES cells differentiation and the proliferation. In the first experiment five adult male monkeys were administered with either peptide-234 (P234) (38.8 μg/kg body weight) or vehicle (0.9% saline). Plasma testosterone and adiponectin levels, as a positive control, were measured using specific ELISA in blood samples collected sequentially at 15 min intervals during a 60 min pre and a 360 min post P234/vehicle injection period. In three monkeys, the experiment was repeated where the animals received a human kisspeptin-10 (50 μg) challenge 30 min after the P234 or vehicle treatment. Our results indicated that systemic administration of P234 did not alter the plasma testosterone levels, but slightly decreased (p<0.01) the plasma adiponectin concentration. Further, kisspeptin dependent testosterone and adiponectin release was significantly (p<0.05) decreased by P234 treatment. For the second experiment, we hypothesized that modulation of expression of Kiss1, Kiss1r and GnRH underlies seasonal changes in reproduction. We examined 1 General Abstract the expression of Kiss1, Kiss1r, GnRH, pDYN, KOR, NKB and NK3R mRNA levels using RT-qPCR and of Kiss1, Kiss1r and GnRH immunoreactivity by using immunocytochemisty in the MBH of adult male rhesus monkeys during breeding season (BS; January; n=3) and non-breeding season (NBS; July; n=3). The animals were maintained under free ranging conditions in the primate breeding colonies of Kunming Institute of Zoology (102.71º longitude, 25.03º latitude). Additionally, we measured CSF (four samples collected at 30 min interval during a period from 9:00-10:30, from the lumbar vertebrae; n=3) kisspeptin and peripheral testosterone levels (blood samples were collected at 20 minutes interval during a period from 1000-1600 hrs; n=4) in BS (November) and NBS (August) monkeys by using specific RIA’s. We observed an increase (p<0.01; p<0.0005; p<0.0001, respectively) in the relative mRNA expression of Kiss1, Kiss1r and GnRH in the MBH of the adult male rhesus monkeys during the BS. The number of Kiss1 positive cells in the arcuate area (ARC) was also increased (p<0.0001) in BS. The Kiss1r positive and Kiss1r positive GnRH cell bodies were also increased (p<0.01) during the BS along with an increase in the number of contacts between Kiss1 and GnRH cell bodies (p<0.01). Kisspeptin levels in the CSF and the peripheral testosterone levels were also increased (p<0.0001) during the BS. In the third experiment, rhesus monkey derived GFP Lyon-ES cells were treated with the human kisspeptin-10. We tested how different doses of kisspeptin-10 treatment would influence specific neuronal differentiation among Lyon ES cells. At the early rosette stage, we incubated rhesus macaque derived tau GFP-Lyon ES cells with 0.1, 1, 10 and 100 nM of kisspeptin-10 or ddH2O as a control, for three days. Different parameters were then assessed at 24, 48 and 72 hr time points. We used tunnel assay for checking apoptotic activity, flow cytometry and cell count to check the effect on proliferation and later immunocytochemistry was done for glial fibrillary acidic protein (GFAP), nesting, β-tubulin III and GnRH expression. The result revealed that kisspeptin treatment affected the cell proliferation in a dose (p<0.001) and time (p<0.0001) dependent manner. The interaction between dose and time was also significant (p<0.01). Lyon-ES cells were also differentiated in to GnRH neuronal type cells in response to kisspeptin exposure. This observation suggests that kisspeptin is an important neurological and stimulatory factor for proliferation and 2 General Abstract differentiation of the Lyon ES cells and specially in inducing GnRH expression. This observation indicates the role of kisspeptin in inducing the GnRH expression in the Lyon ES cells. In summary, we observed that kisspeptin dependent testosterone release was decreased by systemic infusion of P234 which suggests that kisspeptinergic signaling plays key role in regulating the HPG-axis. During breeding season the kisspeptinergic signaling was increased, resulting in the up regulation of the HPG axis. A neurotropic role of kisspeptin was evident in Lyon ES experiment in which kisspeptin treatment induced the GnRH like neuronal expression and GnRH gene expression. This indicates a potential therapeutic use of kisspeptin in future to treat various reproductive disorders i.e., infertility, IHH. Overall from this thesis, we can conclude that kisspeptinergic signaling is playing a key role in regulating the HPG-axis and is being modulated by the season. Additionally, kisspeptin has potential to cause GnRH expression in the pluripotent cells, which is a novel finding and can be used further in the future to cure reproductive disorders i.e., infertility, IHH.