The role of RFRP-3 and Kisspeptin on GnRH secretion in the merino ram

This thesis has used in vitro and in vivo studies to investigate the role of the RF-amides kisspeptin and RFRP-3 in the control of GnRH secretion in the ram. Specifically, it tested the hypothesis that RFRP-3 and kisspeptin are involved in the negative feedback actions of testicular hormones on GnRH and hence LH secretion in the merino rams. To address this hypothesis, the three main aims of the study were:
1. To determine whether RFRP-3 and kisspeptin neurons in the ram hypothalamus are targets for testicular hormones and if RFRP-3 and kisspeptin neurons interact with GnRH neurons.
2. To analyse the fibre interactions between RFRP-3 and kisspeptin with GnRH neurons in the ram hypothalamus and to determine if testicular hormones influenced this relationship.
3. To determine whether RFRP-3 regulates LH secretion in the ram.
Initially the focus for the experiments in this thesis was on RFRP-3. The first study of this thesis used in situ hybridisation to determine the effect of castration on RFRP-3 mRNA expression in the ram hypothalamus. Wether tissue showed significantly fewer numbers of cells containing RFRP-3 mRNA than intact rams, and only in the most caudal region of the DMH. All other regions were not different between the two groups. This suggests that testicular hormones may regulate RFRP-3 gene expression.
To determine whether RFRP-3 neurons are direct target for testicular steroid hormones the second study used dual label fluorescence immunohistochemistry to identify if RFRP-3 immunoreactive (-ir) neurons contained androgen receptors (AR) or oestrogen receptor α (ERα). In addition, both the effect of testicular steroid hormones on this expression as well as the number of RFRP-3-ir cells was examined by comparing the numbers of RFRP-3 cells between rams, short and long term castrated rams. Tissue from ewes in the luteal phase of the oestrous cycle was included for a positive control only. No RFRP-3 cells expressed either AR or ERα in any treatment group, suggesting that testicular steroid hormones do not act directly on RFRP-3 neurons. The number of RFRP-3 cells however, was not significantly different between the male treatment groups. This result casts doubt as to whether RFRP-3 are part of the pathway by which testicular steroid hormones exert their feedback action on GnRH secretion.
In light of this, the same tissue was used to determine whether kisspeptin-ir neurons expressed these sex steroid receptors and whether cell numbers changed with castration. While very few kisspeptin-ir cells were detected in the preoptic area (POA), there was a marked effect on the number of kisspeptin-ir in the arcuate nucleus (ARC). Numbers were near zero in intact rams, but were significantly higher in castrated animals, with the number being in proportion to time after castration. Virtually all of the kisspeptin cells in the ARC expressed both AR and ERα in the ARC of the wether and short term castrated ram. This indicates that testicular hormones act on kisspeptin neurons in the ram hypothalamus, to inhibit kisspeptin expression. This action is likely to be direct, possibly through both AR and ERα.
The third study used immunohistochemistry and the same hypothalamic tissue as used for the previous study to investigate whether GnRH neurons received close appositions from RFRP-3 and kisspeptin fibres, as well as whether reciprocal pathways existed. The use of tissue from both intact and castrated animals allowed a determination of the effect of sex hormones on the level of these fibre appositions. An unexpected finding was a substantial (approximately 80%) co-expression of GnRH and RFRP-3 in the POA. This did not vary across the groups of male sheep. Extensive tests were conducted to determine if there was a methodological explanation but no explanation was found and it was concluded that the double labelling was mostly likely real, although the significance of these cells is unclear.
Another unexpected finding was that despite kisspeptin fibres being observed throughout the POA and anterior hypothalamic area (AHA), no kisspeptin fibres were detected in appositions to GnRH neurons, in any group. In contrast, a small number of kisspeptin-ir cells in the most caudal part of the ARC did receive some contact from GnRH-ir fibres. On the other hand, approximately 50% of GnRH neurons were apposed by RFRP-3 –ir fibres, which did not vary between treatment groups. This indicates that RFRP-3 neurons are likely to regulate GnRH neurons in some way and may regulate LH secretion. In addition, small numbers of RFRP-3 neurons received GnRH fibre appositions, suggestive of a regulatory circuit, although the numbers of cells receiving this input was low, casting doubt on their physiological relevance.

Interactions involving cells and fibres that were double labelled for both GnRH and RFRP-3 were also observed. Only 1-2 GnRH cells per section were observed that were apposed by double labelled fibres, while approximately 20-30% of double labelled cells were apposed by GnRH-ir fibres and 40-60% of double labelled cells were apposed by RFRP-3 –ir fibres. The significance of this is unclear.
The last study of this thesis used in vivo experiments to investigate the extent to which RFRP-3 can regulate LH secretion in the ram. Since studies in other species had revealed both inhibitory and stimulatory actions on LH secretion, two experiments were conducted. In the first experiment intact rams, which had low endogenous circulating LH levels, were given an intravenous infusion with RFRP-3 to determine if it would stimulate LH secretion. The rams were then castrated for the second experiment to produce high circulating LH levels to allow the determination of whether intravenous infusion of RFRP-3 would inhibit LH secretion. Control treatment consisted of a saline infusion. In both experiments neither saline nor RFRP-3 had any effect on mean plasma LH levels or any parameter of pulsatile LH secretion. This suggests that RFRP-3 may not have a significant role in the regulation of LH secretion in the ram.
Overall these studies seem to have raised as many questions as it has asked. A proportion of RFRP-3 neurons in the caudal ARC appear to be regulated by testicular hormones while the remaining RFRP-3 seems to not be. Nonetheless a substantial proportion of GnRH neurons may receive input from RFRP-3 neurons, which provides the opportunity for direct regulation of GnRH secretion, possibly relaying the feedback actions of testicular hormones. On the other hand, whole animal studies suggest that RFRP-3 may not be a significant regulator of LH secretion. The role of RFRP-3 in the regulation of GnRH secretion in the ram, therefore, is far from clear. The studies on kisspeptin neurons, on the other hand, provide clear evidence that in the ram they receive direct regulation by testicular hormones. Unlike data from other species, however, there was no evidence that they interact with, and hence regulate GnRH neurons via the cell bodies. Whether they regulate GnRH neurons at other sites and influence LH secretion in the ram remains unknown.