Kisspeptin neurones do not directly signal to RFRP-3 neurones but RFRP-3 may directly modulate a subset of hypothalamic kisspeptin cells in mice - PubMed (original) (raw)

Kisspeptin neurones do not directly signal to RFRP-3 neurones but RFRP-3 may directly modulate a subset of hypothalamic kisspeptin cells in mice

M C Poling et al. J Neuroendocrinol. 2013 Oct.

Abstract

The neuropeptides kisspeptin (encoded by Kiss1) and RFamide-related peptide-3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP-3 might act directly on each other's neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP-3 systems, we performed double-label in situ hybridisation (ISH) for the RFRP-3 receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurones of adult male and female mice, as well as double-label ISH for the kisspeptin receptor, Kiss1r, in Rfrp-expressing neurones of the hypothalamic dorsal-medial nucleus (DMN). Only a very small proportion (5-10%) of Kiss1 neurones of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co-expression (approximately 25%) existed in Kiss1 neurones in the arcuate nucleus. Thus, RFRP-3 could signal to a small, primarily arcuate, subset of Kiss1 neurones, a conclusion supported by the finding of approximately 35% of arcuate kisspeptin cells receiving RFRP-3-immunoreactive fibre contacts. By contrast to the former situation, no Rfrp neurones co-expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co-expressed with arcuate kisspeptin neurones) was found in <10% of Rfrp neurones. Moreover, kisspeptin-immunoreactive fibres did not readily appose RFRP-3 cells in either sex, further excluding the likelihood that kisspeptin neurones directly communicate to RFRP-3 neurones. Lastly, despite abundant NKB in the DMN region where RFRP-3 soma reside, NKB was not co-expressed in the majority of Rfrp neurones. Our results suggest that RFRP-3 may modulate a small proportion of kisspeptin-producing neurones in mice, particularly in the arcuate nucleus, whereas kisspeptin neurones are unlikely to have any direct reciprocal actions on RFRP-3 neurones.

Keywords: GnIH; Gpr147; Gpr54; Gpr74; Kiss1; Kiss1r; RFRP-3; Tac2; Tacr3; hypothalamus; kisspeptin; neurokinin B; reproduction.

© 2013 British Society for Neuroendocrinology.

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Figures

Figure 1

Figure 1

Expression of Gpr147 and Gpr74 in AVPV/PeN Kiss1 neurons by double label in-situ hybridization. [A] Representative photomicrographs of double label in-situ hybridization of Kiss1 (red fluorescence) and Gpr147 (silver grains) in an intact male. 3V, third ventricle [B] Kiss1 neurons co-expressing Gpr147 (green arrowhead) and Kiss1 neurons with no co-expression of Gpr147 (blue arrows). [C] Quantification of the percent co-expression of Gpr147 in AVPV/PeN Kiss1 neurons between gonadally-intact females (F) and males (M). There were no significant differences in co-expression between any of the groups. [D] Representative photomicrographs of double label in-situ hybridization of Kiss1 (red fluorescence) and Gpr74 (silver grains) in a dioestrous female. [E] Kiss1 neurons co-expressing Gpr74 (green arrowhead) and Kiss1 neurons with no co-expression of Gpr147 (blue arrows). [F] Quantification of the percent co-expression of Gpr147 in Kiss1 neurons between gonadally-intact females (F) and males (M). There were no statistical differences between the sex or gonadal state.

Figure 2

Figure 2

Expression of Gpr147 and Gpr74 in ARC KNDy neurons by double label in-situ hybridization. [A] Representative photomicrographs of double label in-situ hybridization of Tac2 (red fluorescence) and Gpr147 (silver grains) in the ARC of a dioestrous female. [B] ARC Tac2 neurons co-expressing Gpr147 (green arrowhead) and Tac2 neurons with no co-expression of Gpr147 (blue arrows). [C] Quantification of the percent co-expression of Gpr147 in Tac2 neurons between gonadally-intact females (F) and males (M) and gonadectomized (GDX) M and F. There were no significant differences in co-expression between any of the groups. [D] Representative photomicrographs of double label in-situ hybridization of Tac2 (red fluorescence) and Gpr74 (silver grains) in the ARC of a dioestrous female. [E] Tac2 neurons co-expressing Gpr74 (green arrowhead) and a Gpr74 neuron that is not expressing Tac2 (yellow arrow). [F] Quantification of the percent co-expression of Gpr147 in Tac2 neurons between gonadally-intact females (F) and males (M) and gonadectomized (GDX) M and F. These experimental groups were not statistically difference.

Figure 3

Figure 3

Representative photomicrograph of RFRP-3 immunoreactive fibre (green fluorescence) in apposition with ARC kisspeptin neuron (red fluorescence) in a female mouse. Immunohistochemical analysis revealed RFRP-3 contacts with ~35% of kisspeptin cell bodies in the ARC of GDX female mice. Figure is a collapsed stack of several confocal optical sections. Green triangle denotes example RFRP-3 fibre immunoreactivity. Red triangle denotes an ARC kisspeptin cell not receiving RFRP-3 input. Yellow triangle denotes RFRP-3 fibre contacting an ARC kisspeptin cell.

Figure 4

Figure 4

Expression of Kiss1r in Rfrp and GnRH neurons and expression of Tacr3 in Rfrp neurons by double label in-situ hybridization. [A] Representative photomicrographs of double label in-situ hybridization of Rfrp (red fluorescence) and Kiss1r (silver grains) in the DMN of a dioestrous female. Rfrp neurons lacking Kiss1r are marked with blue arrows [B] Quantification of the percent co-expression of Kiss1r in Rfrp neurons between gonadally-intact females and males. There were no significant differences in co-expression between sexes. [C] Representative photomicrographs of double label in-situ hybridization of Kiss1r (silver grains) and GnRH (red fluorescence) in the preoptic area (POA) of an intact male. Green arrowheads identify double-labeled cells [D] Quantification of the percent co-expression of Kiss1r in Rfrp or GnRH neurons in dioestrous females and intact males (percentages averaged across all animals). [E] Representative photomicrographs of double label in-situ hybridization of Rfrp (red fluorescence) and Tacr3 (silver grains) in the DMN of an intact male mouse. [F] Rfrp neurons co-expressing Tacr3 (green arrowhead) and a cell expressing Tacr3 without Rfrp (yellow arrow). [G] Quantification of the percent co-expression of Tacr3 in Rfrp neurons between gonadally-intact females (F) and males (M) and gonadectomized (GDX) M and F. There were no significant differences in co-expression between any of the groups.

Figure 5

Figure 5

Immunohistochemistry for kisspeptin fibres and RFRP-3 cell bodies in the mouse DMN. [A] Representative images of RFRP-3 cell bodies and fibres (green fluorescence) and kisspeptin fibres (red fluorescence) in the DMN of a gonadally-intact male (left) and GDX female (right). [B] Quantification of the percent of RFRP-3 neurons receiving contacts from kisspeptin fibres in the DMN of intact and gonadectomized (GDX) male and female mice. Almost no RFRP-3 cells were contacted and there was no statistical difference between gonadal states.

Figure 6

Figure 6

Expression Tac2 in Rfrp neurons in the DMN by double label in-situ hybridization. [A] Representative photomicrographs of double label in-situ hybridization of Rfrp (red fluorescence) and Tac2 (silver grains) in the DMN of a dioestrous female. [B] Higher magnification of Rfrp neurons co-expressing Tac2 (green arrowhead) and a Tac2 neuron that is not expressing Rfrp (yellow arrow). [C] Quantification of the percent co-expression of Tac2 in Rfrp neurons between gonadally-intact females (F) and males (M) and gonadectomized (GDX) M and F. These experimental groups were not statistically different.

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