Undernutrition impairs reproductive success through suppression of gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. Given that kisspeptin and neurokinin B (NKB) neurons in the arcuate nucleus (ARC) of the hypothalamus are thought to play key stimulatory roles in the generation of GnRH/LH pulses, we hypothesized that feed restriction would reduce the ARC mRNA abundance and protein expression of kisspeptin and NKB in young, male sheep. Fourteen wethers (castrated male sheep five months of age) were either fed to maintain (FM; n = 6) pre-study body weight or feed-restricted (FR; n = 8) to lose 20% of pre-study body weight over 13 weeks. Throughout the study, weekly blood samples were collected and assessed for LH concentration using radioimmunoassay. At Week 13 of the experiment, animals were euthanized, heads were perfused with 4% paraformaldehyde, and brain tissue containing the hypothalamus was collected, sectioned, and processed for detection of mRNA (RNAscope) and protein (immunohistochemistry) for kisspeptin and NKB. Mean LH was significantly lower and LH inter-pulse interval was significantly higher in FR wethers compared to FM wethers at the end of the experiment (Week 13). RNAscope analysis revealed significantly fewer cells expressing mRNA for kisspeptin and NKB in FR wethers compared to FM controls, and immunohistochemical analysis revealed significantly fewer immuno-positive kisspeptin and NKB cells in FR wethers compared to FM wethers. Taken together, this data supports the idea that long-term feed restriction regulates GnRH/LH secretion through central suppression of kisspeptin and NKB in male sheep.
Agouti-related peptide (AgRP) neurons, which relay information from peripheral metabolic signals, may constitute a key central regulator of reproduction. Given that AgRP inhibits luteinizing hormone (LH) secretion and that nutritional suppression of LH elicits an increase in AgRP while suppressing kisspeptin expression in the arcuate nucleus (ARC) of the hypothalamus, we sought to examine the degree to which AgRP could directly regulate ARC kisspeptin neurons. Hypothalamic tissue was collected from four castrated male sheep (10 months of age) and processed for the detection of protein (AgRP input to kisspeptin neurons) using immunohistochemistry and mRNA for melanocortin 3 and 4 receptors (MC3R; MC4R) in kisspeptin neurons using RNAscope. Immunohistochemical analysis revealed that the majority of ARC kisspeptin neurons are contacted by presumptive AgRP terminals. RNAscope analysis revealed that nearly two thirds of the ARC kisspeptin neurons express mRNA for MC3R, while a small percentage (<10%) colocalize MC4R. Taken together, this data provides neuroanatomical evidence for a direct link between orexigenic AgRP neurons and reproductively critical kisspeptin neurons in the sheep, and builds upon our current understanding of the central link between energy balance and reproduction.
Undernutrition limits reproduction through inhibition of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain prestudy body weight (controls) or feed-restricted to lose 20% of pre-study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls.We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin.We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.
Puberty onset is a complex physiological process which enables the capacity for reproduction through increased gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus (ARC) are believed to govern the timing of puberty, the degree to which KNDy neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the ARC. Fluorescent in situ hybridization revealed that the majority (> 74%) of ARC neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer ARC cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of ARC cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and supports the idea that NKB and dynorphin play a role in puberty onset in the female pig.
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