Undernutrition reduces kisspeptin and neurokinin B expression in castrated male sheep

Abstract

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.

The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs

“…Our recent report in male sheep also showed that feed restriction for 13 weeks inhibits the number of ARC cells that express mRNA and protein for NKB 49 . In the present study, using the same undernutrition paradigm that we first established in males, 49 we did not detect a significant reduction in ARC cells that express mRNA for NKB, but did observe a significant reduction in ARC cells expressing NKB protein, albeit not to the degree that we reported in the males. In addition, in the present study, we demonstrate that the mRNA abundance for NKB within KNDy neurons is unaltered with undernutrition.…”

“…Work in female rodents has shown that a 2–4‐week period of chronic undernutrition reduces mRNA for NKB in the ARC 47,48 . Our recent report in male sheep also showed that feed restriction for 13 weeks inhibits the number of ARC cells that express mRNA and protein for NKB 49 . In the present study, using the same undernutrition paradigm that we first established in males, 49 we did not detect a significant reduction in ARC cells that express mRNA for NKB, but did observe a significant reduction in ARC cells expressing NKB protein, albeit not to the degree that we reported in the males.…”

“…Previous reports in rodents 47,48 and in sheep 45 using a single probe for kisspeptin demonstrated that chronic undernutrition reduces ARC mRNA abundance for kisspeptin. More recently, through the use of RNAscope, we have shown that chronic undernutrition in castrated male sheep reduces mRNA for kisspeptin within kisspeptin/NKB expressing cells 49 . However, although only 52%–65% of kisspeptin cells expressed NKB in castrated males, 49 possibly reflecting the lower degree to which males coexpress kisspeptin and NKB in the ARC, 35,61 we were unable to draw a definitive conclusion about the impact of chronic undernutrition on KNDy neurons because we did not assess dynorphin expression.…”

“…However, there has yet to be a report detailing the impact of chronic undernutrition on these reproductively relevant neuropeptides using methods to detect both mRNA and protein within the same animal. Given their stimulatory (kisspeptin and NKB) and inhibitory (dynorphin) roles in GnRH/LH secretion, we hypothesized that chronic undernutrition, which we have shown suppresses LH secretion in young, castrated male sheep, 49 would inhibit expression of kisspeptin and NKB at the same time as stimulating dynorphin expression in the ARC and within KNDy neurons of OVX sheep. In the present study, we used OVX sheep, a longstanding animal model in reproduction, 50 to directly examine the nutritional effects on LH secretion apart from changes in sensitivity to gonadal steroids.…”

Regulation of the gonadotropin‐releasing hormone neuron during stress

“…For psychosocial stress, physical restraint suppressed LH pulses in monkeys, 7 sheep, 8 rats 9 and mice. 10,11 Various models of metabolic stress, including insulininduced hypoglycema, [12][13][14][15][16] glucoprivation, 17,18 lipoprivation 19 and feed restriction, [20][21][22] suppressed LH pulses. Immune/inflammatory stress modeled with either endotoxin (lipopolysaccharide) [23][24][25] or administration of cytokines 26,27 also suppressed LH pulses in many species.…”