The negative impact of undernutrition on reproduction is evident in animal agriculture and human health, but our understanding of the central mechanism that links energy balance and reproduction is far from complete. Given that undernutrition impairs reproduction through the suppression of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion, this review will summarize three main aspects of KNDy neurons as they relate to undernutrition. First, the current understanding of KNDy neurons as the GnRH pulse generator will be considered. Second, various models of undernutrition used to examine the neuropeptides within KNDy neurons (kisspeptin, neurokinin B, and dynorphin) will be discussed. Finally, evidence supporting a role for melanocortin signaling and central inflammation in controlling reproduction during undernutrition will be considered.

The increase in luteinizing hormone (LH) that elicits puberty in many species results from a decrease in sensitivity to estradiol (E2) negative feedback. The neural mechanisms underlying this change are unknown, but do not occur at the gonadotropin-releasing hormone neurons as they lack estrogen receptor alpha (ERalpha). A potentially important area is the arcuate nucleus of the hypothalamus, where neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons) reside. KNDy neurons express ERalpha and while our previous work indicated that KNDy cells were critical for puberty, immunopositive cell numbers went unchanged during development, raising the likelihood of additional neuronal inputs. Herein, we used ovariectomized ewes implanted with E2 at prepubertal, peripubertal, or postpubertal ages to examine whether activation of KNDy neurons changed in association with increased LH secretion. Furthermore, a potential role for the melanocortin system (proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons) was assessed. Activation of KNDy neurons increased with increased LH secretion. Proopiomelanocortin cell numbers were unchanged, but activation of POMC cells and number of POMC-positive contacts onto KNDy neurons increased with age. In contrast, AgRP cell numbers and activity decreased. In addition, the percentage of POMC and AgRP neurons expressing ERalpha did not change. In summary, puberty-related increases in LH secretion are associated with activation of KNDy neurons and POMC neurons, but reduced activation and decreased numbers of AgRP neurons. To this point, no predictable changes in ERalpha expression within KNDy, POMC, or AgRP cell populations have been noted.

The increase in luteinizing hormone (LH) that elicits puberty in many species results from a decrease in sensitivity to estradiol (E2) negative feedback. The neural mechanisms underlying this change are unknown, but do not occur at the gonadotropin-releasing hormone (GnRH) neurons as they lack estrogen receptor-alpha (ERα). A potentially important area is the arcuate nucleus of the hypothalamus (ARC), where neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) reside. KNDy neurons express ERα and while our previous work indicated that KNDy cells were critical for puberty, immunopositive cell numbers went unchanged during development, raising the likelihood of additional neuronal inputs. Herein, we used ovariectomized ewes implanted with E2 at prepubertal, peripubertal, or postpubertal ages to examine whether activation of KNDy neurons changed in association with increased LH secretion. Further, a potential role for the melanocortin system (proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons) was assessed. Activation of KNDy neurons increased with increased LH secretion. POMC cell numbers were unchanged, but activation of POMC cells and number of POMC-positive contacts onto KNDy neurons increased with age. In contrast, AgRP cell numbers and activity decreased. In addition, the percentage of POMC and AgRP neurons expressing Erα did not change. Thus, puberty-related increases in LH secretion are associated with activation of KNDy neurons and POMC neurons, but reduced activation and decreased numbers of AgRP neurons. To this point, no predictable changes in ERα expression within KNDy, POMC, or AgRP cell populations have been noted.

KNDy neurons, located in the hypothalamic arcuate nucleus, coexpress kisspeptin (Kiss), neurokinin B, and dynorphin and play a crucial role in regulating GnRH/LH secretion in midgestation sheep fetuses. We hypothesize that KNDy-GnRH signaling is established during midgestation, with negative feedback acting through KNDy neurons regulating testosterone levels needed for brain masculinization in male fetuses. We used immunofluorescence histochemistry to assess the effect of chemical castration with the GnRH antagonist degarelix on arcuate KNDy neurons in fetal sheep. Fluorescent in situ hybridization demonstrated the presence of steroid receptors in untreated midgestation fetal kisspeptin neurons. Additionally, unanesthetized cannulated midgestation fetal sheep were used to examine the effects of KNDy peptides on LH secretion and characterize receptor specificity. Treatment of male lamb fetuses with degarelix on day 62 of gestation resulted in significantly decreased plasma LH and testosterone concentrations (P < .05), accompanied by a significant increase in arcuate Kiss neurons (P < .05). In unanesthetized cannulated fetuses, bolus administration of KP-10 (a Kiss receptor agonist) and senktide (NK3 receptor agonist) elicited robust LH release within 15 minutes. Pretreatment with the NK3 receptor antagonist SB222200 blocked the LH response to senktide, whereas P271 (Kiss receptor antagonist) did not affect basal LH or block the LH response to KP-10. Blocking κ-opiate receptor with PF4455242 significantly increased LH release. These results support the hypothesis that KNDy neurons regulate GnRH and gonadotropin secretion in midgestation sheep fetuses, acting as targets for negative feedback to maintain a stable androgen environment crucial for brain masculinization.

Lipopolysaccharide (LPS) from Gram-negative bacteria induces an immune response and impairs reproduction through suppression of gonadotropin releasing hormone (GnRH), subsequently luteinizing hormone (LH) secretion. While there is evidence that acute inflammation inhibits kisspeptin, little is known about the impact of chronic inflammation on this key reproductive neuropeptide in livestock species. Thus, we sought to examine a central mechanism whereby LPS suppresses LH secretion in sheep. Twenty wethers were randomly assigned to one of five treatment groups: control (CON; n=4), single acute IV LPS dose (SAD; n=4), daily acute IV LPS dose (DAD; n=4), daily increasing IV LPS dose (DID; n=4), and chronic subcutaneous LPS dose (CSD; n=4). On Days 1 and 7, blood samples were collected every 12 minutes for 360 minutes using jugular venipuncture. Following blood collection on Day 7, all animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and hypothalamic blocks were removed and processed for immunohistochemistry. On Day 1, LH pulse frequency was significantly lower (p=0.02) in SAD (0.25 ± 0.1 pulses/hour), DAD (0.25 ± 0.1 pulses/hour), DID (0.35 ± 0.1 pulses/hour), and CSD (0.40 ± 0.1 pulses/hour) compared to CON (0.70 ±0.1 pulses/hour). On Day 7, only DID animals (0.35 ± 0.1 pulses/hour) had significantly lower (p=0.049) LH pulse frequency compared to controls (0.85 ± 0.1 pulse/hour). Furthermore, only DID animals (33.3 ± 10.9 cells/section/animal) had significantly fewer (p=0.001) kisspeptin-immunopositive cells compared to controls (82.6 ± 13.6 cells/section/animal). Taken together, we suggest that daily increasing doses of LPS is a powerful inhibitor of kisspeptin neurons in young male sheep and a physiologically relevant model to examine the impact of chronic inflammation on the reproductive axis in livestock.

Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 days of age, n = 42; gilts to 160 days of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues were collected for gross morphology and histology, and a fat biopsy was collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/- animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1-/- boars and gilts compared with KISS1+/+ and KISS1+/- animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1-/- boars than in KISS1+/+ and KISS1+/- boars (P < 0.05). Hypogonadism was present in all KISS1-/- gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E-2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 +/- 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Adequate energy intake is essential for reproduction. While we have known for decades that feed restriction inhibits gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion, the central mechanisms that govern this reduction during undernutrition remain to be fully elucidated. The past few years, we have focused on examining central mechanisms whereby chronic feed restriction in young sheep results in the inhibition of LH secretion. This presentation will focus on our current understanding of how feed restriction impacts kisspeptin/neurokinin B/dynorphin (KNDy) neurons in young sheep and will give a glimpse into our next few years as we examine how input to KNDy neurons could play a key role in regulating reproduction during undernutrition.

Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

This review considers three aspects of recent work on the role of KNDy neurons in GnRH pulse generation in ruminants. First, work on basic mechanisms of pulse generation includes several tests of this hypothesis, all of which support it, and evidence that Kiss1r-containing neurons form a positive feedback circuit with the KNDy neural network that strengthen the activity of this network. The second section on pathways mediating external inputs focuses on the influence of nutrition and photoperiod, and describes the evidence supporting roles for proopiomelanocortin (POMC) and agouti-related peptide (AgRP) afferents to KNDy cells in each of these. Finally, we review studies exploring the potential applications of manipulating signaling by kisspeptin, and the other KNDy peptides, to control reproductive function in domestic animals and conclude that, although these approaches show some promise, they do not have major advantages over current practices at this time.

Abstract Disclosure: E.G. Aerts: None. M.J. Griesgraber: None. A. Thomson: None. M. Brown: None. A. Seman: None. S.L. Hardy: None. R.L. Goodman: None. C. Nestor: None. S.M. Hileman: None. The neural mechanisms that regulate the timing of puberty onset are not completely known. In several species, including sheep, puberty onset relies on an increase in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion as a result of decreased sensitivity to estradiol (E2) inhibition. Additionally, it is unclear how E2 acts in the hypothalamus to inhibit GnRH/LH secretion, as GnRH neurons do not contain estrogen receptor alpha (ERα). Neurons within the arcuate nucleus (ARC) of the hypothalamus that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) are critical for the generation of GnRH pulses and contain ERα. Data from our laboratory suggests that KNDy neurons, while critical, may be responding to other inputs that time puberty onset. Proopiomelanocortin (POMC) and Agouti-related peptide (AgRP) neurons in the ARC have been shown to contain ERα and provide input to KNDy neurons, which contain the melanocortin 3 and 4 receptor (MC3R, MC4R). The goal of this work was to assess POMC- and AgRP-positive cell numbers, input onto KNDy neurons, and neuronal activity changes in a puberty-specific manner. Female sheep were ovariectomized and given subcutaneous E2 implants at 5 months (prepubertal, n=4), 8 months (peripubertal, n=5), and 10 months (postpubertal, n=5) of age. Two weeks after surgery, blood samples were collected via jugular venipuncture every 12 minutes for 4 hours and assessed for LH. The sheep were then sacrificed and hypothalamic tissue was perfused, collected, and prepared for staining. The tissue was assessed via immunohistochemistry for POMC, AgRP, kisspeptin, NKB, and c-Fos, a known marker of neuronal activity. Pulsatile LH secretion was, as expected, reduced at 5 months and highest at 10 months. ARC POMC cell numbers did not significantly change over development (p>0.10). The number of ARC POMC close contacts onto ARC kisspeptin neurons increased significantly with development (p<0.05), as well as the percentage of ARC POMC neurons expressing c-Fos (p<0.05). ARC AgRP cell numbers and the percentage of AgRP cells colocalized with c-Fos decreased significantly over time (p<0.05). The number of AgRP close contacts onto ARC NKB cells is currently being analyzed. The percentage of NKB cells colocalizing c-Fos throughout pubertal development also increased significantly (p<0.05). This data supports the hypothesis that the ARC melanocortin system impacts KNDy neurons to regulate GnRH/LH secretion throughout pubertal development. Presentation: Saturday, June 17, 2023

Abstract Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

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 pre‐study 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.

Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in pre- and postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 µm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P < 0.05. We determined mRNA expression for kisspeptin was not different between groups (P > 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P < 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P < 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P < 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin.

Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (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 arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus 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 support the idea that NKB and dynorphin play a role in puberty onset in the female pig. The degree to which KNDy neurons exist in the gilt is characterized for the first time, and the impact of pubertal status on kisspeptin, neurokinin B, and dynorphin in the arcuate nucleus of gilts is examined.

Abstract Undernutrition negatively impacts reproductive success, at least in part, through a central suppression of GnRH secretion. Given that GnRH neurons are devoid of receptors for peripheral metabolic hormones such as leptin and insulin, nutritional regulation of GnRH secretion must be through afferent input. Neurons which co-express kisspeptin, neurokinin B (NKB), and dynorphin, termed KNDy neurons, are a unique population of cells in the arcuate nucleus (ARC) of the hypothalamus and are believed to play a critical role in GnRH/LH pulse generation. With our recent evidence that chronic feed restriction reduced kisspeptin and NKB in young, castrated male sheep, we hypothesized that nutrient restriction would inhibit expression of the KNDy neuron peptides kisspeptin and NKB, while increasing dynorphin expression in young, ovariectomized female sheep. Fifteen ewe lambs were ovariectomized and were fed to maintain body weight (n=7; Fed) or feed-restricted to lose 20% of pre-study body weight (FR; n=8). Blood samples were taken weekly every 12 minutes for 4.5 hours via jugular venipuncture and plasma was stored at -20°C until assessment of LH using radioimmunoassay. Body weights were recorded weekly and feed amounts were adjusted to achieve desired body weights. Following blood collection at Week 13, animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and tissue containing the hypothalamus was collected for assessment of KNDy neuropeptide mRNA abundance by in situ hybridization (RNAscope). At Week 13, the average percent change in body weight was clearly evident (Fed, 7.35 +/- 2.2% vs FR, -21.36 +/- 0.9%), and mean LH concentrations were lower in FR ewes (10.70 +/- 3.1 ng/ml) compared to Fed controls (20.98 +/- 3.8 ng/ml). Data analyzed to date for kisspeptin, NKB, and dynorphin in the ARC show that feed restriction reduced the number of kisspeptin mRNA-expressing cells (Fed, 165 +/- 25 vs FR, 9.25 +/- 6), the number of NKB mRNA-expressing cells (Fed, 141 +/- 28 vs FR, 24 +/- 5), and the number of dynorphin mRNA-expressing cells (Fed, 109 +/- 41 vs FR, 29.5 +/- 24). Together, these findings demonstrate that chronic feed restriction suppresses KNDy neurons and supports a role for these key reproductive neurons in the central mechanism governing GnRH/LH secretion during undernutrition in female sheep.

Abstract Puberty onset depends upon an increase in pulsatile GnRH/LH secretion, which in sheep is the result of reduced sensitivity to estrogen negative feedback. Neurons within the arcuate nucleus of the hypothalamus (ARC) expressing kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy neurons) express estrogen receptors and are believed to play a key role in mediating the effects of estrogen on GnRH/LH secretion. Therefore, the purpose of this study was to assess changes in kisspeptin, NKB, and dynorphin within the ARC across pubertal development in female sheep. Blood samples were collected at 12-minute intervals for 4 hours and assessed for LH secretion in five age groups of ewes: 5 months (n=6), 6 months (n=6), 7 months (n=5), 8 months (n=5), and 10 months (n=6) of age. Following each bleed, ewes were sacrificed, hypothalamic tissue containing the ARC was collected, and then processed for use in dual immunofluorescence and RNAscope. Mean LH and LH pulse frequencies followed the expected patterns: concentrations and frequencies were low during the prepubertal ages (5-7 months of age), intermediate during the peripubertal age (8 months of age), and elevated in the postpubertal age group (10 months of age). Using immunofluorescence, kisspeptin and NKB immuno-positive cell numbers did not change significantly (P > 0.50) over time with cell numbers averaging 235.3±16.8 and 231.3±16.8, respectively. Colocalization of kisspeptin and NKB was greater than 90% for all age groups. Using RNAscope, the total number of cells expressing mRNA for kisspeptin and dynorphin did not change significantly (P > 0.05) over time with cell numbers averaging 46.7±12.0 and 28.3±10.0 cells/hemisection, respectively. Taken together, our data suggest that the increase in LH secretion that drives puberty onset is not limited by changes in kisspeptin, NKB, or dynorphin expression, but may instead depend on other factors such as changes in receptor expression or changes in KNDy neuron activity via a reduction in inhibitory and/or an increase in stimulatory afferent inputs.

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty.

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.