Evidence that pubertal status impacts KNDy neurons in the gilt

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.

Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep

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.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirm they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Effect of Yuzu (Citrus junos) Seed Limonoids and Spermine on Intestinal Microbiota and Hypothalamic Tissue in the Sandhoff Disease Mouse Model

The effect of limonoids and spermine (Spm) extracted from yuzu (Citrus junos) seeds on the gut and the brain in a mouse model with Sandhoff disease (SD) was investigated. Wild-type and SD mice were fed a normal diet, or a diet supplemented with limonoid, Spm, or limonoid + Spm for 14–18 weeks, and then 16S rRNA gene amplicon sequencing with extracted DNA from their feces was executed. For SD control mice, intestinal microbiota was mostly composed of Lactobacillus and linked to dysbiosis. For SD and wild-type mice fed with limonoids + Spm or limonoids alone, intestinal microbiota was rich in mucin-degrading bacteria, including Bacteroidetes, Verrucomicrobia, and Firmicutes, and displayed a higher production of short-chain fatty acids and immunoglobulin A. Additionally, SD mice fed with limonoids + Spm or limonoids alone had less inflammation in hypothalamic tissues and displayed a greater number of neurons. Administration of limonoids and/or Spm improved the proportions of beneficial intestinal microbiota to host health and reduced neuronal degeneration in SD mice. Yuzu seed limonoids and Spermine may help to maintain the homeostasis of intestinal microbiota and hypothalamic tissue in the SD mouse model.

Evidence That Agouti-Related Peptide May Directly Regulate Kisspeptin Neurons 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.

41 Diet-induced alterations in the hypothalamic and arcuate nucleus transcriptome in prepubertal heifers

Early onset of puberty is a key trait underpinning economically efficient cattle production systems. Although prepubertal plane of nutrition has been positively associated with earlier onset of puberty, the underlying biochemical mechanisms remain unknown. The hypothalamus, and particularly the arcuate nucleus (ARC), has a pivotal role during this developmental stage because it is a central regulator of homeostasis, mediating various neuroendocrine and physiological functions, including feed intake and reproduction. Thus, we hypothesised that a high plane of nutrition during early calfhood would affect the transcriptional profile of hypothalamic tissue, and in particular, elicit differential expression of biochemical pathways consistent with earlier sexual development in beef heifers. Angus×Holstein-Friesian heifer calves (19±5 days of age, 51.2±7.8kg, mean±s.d.) were offered a high (HP, n=14) or moderate plane of nutrition (MP, n=15) from 3 to 21 weeks of age to achieve target growth rates of 1.2 and 0.5 kg/d, respectively. At 21 weeks of age, calves were killed and the brain recovered. The ARC region was separated from the remainder of the hypothalamus, with both tissues flash frozen and subsequently used for RNA sequencing. Differential expression of genes (DEGs) was determining using R Bioconductor package EdgeR (version 3.20.9). The resultant list of DEGs was then submitted to Ingenuity Pathway Analysis and Weighted Gene Co-expression Network Analysis (WGCNA). At slaughter, bodyweight was higher in HP calves (189.6 vs. 113.0 kg; P&lt;0.001). Compared with calves on MP, a high plane of nutrition altered the expression of 39 and 80 transcripts in the hypothalamus and ARC, respectively (P&lt;0.05). COL15A1, CDH17, and IL20RA were among the most upregulated transcripts in both hypothalamic tissue sections. The HP treatment induced downregulation of AGRP and NPY and upregulation of POMC in the ARC, which have previously been associated with early onset of puberty in heifers. Functional analysis highlighted the importance of DEGs in the hypothalamus and ARC related to diets, with stress-signalling pathways among the most highly dysregulated pathways in both tissues. Through pathway analysis of the ARC DEGs, a total of 7 networks were derived, including one involved in organ morphology, reproductive system development and function, and developmental disorder. Interestingly, CGA, the most upregulated DEG in the ARC and a potential upstream regulator in this brain region, was one of the most connected genes within this network. Furthermore, WGCNA revealed that expression of some hub genes in networks of co-expressed genes, previously associated with puberty in cattle, was affected by diet in the ARC (POMC, CBLN2, and CHGA) and the remainder of the hypothalamus (PENK). The results of this study indicate that an enhanced plane of nutrition during early calfhood alters the biochemical regulation of the hypothalamus consistent with advanced sexual development in the prepubertal heifer. This research was funded by Irish Department of Agriculture, Food and the Marine (RSF 13/S/515).

Kisspeptin Neurons in the Infundibular Nucleus of Ovariectomized Cats and Dogs Exhibit Unique Anatomical and Neurochemical Characteristics

Neurons co-synthesizing kisspeptin (KP), neurokinin B (NKB), and dynorphin (“KNDy neurons”) in the hypothalamic arcuate/infundibular nucleus (INF) form a crucial component of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) “pulse generator.” The goal of our study was to characterize KP neuron distribution, neuropeptide phenotype and connectivity to GnRH cells in ovariectomized (OVX) dogs and cats with immunohistochemistry on formalin-fixed hypothalamic tissue sections. In both species, KP and NKB neurons occurred in the INF and the two cell populations overlapped substantially. Dynorphin was detected in large subsets of canine KP (56%) and NKB (37%) cells and feline KP (64%) and NKB (57%) cells; triple-labeled (“KNDy”) somata formed ∼25% of all immunolabeled neurons. Substance P (SP) was present in 20% of KP and 29% of NKB neurons in OVX cats but not dogs, although 26% of KP and 24% of NKB neurons in a gonadally intact male dog also contained SP signal. Only in cats, cocaine- and amphetamine regulated transcript was also colocalized with KP (23%) and NKB (7%). In contrast with reports from mice, KP neurons did not express galanin in either carnivore. KP neurons innervated virtually all GnRH neurons in both species. Results of this anatomical study on OVX animals reveal species-specific features of canine and feline mediobasal hypothalamic KP neurons. Anatomical and neurochemical similarities to and differences from the homologous KP cells of more extensively studied rodent, domestic and primate species will enhance our understanding of obligate and facultative players in the molecular mechanisms underlying pulsatile GnRH/LH secretion.

Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.

Stress-induced increased expression of TLR2, TLR3, and TLR4 genes in hypothalamic tissue

The aim of this work was to study the expression of Toll-like receptors (TLRs) genes in the hypothalamic structures of the brain, after the application of acute stressful effects. A hypothesis has been put forward about Toll-like receptors as a key link in the mechanisms of the implementation of a stress reaction, including at the level of the central nervous system (CNS). The important role of TLR in the pathogenesis of stress-mediated diseases of the central nervous system is assumed. The expression of TLR2, TLR3, and TLR4 genes in rat hypothalamus was studied for after 3 hours after the application of acute emotional-physical stress. A reliable increase in the level of gene expression of all three receptors at the mRNA level in stressed animals was established compared with the control. The obtained experimental data indicate the activation of the system of Toll-like receptors at the level of the central nervous system under stress. The activation of several receptors of the TLR family with different specificities in the absence of an increase in microbial load, including at the level of pathogen-associated molecular factors (PAMP), may also indicate the likely significant role of endogenous TLR ligands in the described processes.