ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Sensitivity to phosphorus limitation increases with ploidy level in a New Zealand snail.

Evolutionary and ecological factors that explain natural variation in ploidy level remain poorly understood. One intriguing possibility is that nutrient costs associated with higher per-cell nucleic acid content could differentially influence the fitness of different ploidy levels. Here, we test this hypothesis by determining whether access to phosphorus (P), a main component of nucleic acids, differentially affects growth rate in asexual freshwater snails (Potamopyrgus antipodarum) that differ in ploidy. As expected if larger genomes generate higher dietary P requirements, tetraploid P. antipodarum experienced a more than twofold greater reduction in growth rate in low-P versus high-P conditions relative to triploids. Mirroring these results, tetraploid P. antipodarum also had a significant reduction in body P content under low P relative to high P, whereas triploid body P content was unaffected. Taken together, these results set the stage for the possibility that P availability could influence the distribution and relative frequency of P. antipodarum of different ploidy levels. These findings could be applicable to many other animal taxa featuring ploidy-level variation, which includes many mixed sexual/asexual taxa.

Adverse effects of excessive leucine intake depend on dietary protein intake: a transcriptomic analysis to identify useful biomarkers.

The present study was conducted to identify reliable gene biomarkers for the adverse effects of excessive leucine (Leu) in Sprague-Dawley rats by DNA microarray. It has long been known that the adverse effects of excessive amino acid intake depend on dietary protein levels. Male rats were divided into 12 groups (n=6) and fed for 1 wk a diet containing low (6%), moderate (12%) or high (40%) protein. Different levels of Leu (0, 2, 4, and 8%) were added to the diets. Consumption of diets containing more than 4% Leu in 6% protein resulted in growth retardation and reduced liver weight, whereas the administration of the same dose of Leu with 12% or 40% protein did not affect them. By a process of systematic data extraction, 6 candidate gene markers were identified. The liver gene expression data obtained from another experiment with 0, 2, 3, 4, and 8% Leu in a low-protein diet was used to examine the validity of these biomarker candidates with receiver operating characteristic (ROC) curve analysis. All of AUC values of the biomarker candidates were more than 0.700, suggesting the effectiveness of the marker candidates as the indices of Leu excess. The cut-off value for the ROC curve of the gene-marker panel, which was obtained by multiple regression analysis of gene markers, indicated that Leu levels higher than 3% have adverse effects. In conclusion, the gene-marker panel suggested that for male rats dietary Leu supplementation of 2% is the NOAEL dose in low-protein (6%) diets.

Maternal micronutrient supplementation suppresses T cell chemokine receptor expression and function in F1 mice.

Prenatal environmental exposures play a critical role in determining late-life chronic disease susceptibility. However, the mechanisms linking the in utero environment and disease development in the offspring are poorly understood. Recent investigations have confirmed a central pathogenic role of T cell chemokine receptors, particularly C-C chemokine receptor (CCR) 2 and CCR5, in chronic inflammatory conditions. This study was designed to determine the effect of a synthetic prenatal micronutrient supplementation (MS) diet rich in methionine pathway metabolites on the T cell chemokine system in F1 C57Bl/6 mice. Female mice were fed either an MS or control diet 3 wk prior to mating, during pregnancy, and lactation. At 4 wk of age, F1 mice were killed for experiments or were fed the standard NIH-31 diet and allowed to age. Food consumption, maternal weight gain, and litter size were similar in dams fed the control and MS diets. However, the F1 offspring of dams fed the MS diet were smaller in size (P < 0.001). T cells from the MS F1 offspring had global hypermethylation compared with control F1 offspring (P < 0.005), corresponding to lower T cell chemokine receptor expression [CCR2 (P < 0.001), CCR5 (P < 0.001), and C-x-C chemokine receptor 3 (P < 0.01)] and cytokine expression [TNFα (P < 0.05), IL-2 (P < 0.001), and IL-4 (P < 0.01)]. Reduced T cell chemokine receptor gene expression in MS F1 mice was associated with decreased chemotaxis in vitro to C-C chemokine ligand (CCL) 2 and C-X-C chemokine ligand 10 (P < 0.01) and in vivo to CCL2 (P < 0.01). Taken together, the results suggest that epigenetic alteration through prenatal diet manipulation reduces the response to proinflammatory signals in mice.

Evaluation of the effects of dietary fat, conjugated linoleic acid, and ractopamine on growth performance, pork quality, and fatty acid profiles in genetically lean gilts.

An 8-wk study of the effects of CLA, rendered animal fats, and ractopamine, and their interactive effects on growth, fatty acid composition, and carcass quality of genetically lean pigs was conducted. Gilts (n = 228; initial BW of 59.1 kg) were assigned to a 2 x 2 x 3 factorial arrangement consisting of CLA, ractopamine, and fat treatments. The CLA treatment consisted of 1% CLA oil (CLA-60) or 1% soybean oil. Ractopamine levels were either 0 or 10 ppm. Fat treatments consisted of 0% added fat, 5% choice white grease (CWG), or 5% beef tallow (BT). The CLA and fat treatments were initiated at 59.1 kg of BW, 4 wk before the ractopamine treatments. The ractopamine treatments were imposed when the gilts reached a BW of 85.7 kg and lasted for the duration of the final 4 wk until carcass data were collected. Lipids from the belly, outer and inner layers of backfat, and LM were extracted and analyzed for fatty acid composition from 6 pigs per treatment at wk 4 and 8. Feeding CLA increased (P < 0.02) G:F during the final 4 wk. Pigs fed added fat as either CWG or BT exhibited decreased (P < 0.05) ADFI and increased (P < 0.01) G:F. Adding ractopamine to the diet increased (P < 0.01) ADG, G:F, and final BW. The predicted carcass lean percentage was increased (P < 0.05) in pigs fed CLA or ractopamine. Feeding either 5% fat or ractopamine increased (P < 0.05) carcass weight. Adding fat to the diets increased (P < 0.05) the 10th rib backfat depth but did not affect predicted percent lean. Bellies of gilts fed CLA were subjectively and objectively firmer (P < 0.01). Dietary CLA increased (P < 0.01) the concentration of saturated fatty acids and decreased (P < 0.01) the concentration of unsaturated fatty acids of the belly fat, both layers of backfat, and LM. Ractopamine decreased (P < 0.01) the i.m. fat content of the LM but had relatively little effect on the fatty acid profiles of the tissues compared with CLA. These results indicate that CLA, added fat, and ractopamine work mainly in an additive fashion to enhance pig growth and carcass quality. Furthermore, these results indicate that CLA results in more saturated fat throughout the carcass.

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids.

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.

Effect of supplemental oxygen on blood plasma organic acids within embryos from selected lines of turkeys.

The hypothesis was tested that increased oxygen tensions during the plateau stage of oxygen consumption (25 and 26 d of incubation) would cause different metabolic responses from embryos selected for increased egg production or growth. Embryos were exposed to 171 or 152 mm Hg partial pressure of oxygen from 25 to 28 d of incubation, a time when the oxygen conductance properties of the eggshell are exceeded by the embryonic tissue demands for oxygen. Carbohydrate and lipid metabolism were observed by measuring plasma organic acids in embryos from selected lines and randombred controls. (E was selected for increased egg production; RBC1 is the randombred line from which it was selected. F was selected for increased BW; RBC2 is the randombred line from which it was selected.) During the plateau stage in oxygen consumption, RBC2 embryos responded to added oxygen by utilizing fat rather than carbohydrate, whereas F embryos responded by using less fat as well as less carbohydrate from the liver and kidney. The response of F embryos to added oxygen is the opposite that might be expected for aerobic metabolism. The reason that selection for growth has resulted in such a metabolism is unknown. The E embryos displayed depressed lactate and beta-hydroxybutyrate levels, but plasma urates were elevated compared with RBC1, suggesting that the selection for egg production has also resulted in a unique metabolism. The embryonic processes described in the current study suggest that selected embryos are unable to respond to elevated partial pressure of oxygen by adjusting energy metabolism, which may result in increased embryonic mortality during this stage.

Sp4, a member of the Sp1-family of zinc finger transcription factors, is required for normal murine growth, viability, and male fertility.

We report the cloning, characterization, and targeting of an Sp1-related zinc finger transcription factor gene from the distal arm of mouse chromosome 12. This gene, previously identified in rats and humans and designated sp4, is homologous to the Drosophila buttonhead (btd) gene, which is expressed in the head region of developing flies. Similarly, in situ hybridizations show that sp4 is highly expressed in mouse embryos in the developing central nervous system (CNS). Expression of sp4 is seen as early as Day 9 of development, where transcripts are abundant in the posterior neuropore. Expression in later embryos is detected throughout the CNS as well as in other structures, including the nasal mucosa, the vomeronasal organ, the epithelium of the lung and intestinal tract, the testes, and the developing teeth. Northern blot analysis showed sp4 expression in the adult brain and other tissues. Gene targeting by homologous recombination was used to determine the role of sp4 during mouse development. Two-thirds of homozygous mutants die within the first few days after birth and those that survive are smaller than their wild-type littermates. While fertility of the female mutants appears normal, homozygous mutant males do not breed, despite having histologically intact testes containing mature sperm. sp4/sp4 mutant males fail to copulate, indicating that this gene is required for normal male reproductive behavior.