In ovaries with high or low variation in follicle size, granulosa cells of antral follicles exhibit distinct size-related processes.

Antral follicle size might be a valuable additive predictive marker for IVF outcome. To better understand consequences of antral follicle size as a marker for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates. Using the pig as a model, we compared gene expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool of sows, which had a high variation versus low variation in follicle size. Selected gene expression was confirmed at the protein level. Granulosa cells of smaller antral follicles showed increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of the androgen receptor and the epidermal growth factor receptor, which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool are more proliferative, granulosa cells of larger follicles express more maturation markers such as insulin-like growth factor-1 (IGF1) and angiopoietin 1 (ANGPT1) and are therefore more differentiated. As both higher IGF1 and ANGPT1 have been associated with better IVF outcomes, the results of our study imply that including smaller follicles for oocyte aspiration might have negative consequences for IVF outcome.

Isocaloric high-fat feeding directs hepatic metabolism to handling of nutrient imbalance promoting liver fat deposition.

BACKGROUND/OBJECTIVES: Consumption of fat-rich foods is associated with obesity and related alterations. However, there is a group of individuals, the metabolically obese normal-weight (MONW) subjects, who present normal body weight but have metabolic features characteristic of the obese status, including fat deposition in critical tissues such as liver, recognized as a major cause for the promotion of metabolic diseases. Our aim was to better understand metabolic alterations present in liver of MONW rats applying whole genome transcriptome analysis. METHODS: Wistar rats were chronically fed a high-fat diet isocaloric relative to Control animals to avoid the hyperphagia and overweight and to mimic MONW features. Liver transcriptome analysis of both groups was performed. RESULTS: Sustained intake of an isocaloric high-fat diet had a deep impact on the liver transcriptome, mainly affecting lipid metabolism. Although serum cholesterol levels were not affected, circulating triacylglycerols were lower, and metabolic adaptations at gene expression level indicated adaptation toward handling the increased fat content of the diet, an increased triacylglycerol and cholesterol deposition in liver of MONW rats was observed. Moreover, gene expression pointed to increased risk of liver injury. One of the top upregulated genes in this tissue was Krt23, a marker of hepatic disease in humans that was also increased at the protein level. CONCLUSION: Long-term intake of a high-fat diet, even in the absence of overweight/obesity or increase in classical blood risk biomarkers, promotes a molecular environment leading to hepatic lipid accumulation and increasing the risk of suffering from hepatic diseases.

Effects of early-life stress on peripheral and central mitochondria in male mice across ages.

Exposure to early-life stress (ES) increases the vulnerability to develop metabolic diseases as well as cognitive dysfunction, but the specific biological underpinning of the ES-induced programming is unknown. Metabolic and cognitive disorders are often comorbid, suggesting possible converging underlying pathways. Mitochondrial dysfunction is implicated in both metabolic diseases and cognitive dysfunction and chronic stress impairs mitochondrial functioning. However, if and how mitochondria are impacted by ES and whether they are implicated in the ES-induced programming remains to be determined. ES was applied by providing mice with limited nesting and bedding material from postnatal day (P)2-P9, and metabolic parameters, cognitive functions and multiple aspects of mitochondria biology (i.e. mitochondrial electron transport chain (ETC) complex activity, mitochondrial DNA copy number, expression of genes relevant for mitochondrial function, and the antioxidant capacity) were studied in muscle, hypothalamus and hippocampus at P9 and late adulthood (10-12 months of age). We show that ES altered bodyweight (gain), adiposity and glucose levels at P9, but not in late adulthood. At this age, however, ES exposure led to cognitive impairments. ES affected peripheral and central mitochondria in an age-dependent manner. At P9, both muscle and hypothalamic ETC activity were affected by ES, while in hippocampus, ES altered the expression of genes involved in fission and antioxidant defence. In adulthood, alterations in ETC complex activity were observed in the hypothalamus specifically, whereas in muscle and hippocampus ES affected the expression of genes involved in mitophagy and fission, respectively. Our study demonstrates that ES affects peripheral and central mitochondria biology throughout life, thereby uncovering a converging mechanism that might contribute to the ES-induced vulnerability for both metabolic diseases and cognitive dysfunction, which could serve as a novel target for intervention.

Further localization of the gene for hereditary paragangliomas and evidence for linkage in unrelated families.

Paragangliomas of the head and neck are slow-growing tumors that rarely show malignant progression. Familial transmission has been described, consistent with an autosomal dominant gene that is maternally imprinted. Clinical manifestations of hereditary paraganglioma are determined by the sex of the transmitting parent. All affected individuals have inherited the disease gene from their father, expression of the phenotype is not observed in the offspring of an affected female or female gene carrier until subsequent transmittance of the gene through a male gene carrier. Recently, we assigned the gene responsible for paragangliomas (PGL) to chromosome 11q23-qter by linkage in a single large Dutch kindred. We now report confirmation of this localization in five unrelated Dutch families with hereditary paragangliomas. On the basis of segregation of haplotypes in the available family material, we localize the PGL locus between markers STMY and CD3D on chromosome 11q22.3-q23.

Confinement of PGL, an imprinted gene causing hereditary paragangliomas, to a 2-cM interval on 11q22-q23 and exclusion of DRD2 and NCAM as candidate genes.

Paragangliomas of the head and neck region, also known as glomus tumours, are mostly benign tumours of neuro-ectodermal origin. We mapped the familial form by linkage analysis in 6 families to chromosome region 11q22-q23, between the markers STMY and CD3D which currently span a 16-cM interval. Here, we performed detailed haplotype analysis of this region in a single Dutch multibranch 7-generation family. A region of 2 cM between the markers D11S938/D11S4122 and D11S1885 was shared between all patients of whom disease haplotypes could be reconstructed. In support of this localization, a recombination observed in a small French family with 2 affected nieces places the PGL gene proximal to marker D11S908, genetically coincident with D11S1885.

Genomic structure of the human PLZF gene.

The human PLZF (promyelocytic leukaemia zinc finger) gene encodes a Krüppel-like zinc finger protein, which was identified via the reciprocal translocation t(11;17)(q23;q21) fusing it to the retinoic acid receptor alpha (RARalpha) gene in promyelocytic leukaemia. To determine its complete genomic organisation, we constructed a cosmid-map fully containing the hPLZF gene. The gene has seven exons, including a novel 5' untranslated exon, varying in size from 87 to 1358bp and spans at least 120kb. Flanking intronic sequences were identified and all splice acceptor and donor sites conformed to the gt/ag rule. Five polymorphic markers could be fine located in its vicinity. These data will facilitate mutation analysis of hPLZF in t(11;17) leukaemia cases, as well as assist mapping and loss-of-heterozygosity analysis. Here we have tested hPLZF as a possible candidate for the PGL1 locus involved in hereditary head and neck paragangliomas. However, mutation analysis revealed no aberration in 12 paraganglioma patients from different families.

Paragangliomas of the head and neck region show complete loss of heterozygosity at 11q22-q23 in chief cells and the flow-sorted DNA aneuploid fraction.

Nonchromaffin paragangliomas of the head and neck region, also known as glomus tumors, are usually benign neoplasms consisting of clusters of chief cells surrounded by sustentacular cells arranged in so-called 'Zellballen.' Most of the patients have a familial background. In a previous study, examining all chromosome arms, we found loss of heterozygosity (LOH) predominantly at the chromosome 11q22-q23 region, where the disease causing gene PGL1 has been located by linkage analysis. However, all tumors showed only partial loss of allele signal intensities, and it was not clear whether this represented allelic imbalance or cellular heterogeneity. In the current study, we have performed LOH analysis for the 11q22-q23 region on DNA-aneuploid tumor cells, enriched by flow sorting, and on purified chief cell fractions obtained by single-cell microdissection. Complete LOH was found for two markers (D11S560 and CD3D) in the flow-sorted aneuploid fractions, whereas no LOH was found in the diploid fractions of three tumors. The microdissected chief cells from two of these tumors also showed complete LOH for both markers, indicating that the chief cells are clonal proliferated tumor cells. These results indicate that the PGL1 gene is likely to be a tumor suppressor gene, which is inactivated according to the two-hit model of Knudson. Furthermore, it shows that chief cells are a major if not the sole neoplastic component of paragangliomas.

Identification of early transcriptome-based biomarkers related to lipid metabolism in peripheral blood mononuclear cells of rats nutritionally programmed for improved metabolic health.

Moderate maternal calorie restriction during lactation protects rat offspring against obesity development in adulthood, due to an improved ability to handle and store excess dietary fuel. We used this model to identify early transcriptome-based biomarkers of metabolic health using peripheral blood mononuclear cells (PBMCs), an easily accessible surrogate tissue, by focusing on molecular markers of lipid handling. Male and female offspring of control and 20 % calorie-restricted lactating dams (CR) were studied. At weaning, a set of pups was killed, and PBMCs were isolated for whole-genome microarray analysis. The remaining pups were killed at 6 months of age. CR gave lower body weight, food intake and fat accumulation, and improved levels of insulin and leptin throughout life, particularly in females. Microarray analysis of weaned rat PBMCs identified 278 genes significantly differentially expressed between control and CR. Among lipid metabolism-related genes, expression of Cpt1a, Lipe and Star was increased and Fasn, Lrp1 and Rxrb decreased in CR versus control, with changes fully confirmed by qPCR. Among them, Cpt1a, Fasn and Star emerged as particularly interesting. Transcript levels of Cpt1a in PBMCs correlated with their levels in WAT and liver at both ages examined; Fasn expression levels in PBMCs at an early age correlated with their expression levels in WAT; and early changes in Star expression levels in PBMCs correlated with their expression levels in liver and were sustained in adulthood. These findings reveal the possibility of using transcript levels of lipid metabolism-related genes in PBMCs as early biomarkers of metabolic health status.

Quercetin decreases high-fat diet induced body weight gain and accumulation of hepatic and circulating lipids in mice.

Dietary flavonoids may protect against cardiovascular diseases (CVD). Increased circulating lipid levels and hepatic lipid accumulation are known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the flavonoid quercetin on hepatic lipid metabolism in mice with high-fat diet induced body weight gain and hepatic lipid accumulation. Adult male mice received a 40 energy% high-fat diet without or with supplementation of 0.33 % (w/w) quercetin for 12 weeks. Body weight gain was 29 % lower in quercetin fed mice (p < 0.01), while the energy intake was not significantly different. Quercetin supplementation lowered hepatic lipid accumulation to 29 % of the amount present in the control mice (p < 0.01). (1)H nuclear magnetic resonance serum lipid profiling revealed that the supplementation significantly lowered serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3), were downregulated. Quercetin decreased high-fat diet induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are possibly under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content of the diet.

Chronic quercetin exposure affects fatty acid catabolism in rat lung.

Dietary quercetin intake is suggested to be health promoting, but this assumption is mainly based on mechanistic studies performed in vitro. Previously, we identified rat lung as a quercetin target tissue. To assess relevant in vivo health effects of quercetin, we analyzed mechanisms of effect in rat lungs of a chronic (41 weeks) 1% quercetin diet using whole genome microarrays. We show here that fatty acid catabolism pathways, like beta-oxidation and ketogenesis, are up-regulated by the long-term quercetin intervention. Up-regulation of genes (Hmgcs2, Ech1, Acox1, Pcca, Lpl and Acaa2) was verified and confirmed by quantitative real time PCR. In addition, free fatty acid levels were decreased in rats fed the quercetin diet, confirming that quercetin affects fatty acid catabolism. This in vivo study demonstrates for the first time that fatty acid catabolism is a relevant process that is affected in rats by chronic dietary quercetin.

A high-resolution STS, EST, and gene-based physical map of the hereditary paraganglioma region on chromosome 11q23.

The genes responsible for hereditary paragangliomas (glomus tumors, MIM No. 168000) have been mapped to two distinct loci on the long arm of chromosome 11. Most of the informative families appear to be linked to the distal locus on chromosome 11q23 (PGL1), which has been previously confined to a 2-cM interval by haplotype analysis in an extended Dutch pedigree. To facilitate the identification of the PGL1 disease gene, we constructed an approximately 4-Mb ordered clone contig map of Sequence tagged sites, expressed sequence tags (ESTs), and known genes that spans the PGL1 critical region on chromosome 11q23. Among 29 new positional candidate ESTs, only two (EST100999 and EST241777) mapped within the PGL1 critical region. We further characterized the genomic organization of the promyelocytic leukemia zinc finger (PLZF) gene that maps within the PGL1 critical region and physically excluded the serotonin receptor type 3 (5HT3R) gene. Finally, we identified a common, silent, single-base substitution polymorphism in the 5HT3R gene and characterized the allele sets of two new highly polymorphic microsatellite repeats within the PGL1 critical region.

Lack of Bcl10 mutations in testicular germ cell tumours and derived cell lines.

Aberrations within Bcl10, a gene involved in execution of apoptosis, has most recently been found in a variety of cancers, including cell lines of testicular germ cell tumours of adolescents and adults (TGCTs). To study this in more detail, we screened exons 2 and 3 of this gene for mutations in a larger series of cell lines as well as primary TGCTs by single-strand conformation polymorphism and endonuclease restriction analysis. Because no aberrations were detected, we conclude that inactivation of Bcl10 by mutation is at least far less important in the development of TGCTs than proposed.

First experiences with genetic counselling based on predictive DNA diagnosis in hereditary glomus tumours (paragangliomas).

Hereditary glomus tumour (MIM 168,000) or paraganglioma (PGL) is a slowly progressive disorder causing benign tumour growth predominantly in the head and neck region. Though benign in nature the tumours can lead to severe morbidity. Inheritance of PGL is autosomal dominant and is strongly modified by genomic imprinting; only a paternally transmitted PGL gene leads to symptoms. A gene for PGL has recently been mapped to 11q22.3-q23. Genetic counselling on the basis of DNA linkage diagnosis was offered in an extended Dutch pedigree. Thirty-two subjects opted for further counselling, of whom 20 applied for DNA testing and participated in a standardised protocol. Sixteen cases had presymptomatic testing (paternal allele); four of these appeared to have the at risk haplotype and in two of them a glomus tumour was subsequently detected on MRI. In one case linkage results were inconclusive (recombination) and one person did not want to learn his test result. Four cases had testing for carrier status (maternal allele) of which one appeared to be a carrier. Our data show that genetic counselling gains significant accuracy when based on parent of origin, sex of the counsellee, and DNA linkage diagnosis. Moreover, a normal DNA result may prevent unnecessary worry and investigations, while an established presymptomatic diagnosis will guide adequate clinical management. The psychological impact of counselling and predictive DNA testing is unclear as yet. Further investigations into the natural history of PGL in gene carriers and into the psychological impact of DNA testing is desirable.

Allelotype of head and neck paragangliomas: allelic imbalance is confined to the long arm of chromosome 11, the site of the predisposing locus PGL.

Paragangliomas of the head and neck region are usually slow growing, benign tumors. A considerable fraction has a positive family history, and the predisposing locus, PGL, has recently been assigned to 11q22-q23. The inheritance pattern of the disease suggests that PGL undergoes maternal genomic imprinting. We have investigated 26 tumor samples from 22 patients with head and neck paragangliomas for the occurrence of loss of heterozygosity (LOH) on all non-acrocentric autosome arms. LOH was found only on chromosome 11, with a marked clustering on the distal half of the q-arm. However, in many cases the resulting allelic imbalance relative to normal DNA was weak, suggesting that only part of the tumor showed this abnormality. In all eight cases where we were able to determine the parental origin, the allele undergoing loss was maternally derived. Clonality analysis with a polymorphic marker for the X-chromosome indicated that two of three informative female cases were polyclonal, although a number of tumors carry aneuploid stemlines in DNA flow cytometry. We conclude that either tumor heterogeneity or polyclonality may explain the partial allele loss events seen in certain cases.

Repositioning the hereditary paraganglioma critical region on chromosome band 11q23.

Hereditary paragangliomas (PGL, glomus tumors, MIM no.168000) are mostly benign, slow-growing tumors of the head and neck region. The gene (or genes) affecting risk to PGL are subject to genomic imprinting: children of affected fathers exhibit an autosomal dominant pattern of disease inheritance, whereas children of affected mothers rarely if ever develop the disease through maternal transmission. We previously confined the disease gene to an approximately 6 Mb critical region on chromosome band 11q23 (PGL1). Based on haplotype analysis of an extended Dutch pedigree, a 2 Mb sub-region between D11S938 and D11S1885 was proposed as the PGL1 critical interval. In this study, we excluded this interval by analysis of two new single tandem repeat polymorphisms (STRP) contained therein. Instead, we predicted a non-overlapping, more proximal 2 Mb critical interval between D11S1647 and D11S897, and evaluated this new region using nine STRP (D11S1986, five new, closely-linked STRP, D11S1347, D11S3178, and D11S1987). Consistent with our prediction, we observed substantial haplotype-sharing within the Dutch pedigree. We also analyzed four new American PGL families. A recombination event detected in one family further defined D11S1347 as the new telomeric border. We observed significant haplotype-sharing within this new interval among three unrelated American PGL families, strongly suggesting that they originated from a common ancestor. Thus, we confined PGL1 to an approximately 1.5 Mb region between D11S1986 and D11S1347, and showed identity-by-descent sharing for a group of American PGL families.

Founder effect at PGL1 in hereditary head and neck paraganglioma families from the Netherlands.

PGL1, a gene responsible for hereditary paragangliomas of the head and neck, recently was mapped to a 2-cM interval on chromosome 11q22-q23, by linkage and haplotype-sharing analysis of a large multibranch Dutch family. We determined the disease-linked haplotype, as defined by 13 markers encompassing a large interval on 11q21-q23, in 10 additional families ascertained from the same geographical locale. Alleles were identical for six contiguous markers, spanning a genetic distance of 6 cM and containing PGL1. Despite this strong indication of a common ancestor, no kinships between the families could be demonstrated through genealogical surveys going back to 1800 a.d. We conclude that a single ancestral mutation is responsible for most, if not all, hereditary paragangliomas, in this region of The Netherlands, and that strong founder effects may exist at the PGL1 locus.