Molecular and Structural Bases of Hypothalamic Puberty
ABSTRACT: To compare the global profile of hypothalamic gene expression in agonadal male Rhesus Monkeys before and after reactivation of the pulsatile GnRH release during the pubertal phase of development. 20 Samples looking at Cortex and Mediobasal hypothalamus in 3 stages of Rhesus Monkeys (early pubertal, late juvenile, and late pubertal).
Project description:To compare the global profile of hypothalamic gene expression in agonadal male infants before and after activation of the neurobiologic brake that arrests pulsatile GnRH release during the juvenile phase of development. Affymetrix arrays were used to detect global changes in gene expression in the hypothalamus of of the agonadal rhesus monkey during development.
Project description:C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation. There are 5 male and 5 female C57BL/6J animals and 5 male and 5 female DBA/2J. These are adult, naïve animals. One array is run for each animal for a total of 20 Mouse MOE430 2.0 arrays. The purpose is to determine baseline (naïve) gene expression differences in striatum for these two inbred strains. There are 12 male C57BL/6J animals and 12 male DBA/2J. These are adult, naïve animals. One array is run for each animal for a total of 24 Mouse Ref8 v2 arrays. The purpose is to determine baseline (naïve) gene expression differences in striatum for these two inbred strains. ***This submission represents the microarray component of the study
Project description:This study was designed to provide a genome-wide analysis of the effects of luteinizing hormone (LH) ablation/replacement versus steroid ablation/replacement on gene expression in the developed corpus luteum (CL) in primates during the menstrual cycle. Naturally cycling, female rhesus monkeys were left untreated (Control; n = 4) or received one of the following treatments for three days beginning on Day 9 of the luteal phase: daily injection of the gonadotropin-releasing hormone (GnRH) antagonist (Antide; n = 5), Antide + recombinant human LH (A+LH; n = 4), Antide + LH + the 3b-HSD antagonist Trilostane (A+LH+TRL; n = 4), and Antide + LH + TRL + progesterone replacement with a synthetic progestin R5020 (A+LH+TRL+ R5020; n = 5). On Day 12 of the luteal phase, CL were removed and samples of RNA from individual CL were fluorescently labeled and hybridized to Affymetrix™ rhesus macaque total genome microarrays. The greatest number of altered transcripts was associated with the ablation/replacement of LH, while ablation/replacement of progestin affected fewer transcripts. Replacement of LH during Antide treatment restored expression of most transcripts to control levels. Real-time PCR validation of a subset of transcripts revealed that most expression patterns were similar between microarray and real-time PCR. Analysis of protein levels were subsequently determined for 2 of the transcripts differentially expressed by real-time PCR. This is the first genome-wide analysis of LH and steroid regulation of gene transcription in the developed primate CL. Further analysis of novel transcripts identified in this data set can clarify the relative role for LH and steroids in CL maintenance and luteolysis. Keywords: LH/steroid ablation/replacement in primate mid-late luteal phase corpora lutea 22 samples from Rhesus Macaque corpus luteum hybridized to individual Rhesus Affymentrix Gene Chip Arrays. 5 treatment groups, with at least 4 replicates per treatment.
Project description:Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase, causing a toxic buildup of acetylcholine at muscarinic and nicotinic receptors. Current medical countermeasures to nerve agent intoxication increase survival if administered within a short period of time following exposure but may not fully prevent neurological damage. Therefore, there is a need to discover drug treatments that are effective when administered after the onset of seizures and secondary responses that lead to brain injury. Methods To determine potential therapeutic targets for such treatments, we analyzed gene expression changes in the rat piriform cortex following sarin (O-isopropyl methylphosphonofluoridate) exposure. Male Sprague-Dawley rats were challenged with 1.0 x LD50 sarin and subsequently treated with atropine sulfate, 2-pyridine aldoxime methylchloride (2-PAM), and the anticonvulsant diazepam. Control animals received an equivalent volume of vehicle and drug treatments. The piriform cortex, a brain region particularly sensitive to neural damage from sarin-induced seizures, was extracted at 0.25, 1, 3, 6, and 24 h after seizure onset, and total RNA was processed for microarray analysis. Principal component analysis identified sarin-induced seizure occurrence and time point following seizure onset as major sources of variability within the dataset. Based on these variables, the dataset was filtered and analysis of variance was used to determine genes significantly changed in seizing animals at each time point. The calculated p-value and geometric fold change for each probeset identifier were subsequently used for gene ontology analysis to identify canonical pathways, biological functions, and networks of genes significantly affected by sarin-induced seizure over the 24-h time course. Results A multitude of biological functions and pathways were identified as being significantly altered following sarin-induced seizure. Inflammatory response and signaling pathways associated with inflammation were among the most significantly altered across the five time points examined. Conclusions This analysis of gene expression changes in the rat brain following sarin-induced seizure and the molecular pathways involved in sarin-induced neurodegeneration will allow a better identification of potential therapeutic targets for the development of effective neuroprotectants to treat nerve agent exposure. Three animals were used for each experimental group (naïve, sarin-exposed non-seizure, non-seizure control [saline], sarin-exposed seizure, and seizure control [saline]) at each time point (0.25, 1, 3, 6, 24 h), with the exception of 1 h seizure control, 3 h sarin-exposed seizure, 24 h sarin-exposed non-seizure, and 24 h sarin-exposed seizure (n=4).
Project description:Pregnancy has been shown to decrease the risk of mammary carcinogenesis in human rretrospective epidemiological studies. In rodents, pregnancy prior to carcinogen administration or after carcinogen challenge has also been shown to reduce the incidence of palpable carcinomas. In this study our objective to determine the underlying genomic signature of the pregnancy and reproductive hormones on the mammary gland that contribute to the protection against mammary gland carcinogenesis. We used the rat microarray technology to observe total transcriptome changes after the pregnancy and exogenous reproductive hormone stimulation of the mammary gland. Fifteen 3 month old post-pubertal virgin Lewis rats were randomly assigned to three groups (5 rats per group): control (C), pregnancy (P) and hormone treatment (H). The P group animals had a full-term pregnancy (21-23 days) and rats in the group H were implanted subcutaneously on the dorsal midline with two silastic capsules [(0.078 inch inner diameter, 0.125 inch outer diameter) x 2 cm long; Dow Corning, Midland, MI) filled separately with 100 μg ethynyl estradiol (Sigma, St. Louis, MO) packed in a cellulose matrix (Sigma) and 30 mg of megesterol acetate (Sigma) for 21 days. The control animals had neither the hormone treatment nor being pregnant. The animals in C and P groups were also implanted with sham capsules filled with cellulose matrix only. The capsules were surgically implanted at the beginning of the experiment and removed from all animals after 21 days except that the capsules were removed from the P group following parturition (21-23 days). The delivered pups in the P group were euthanized within 4-6 hours of delivery to avoid suckling. After the removal of capsules all groups were rested a total of ~49 days before euthanasia. All animals were euthanized during metestrus stage, determined by vaginal cytology and total RNA was extracted from the mammary gland tissues using Trizol reagent.
Project description:The Sertoli cells (Sc) of 5 days (infant) and 12 days (pubertal) old rat were isolated and cultured in triplicates. Nuclear and cytoplasmic fractionation was done for both the cases. All the four protein fractions (nuclear and cytoplasm of infant and pubertal Sc) were anaysed using Lc-MS/MS. SWATH analysis was done for all the four samples in biological and technical replicates. The objective was to quantity the proteins of all the samples with respect to each other at whole proteome level.
Project description:In order to study the heart disorder that the long term, high energy diet caused, Bama miniature pigs were fed a high-fat, high-sucrose diet for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. The control group consisted of 6 Bama pigs fed a control diet, and the HFHSD group comprised 6 pigs that were induced with a HFHS diet, which included 37% sucrose, 53% control diet and 10% pork lard. The pigs were fed twice every day and provided water ad libitum for 23 months. The pigs were fasted for 12 hours and euthanized with ketamine and xylazine. Pig hearts from the HFHSD group pigs (120, 126, 138, 140, 144, and 146) and three control group pigs (157, 159, and 161) were sampled and preserved in liquid nitrogen and then for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Morphogenesis of the mammary gland relies on the precise developmental control of morphological elements including TEBs, ducts and lobules. In the peripubertal mammary gland, rising levels of ovarian hormones control this development through a tightly controlled genetic program where specific sets of genes are up-regulated. We used microarrays to detail the program of gene expression underlying different classes of up-regulated genes during the peripubertal process after administration of endocrine disruptors during the fetal and neonatal development. Rat mammary glands were selected at two peripubertal periods (days 35 and 50), after administration of genistein and vinclozolin during the fetal and neonatal development, for RNA extraction and hybridization on Affymetrix microarrays.. We sought to obtain homogeneous populations of mammary gland for each treatment, and at each developmental stage, in order to increase the temporal and specific effect resolution of time course and endocrine disruption.
Project description:Sustained caloric restriction (CR) extends lifespan in animal models but the mechanism and primary tissue target(s) have not been identified. Gene expression changes with aging and CR were examined in both heart and subcutaneous white adipose tissue (WAT) of F344 male rats using Affymetrix® RAE 230 arrays and validated by qRT-PCR on 18 genes. In heart, age- associated changes but not CR-associated changes in old. In WAT, genes were identified where the aging change is suppressed by CR (candidate markers of healthy aging) and those affected by CR but not normal aging (candidate longevity assurance genes). 10-21% of age-associated genes were regulated in common between tissues. Gene set enrichment analysis (GSEA) revealed coordinate small magnitude changes in ribosomal, proteasomal, and mitochondrial genes with similarities between heart and WAT. Further analysis revealed PPARgamma as a potential upstream regulator of altered gene expression in old CR WAT. These results demonstrate a reduced mRNA response to CR with age in heart relative to WAT. In WAT, we identified candidate CR mimetic targets and candidate markers of healthy aging. These data suggest a role for subcutaneous WAT in the effects of CR and strengthen the role for PPAR signaling in aging and CR while indicating that the effects of CR in heart can occur independent of global changes in mRNA level. Experiment Overall Design: Tissues (n=5-7 animals per group) were obtained from the NIH-NIA aging rodent tissue bank. According to supplier records, animals were housed in a specific pathogen free barrier facility under contract with BioReliance. AL animals were housed 3 per cage and CR animals were housed singly. CR (60% of AL) was initiated at 4 months of age with a switch from the NIH 31 to NIH fortified diet. F344 male rats were sacrificed at 4 months (4AL) or 28 months of age (28AL, 28CR). Animals with gross tumor pathologies were excluded from the study. All animals were euthanized by CO2 asphixiation and tissues were frozen in liquid nitrogen then stored at –80°C. Subcutaneous WAT was collected from the abdominal region. Recorded body weights differed dramatically between the 3 groups (4AL = 253±8 g, 28AL = 382±20 g, 28CR = 288±7 g) and heart weight as a fraction of body weight was not significantly different between the groups (data not shown). The apical ~1/3 of the heart was homogenized for the expression studies. Sections from the cut face were stained with haematoxylin and eosin to confirm expected aging-associated pathology changes.