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1.
J Endocrinol ; 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189133

RESUMO

Human offspring encounter high amounts of phytoestrogens, such as genistein (GEN), through maternal diet and soy-based formulas. Such chemicals can exert estrogenic activity and thereby disrupt neurobehavioral programming. Besides inducing direct host effects, GEN might cause gut dysbiosis and alter gut metabolites. To determine whether exposure to GEN affects these parameters, California mice (Peromyscus californicus) dams were placed two weeks prior to breeding and throughout gestation and lactation on a diet supplemented with GEN (250 mg/kg feed weight) or AIN93G phytoestrogen-free control diet (AIN). At weaning, offspring socio-communicative behaviors, gut microbiota and metabolite profiles were assayed. Exposure of offspring to GEN induced sex-dependent effects on gut microbiota and metabolites. GEN exposed females were less likely to investigate a novel female mouse when tested in a three-chamber social test. When isolated, GEN males and females exhibited increased latency to elicit their first call, suggestive of reduced motivation to communicate with other individuals. Correlation analyses revealed interactions between GEN-induced microbiome, metabolome, and socio-communicative behaviors. For instance, comparison of GEN males with AIN males revealed the fraction of calls above 20 kHz was associated with daidzein, tocopherol, Flexispira spp., and Odoribacter spp. Results suggest early GEN exposure disrupts normal socio-communicative behaviors in California mice, which are otherwise evident in these social rodents. Such effects may be due to GEN disruptions on neural programming but might be attributed to GEN-induced microbiota shifts and resultant changes in gut metabolites. Findings indicate cause for concern that perinatal exposure to GEN might detrimentally affect offspring microbiome-gut-brain axis.

2.
BMC Genomics ; 20(1): 87, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683046

RESUMO

BACKGROUND: Brain sexual differentiation is sculpted by precise coordination of steroid hormones during development. Programming of several brain regions in males depends upon aromatase conversion of testosterone to estrogen. However, it is not clear the direct contribution that Y chromosome associated genes, especially sex-determining region Y (Sry), might exert on brain sexual differentiation in therian mammals. Two species of spiny rats: Amami spiny rat (Tokudaia osimensis) and Tokunoshima spiny rat (T. tokunoshimensis) lack a Y chromosome/Sry, and these individuals possess an XO chromosome system in both sexes. Both Tokudaia species are highly endangered. To assess the neural transcriptome profile in male and female Amami spiny rats, RNA was isolated from brain samples of adult male and female spiny rats that had died accidentally and used for RNAseq analyses. RESULTS: RNAseq analyses confirmed that several genes and individual transcripts were differentially expressed between males and females. In males, seminal vesicle secretory protein 5 (Svs5) and cytochrome P450 1B1 (Cyp1b1) genes were significantly elevated compared to females, whereas serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) was upregulated in females. Many individual transcripts elevated in males included those encoding for zinc finger proteins, e.g. zinc finger protein X-linked (Zfx). CONCLUSIONS: This method successfully identified several genes and transcripts that showed expression differences in the brain of adult male and female Amami spiny rat. The functional significance of these findings, especially differential expression of transcripts encoding zinc finger proteins, in this unusual rodent species remains to be determined.


Assuntos
Encéfalo/metabolismo , Murinae/genética , Caracteres Sexuais , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Murinae/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Cromossomo Y
3.
Sci Rep ; 7(1): 2822, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588204

RESUMO

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are being used in non-edible and edible consumer products. It is not clear though if exposure to these chemicals can exert toxic effects on the host and gut microbiome. Conflicting studies have been reported on whether AgNPs result in gut dysbiosis and other changes within the host. We sought to examine whether exposure of Sprague-Dawley male rats for two weeks to different shapes of AgNPs, cube (AgNC) and sphere (AgNS) affects gut microbiota, select behaviors, and induces histopathological changes in the gastrointestinal system and brain. In the elevated plus maze (EPM), AgNS-exposed rats showed greater number of entries into closed arms and center compared to controls and those exposed to AgNC. AgNS and AgNC treated groups had select reductions in gut microbiota relative to controls. Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus were decreased in AgNC exposed group, whereas, Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica were reduced in AgNS exposed group. Bacterial reductions correlated with select behavioral changes measured in the EPM. No significant histopathological changes were evident in the gastrointestinal system or brain. Findings suggest short-term exposure to AgNS or AgNC can lead to behavioral and gut microbiome changes.


Assuntos
Disbiose/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Aggregatibacter/efeitos dos fármacos , Animais , Bacteroides/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Clostridium/efeitos dos fármacos , Corynebacterium/efeitos dos fármacos , Disbiose/induzido quimicamente , Disbiose/fisiopatologia , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/fisiopatologia , Humanos , Nanopartículas Metálicas/administração & dosagem , Peptococcus/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
4.
J Biomol Tech ; 28(1): 31-39, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337070

RESUMO

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.


Assuntos
Microbiologia Ambiental , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ambientes Extremos , Metagenoma , Tipagem Molecular/normas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA/normas
5.
Physiol Rep ; 5(3)2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28196854

RESUMO

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) prevalent in many household items. Rodent models and human epidemiological studies have linked this chemical to neurobehavior impairments. In California mice, developmental exposure to BPA results in sociosexual disorders at adulthood, including communication and biparental care deficits, behaviors that are primarily regulated by the hypothalamus. Thus, we sought to examine the transcriptomic profile in this brain region of juvenile male and female California mice offspring exposed from periconception through lactation to BPA or ethinyl estradiol (EE, estrogen present in birth control pills and considered a positive estrogen control for BPA studies). Two weeks prior to breeding, P0 females were fed a control diet, or this diet supplemented with 50 mg BPA/kg feed weight or 0.1 ppb EE, and continued on the diets through lactation. At weaning, brains from male and female offspring were collected, hypothalamic RNA isolated, and RNA-seq analysis performed. Results indicate that BPA and EE groups clustered separately from controls with BPA and EE exposure leading to unique set of signature gene profiles. Kcnd3 was downregulated in the hypothalamus of BPA- and EE-exposed females, whereas Tbl2, Topors, Kif3a, and Phactr2 were upregulated in these groups. Comparison of transcripts differentially expressed in BPA and EE groups revealed significant enrichment of gene ontology terms associated with microtubule-based processes. Current results show that perinatal exposure to BPA or EE can result in several transcriptomic alterations, including those associated with microtubule functions, in the hypothalamus of California mice. It remains to be determined whether these genes mediate BPA-induced behavioral disruptions.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Etinilestradiol/toxicidade , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Fenóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Transcriptoma/efeitos dos fármacos , Animais , Regulação para Baixo , Feminino , Masculino , Exposição Materna , Peromyscus , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Regulação para Cima
6.
Physiol Genomics ; 49(4): 201-215, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28159858

RESUMO

Developmental exposure of turtles and other reptiles to endocrine-disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), can stimulate partial to full gonadal sex-reversal in males. We have also recently shown that in ovo exposure to either EDC can induce similar sex-dependent behavioral changes typified by improved spatial learning and memory or possibly feminized brain responses. Observed behavioral changes are presumed to be due to BPA- and EE-induced brain transcriptomic alterations during development. To test this hypothesis, we treated painted turtles (Chrysemys picta) at developmental stage 17, incubated at 26°C (male-inducing temperature), with 1) BPA (1 ng/µl), 2) EE (4 ng/µl), or 3) vehicle ethanol (control group). Ten months after hatching and completion of the behavioral tests, juvenile turtles were euthanized, brains were collected and frozen in liquid nitrogen, and RNA was isolated for RNA-Seq analysis. Turtles exposed to BPA clustered separately from EE-exposed and control individuals. More transcripts and gene pathways were altered in BPA vs. EE individuals. The one transcript upregulated in both BPA- and EE-exposed individuals was the mitochondrial-associated gene, ND5, which is involved in oxidative phosphorylation. Early exposure of turtles to BPA increases transcripts linked with ribosomal and mitochondrial functions, especially bioenergetics, which has been previously linked with improved cognitive performance. In summary, even though both BPA and EE resulted in similar behavioral alterations, they diverge in the pattern of neural transcript alterations with early BPA significantly upregulating several genes involved in oxidative phosphorylation, mitochondrial activity, and ribosomal function, which could enhance cognitive performance.


Assuntos
Compostos Benzidrílicos/toxicidade , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Etinilestradiol/toxicidade , Fenóis/toxicidade , Transcriptoma/efeitos dos fármacos , Tartarugas/genética , Animais , Feminino , Masculino
7.
Sci Total Environ ; 579: 1804-1814, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27932218

RESUMO

Bisphenol A (BPA) is a widely present endocrine disruptor chemical found in many household items. Moreover, this chemical can bioaccumulate in various terrestrial and aquatic sources; thereby ensuring continual exposure of animals and humans. For most species, including humans, diet is considered the primary route of exposure. However, there has been little investigation whether commercial-brands of dog foods contain BPA and potential health ramifications of BPA-dietary exposure in dogs. We sought to determine BPA content within dog food, whether short-term consumption of these diets increases serum concentrations of BPA, and potential health consequences, as assessed by potential hematological, serum chemistry, cortisol, DNA methylation, and gut microbiome changes, in dogs associated with short-term dietary exposure to BPA. Fourteen healthy privately-owned dogs were used in this study. Blood and fecal samples were collected prior to dogs being placed for two-weeks on one of two diets (with one considered to be BPA-free), and blood and fecal samples were collected again. Serum/plasma samples were analyzed for chemistry and hematology profiles, cortisol concentrations, 5-methylcytosine in lymphocytes, and total BPA concentrations. Fecal samples were used for microbiome assessments. Both diets contained BPA, and after two-weeks of being on either diet, dogs had a significant increase in circulating BPA concentrations (pre-samples=0.7±0.15ng/mL, post-samples=2.2±0.15ng/mL, p<0.0001). Elevated BPA concentrations positively correlated with increased plasma bicarbonate concentrations and associated with fecal microbiome alterations. Short-term feeding of canned dog food increased circulating BPA concentrations in dogs comparable to amounts detected in humans, and greater BPA concentrations were associated with serum chemistry and microbiome changes. Dogs, who share our internal and external environments with us, are likely excellent indicators of potential human health concerns to BPA and other environmental chemicals. These findings may also have relevance to aquatic and terrestrial wildlife.


Assuntos
Compostos Benzidrílicos/sangue , Exposição Dietética/análise , Disruptores Endócrinos/sangue , Contaminação de Alimentos/análise , Alimentos em Conserva/análise , Fenóis/sangue , Animais , Compostos Benzidrílicos/toxicidade , Cães/sangue , Disruptores Endócrinos/análise , Disruptores Endócrinos/toxicidade , Contaminação de Alimentos/estatística & dados numéricos , Animais de Estimação/sangue , Fenóis/toxicidade
8.
Reprod Fertil Dev ; 29(8): 1602-1612, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27569192

RESUMO

Our prior work showed that a novel microbiome resides in the seminal vesicles of wild-type and oestrogen receptor α (Esr1) knock-out mice and is impacted by the presence of functional Esr1 genes. The seminal fluid microbiome (SFM) may influence the health and reproductive status of the male, along with that of his partner and offspring. A high-fat diet (HFD) alters metabolites and other factors within seminal fluid and might affect the SFM. Adult (~15 weeks old) male mice were placed for 4 weeks on a control or high-fat diet and seminal fluid and fecal samples were collected, bacterial DNA isolated and subjected to 16s rRNA sequencing. Corynebacterium spp. were elevated in the seminal fluid of HFD males; however, Acinetobacter johnsonii, Streptophyta, Ammoniphilus spp., Bacillus spp. and Propionibacterium acnes were increased in control males. Rikenellaceae was more abundant in the fecal samples from HFD males. However, Bacteroides ovatus and another Bacteroides species, Bilophila, Sutterella spp., Parabacteroides, Bifidobacterium longum, Akkermansia muciniphila and Desulfovibrio spp. were greater in control males. Thus, short-term consumption of a HFD influences the seminal fluid and fecal microbiomes, which may have important health consequence for males and developmental origins of health and disease effects in resulting offspring.


Assuntos
Dieta Hiperlipídica , Microbioma Gastrointestinal/fisiologia , Sêmen/fisiologia , Animais , Fezes/microbiologia , Masculino , Camundongos
9.
Gut Microbes ; 7(6): 471-485, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27624382

RESUMO

Gut dysbiosis may result in various diseases, such as metabolic and neurobehavioral disorders. Exposure to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE), especially during development, may also increase the risk for such disorders. An unexplored possibility is that EDC-exposure might alter the gut microbial composition. Gut flora and their products may thus be mediating factors for the disease-causing effects of these chemicals. To examine the effects of EDCs on the gut microbiome, female and male monogamous and biparental California mice (Peromyscus californicus) were exposed to BPA (50 mg/kg feed weight) or EE (0.1 ppb) or control diet from periconception through weaning. 16s rRNA sequencing was performed on bacterial DNA isolated from fecal samples, and analyses performed for P0 and F1 males and females. Both BPA and EE induced generational and sex-dependent gut microbiome changes. Many of the bacteria, e.g. Bacteroides, Mollicutes, Prevotellaceae, Erysipelotrichaceae, Akkermansia, Methanobrevibacter, Sutterella, whose proportions increase with exposure to BPA or EE in the P0 or F1 generation are associated with different disorders, such as inflammatory bowel disease (IBD), metabolic disorders, and colorectal cancer. However, the proportion of the beneficial bacterium, Bifidobacterium, was also elevated in fecal samples of BPA- and EE-exposed F1 females. Intestinal flora alterations were also linked to changes in various metabolic and other pathways. Thus, BPA and EE exposure may disrupt the normal gut flora, which may in turn result in systemic effects. Probiotic supplementation might be an effective means to mitigate disease-promoting effects of these chemicals.


Assuntos
Bactérias/efeitos dos fármacos , Compostos Benzidrílicos/toxicidade , Modelos Animais de Doenças , Etinilestradiol/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Peromyscus/microbiologia , Fenóis/toxicidade , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Feminino , Masculino
11.
Sci Rep ; 6: 23027, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26971397

RESUMO

Bacteria harbored in the male reproductive system may influence reproductive function and health of the male and result in developmental origins of adult health and disease (DOHaD) effects in his offspring. Such effects could be due to the seminal fluid, which is slightly basic and enriched with carbohydrates; thereby, creating an ideal habitat for microbes or a potential seminal fluid microbiome (SFM). Using wild-type (WT) and estrogen receptor-alpha (ESR1) knockout (KO) male mice, we describe a unique SFM whose inhabitants differ from gut microbes. The bacterial composition of the SFM is influenced according to whether mice have functional Esr1 genes. Propionibacterium acnes, causative agent of chronic prostatitis possibly culminating in prostate cancer, is reduced in SFM of ESR1 KO compared to WT mice (P ≤ 0.0007). In certain genetic backgrounds, WT mice show a greater incidence of prostate cancer than ESR1 KO, which may be due to increased abundance of P. acnes. Additionally, select gut microbiome residents in ESR1 KO males, such as Lachnospiraceae and Christensenellaceae, might contribute to previously identified phenotypes, especially obesity, in these mutant mice. Understanding how genetics and environmental factors influence the SFM may provide the next frontier in male reproductive disorders and possibly paternal-based DOHaD diseases.


Assuntos
Bactérias/metabolismo , Receptor alfa de Estrogênio/deficiência , Microbiota/fisiologia , Sêmen/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Receptor alfa de Estrogênio/genética , Fezes/microbiologia , Firmicutes/fisiologia , Genótipo , Interações Hospedeiro-Patógeno , Masculino , Redes e Vias Metabólicas/genética , Camundongos Knockout , Microbiota/genética , Obesidade/genética , Obesidade/microbiologia , Propionibacterium acnes/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
12.
Curr Protoc Plant Biol ; 1(1): 197-215, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-31725988

RESUMO

Next-generation sequencing (NGS) technologies have revolutionized the study of genomics with an ever-expanding list of applications. RNA-Seq has emerged as a powerful method, applying transcriptome analysis to a wider range of organisms-most significantly, non-model organisms lacking prior genomic sequencing. Whereas an initial concern of NGS datasets was the potential limitation of short read lengths, short read sequences have been successfully employed in creation of de novo transcriptome assemblies that allow for subsequent mapping of reads for expression analysis. Prior genomic sequence knowledge is no longer a requirement for identification of functional transcriptional elements and for global gene expression characterization. Significant cost reductions in generating RNA-Seq data, and improvements in de novo assemblers, has allowed the analysis of transcriptomes in heretofore unsequenced plant species. These protocols describe standard methods for constructing RNA-Seq libraries to be sequenced on Illumina sequencing platforms for comprehensive transcriptome analysis. © 2016 by John Wiley & Sons, Inc.

13.
Genome Announc ; 3(3)2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25953181

RESUMO

The assembly and annotation of the draft genome sequences for Pseudoalteromonas strains ATCC BAA314, ATCC 700518, and ATCC 700519 reveal candidates for promoting symbiosis between Pseudoalteromonas strains and eukaryotes. Groups of genes generally associated with virulence are present in all three strains, suggesting that these bacteria may be pathogenic under specific circumstances.

14.
J Biomol Tech ; 26(1): 4-18, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25649271

RESUMO

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/normas , Análise de Sequência de RNA/normas , Animais , Sequência de Bases , DNA Complementar/genética , Humanos , Limite de Detecção , Camundongos , Poliadenilação , RNA/genética , Ratos , Padrões de Referência
15.
PLoS One ; 10(2): e0116704, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25675094

RESUMO

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.


Assuntos
Animais de Laboratório , Fezes/microbiologia , Camundongos Endogâmicos/genética , Camundongos Endogâmicos/microbiologia , Microbiota , Animais , Biodiversidade , Análise por Conglomerados , Feminino , Trato Gastrointestinal/microbiologia , Metagenoma , Camundongos , RNA Ribossômico 16S/genética
16.
Biol Reprod ; 71(6): 1991-2002, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15306552

RESUMO

One method to identify the factors that control ovarian function is to characterize the genes that are expressed in ovary. In the present study, cDNA libraries from fetal, neonatal, and prepubertal porcine ovaries, pubertal ovaries on different days of the estrous cycle (Days 0 [follicle], 5, and 12 [follicle and corpus luteum]), and follicles isolated from weaned sows (diameter, 2, 4, 6, and 8 mm) were constructed and sequenced. A total of 22 176 cDNAs were sequenced, of which 15 613 were of sufficient quality for clustering. Clustering of cDNAs resulted in 8507 contigs, 6294 (74%) of which were comprised of a single sequence. Sixty-eight percent of the contigs had consensus sequences that were homologous to existing Tentative Consensus (TC) sequences or mature transcripts (ET) in The Institute for Genomic Research Porcine Gene Index. The consensus sequences were classified according to the Gene Ontology Index. Most cDNA-encoded proteins were components of the nucleus, ribosome, or mitochondrion. The proteins primarily functioned in binding, catalysis, and transport. Nearly 75% of the proteins were involved in metabolism and cell growth and/or maintenance. Analysis of the cDNA frequency across different libraries demonstrated differential gene expression within different-size follicles, between follicles and corpora lutea, and across developmental time-points. The expression of selected genes (analyzed by ribonuclease protection assay and Northern blotting) was consistent with the frequency of their respective cDNA in the individual libraries. This porcine ovary unigene set will be useful for identifying factors and mechanisms controlling ovarian follicular development in a variety of species.


Assuntos
Etiquetas de Sequências Expressas/metabolismo , Ovário/metabolismo , Suínos/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Northern Blotting , Mapeamento de Sequências Contíguas , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Biblioteca Gênica , Família Multigênica , Ovário/embriologia , RNA Mensageiro/metabolismo , Maturidade Sexual/fisiologia
17.
Biol Reprod ; 71(4): 1230-43, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15175238

RESUMO

Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3' end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5' end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.


Assuntos
Desenvolvimento Embrionário/genética , Etiquetas de Sequências Expressas , Biblioteca Gênica , Família Multigênica/genética , RNA Mensageiro/análise , Suínos/embriologia , Suínos/genética , Animais , Blastocisto/fisiologia , Feminino , Técnicas In Vitro , Oócitos/fisiologia , Distribuição Aleatória
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