Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

In vivo gene expression in granulosa cells during pig terminal follicular development.

ABSTRACT: The aim of this study is the identification of genes and gene networks involved in pig ovarian follicular development. cDNA nylon micro-arrays (2849 sequenced clones) were designed from different libraries : four subtractive suppressive hybridization libraries (generated from small versus large and small versus medium follicles) and a pig multi-tissue cDNA library. Granulosa cells were isolated from healthy follicles (small, medium or large), 24 h or 96 h after the end of a progestagen treatment. The RNA isolated from these cells was used to hybridize the micro-arrays and hybridisations were performed in duplicate. The images were quantified using Bzscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Keywords: cell type comparison Granulosa cells were isolated from healthy follicles (small, medium or large). The RNA isolated from these cells was used to hybridize nylon micro-arrays. The arrays contained PCR products from 2848 pig cDNA clones (GPL3978), spotted in duplicate on two separate fields of the same membrane (last number 1 of the local ID corresponds to the left field and 2 corresponds to right field). The data have been generated from 14 RNA follicle pools (five Large, five Medium and four Small follicles pools). For each follicle pool, 2 radioactive labellings were performed (before last number of the local ID corresponds to the duplicate number). Each membrane was exposed 6, 12 or 24 hours (depending on the saturation signal) to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 µm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The images were quantified using semi-automated software, BZScan (Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. BMC Genomics 5: 38, 2004). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. A new version of BZsan (AGscan solfware) is available at : http://mulcyber.toulouse.inra.fr/projects/agscan/ The experimental data were managed using the free BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002.) modified by Sigenae team to accept radioactive experiments. Finally, data consisted in (14 RNA x 2 labellings x 2 fields) 14 probes, 28 hybridisations, 42 images (14 images were filtred out for background too hight).

ORGANISM(S): Sus scrofa

SUBMITTER: Agnes Bonnet

PROVIDER: E-GEOD-5798 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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In vivo gene expression in granulosa cells during pig terminal follicular development.

Bonnet A A Lê Cao K A KA Sancristobal M M Benne F F Robert-Granié C C Law-So G G Fabre S S Besse P P De Billy E E Quesnel H H Hatey F F Tosser-Klopp G G

Reproduction (Cambridge, England) 20080502 2

Ovarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive vi ...[more]

PMID: 18456903

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Project description:These vector oligonucleotide hybridizations were performed to determine the quality of the spotting process. EST with low signal values of the vector array were excluded from the analysis, since they probably were mis-amplified or mis-spotted Keywords: spotting control cDNA arrays (GPL3978) were first hybridized with a vector oligonucleotide labeled (T7 probe) with 33P-ATP. After washing, the arrays were exposed 6 hours to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 µm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The images were quantified using semi-automated software, BZScan (Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. BMC Genomics 5: 38, 2004). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. A new version of BZsan (AGscan solfware) is available at : http://mulcyber.toulouse.inra.fr/projects/agscan/ The experimental data were managed using the free BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002.) modified by Sigenae team to accept radioactive experiments. After stripping, the arrays were hybridized with a complex target. The data correspond to the hybridization of the first membrane DEV-1 and the last one DEV-20. The arrays contained PCR products from 2848 pig cDNA clones (GPL3978), spotted in duplicate on two separate fields of the same membrane (last number 1 of the local ID corresponds to the left field and 2 corresponds to right field). Finally, data consisted in four data files and were used to delete clones with a bad quality of spotting.

Project description:Folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration (a process called atresia). Even if atresia involves an apoptosis process, this mechanism is not well understood. The objective of this experiment was : 1) to analyse gene expression in pig granulosa cells of ovarian follicles during atresia using transcriptome analysis with a 9 024 cDNAs microarray, 2) the identification of gene networks involved in pig ovarian follicular atresia. Granulosa cells were isolated from atretic follicles (small, medium or large). Gene expression was analysed by hybridization of nylon cDNA microarrays. The images were quantified using Bzscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Keywords: pig ovary, folliculogenesis, atresia, gene expression, cDNA microarray, bio-analysis Calibrated follicles were isolated from porcine ovaries after ovariectomy (24h ou 96h after the end of progestagen treatment: Regumate 18 days, 20 mg/days). The follicular status was determined by histological criteria using Feulgen coloration (Monget et al. 1993, Besnard et al. 1996). Healthy follicle is characterized by frequent mitosis and pyknosis absence in granulosa cells. Presence of frequent pyknotic bodies in granulosa cells and no mitosis were allocated to atretic cells. Two cross-classified biological factors of pig ovarian follicles are considered : 1) the stage of development of the follicular growth : small (1-2 mm of diameter), medium (3 mm) and large (>5 mm) follicles; 2) the follicular status: healthy (S1) vs beginning (A2) or advanced (A3) atresia. Gene expression was analysed by hybridization of nylon cDNA microarrays (GPL3729). After hybridisation the data analysis using BASE software and statistical analysis was performed to identify regulated genes. For each of three follicle class and three follicle status combination 4 biological replicates were considered. Total 36 membranes were first hybridized with a vector oligonucleotide labeled (T7 probe 5'-taatacgactcactataggga-3') with gamma33P ATP at 42Â°C during 12h to determine the quality of the spotting process. After being washed, the arrays were exposed 6 or 24 hours to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 ÃÂµm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). Data were acquired with help of a microarray image processing software - AGScan (Cathelin, 2006, http://mulcyber.toulouse.inra.fr/projects/agscan/). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. Arrays images and data were stored using BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002) adapted by Sigenae (http://www.sigenae.org) for analysis and storage of radioactitive microarray data

Project description:In mammals, ovarian folliculogenesis leading to the ovulation of completely mature oocytes is a long and complex process that is regulated at different levels. The mechanisms that underlie the selection of one or several dominant follicles as well as the regulation of the number of ovulating follicles are largely unknown. In this project, we proposed to study the genetic determinism that underlies the difference of ovulation rate between species (cattle and pigs), by studying two processes that exist in ovary: follicular development and follicular atresia. Towards this purpose, we made a comparative transcriptomics study on granulosa cells. Pig and cattle comparison was achieved by a transcriptome analysis with a 9K nylon pig microarray (GPL3729) on granulosa cells from either small healthy antral follicles (SHF), small atretic follicles (SAF) or large healthy antral follicles (LHF). The images were quantified using AGscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Transcriptomic analysis on pig and cattle evidenced 997 differentially expressed genes (FDR1%) between the three follicle classes and/or the two species. This research project which implicated three laboratories from INRA: "Laboratoire de Genetique Cellulaire" (UMR444-LGC) , "Station d'Amelioration Genetique des Animaux" (UR 631-SAGA) and "Physiologie des Comportements et de la reproduction" (UMR 85-PRC) benefited from both European funding through SABRE project and French ANR funding through GenOvul project. Keywords: transcriptome analysis, pig, cattle, ovary, folliculogenesis, gene expression, cDNA microarray The data were obtained from 34 RNA samples: 10 small healthy follicles samples (6 for sows and 4 for cows), 13 small atretic follicles samples (7 for sows and 6 for cows) and 11 samples for large healthy follicles (6 for sows and 5 for cows). They were hybridized on a 9K pig nylon microarray (GPL3729).

Project description:Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. Late vitellogenesis: 3 samples Post-vitellogenesis: 4 samples Oocyte maturation (germinal vesicle breakdown): 6 samples

Project description:Background - Mammalians gamete production takes place in the testis but when they exit this organ, although spermatozoa have a specialized and distinct morphology, they are immotile and infertile. It is only after their travel in the epididymis that sperm acquire their motility and fertility. Epididymis can be divided in three gross morphological regions, head (caput), body (corpus) and tail (cauda), containing a long and unique convoluted tubule connecting the testis to the vas deferens. Results - In this study, the testis, the efferent ducts (vas efferens, VE), nine distinct successive epididymal segments and the deferent duct (vas deferens, VD) of four adult boars of known fertility were isolated and their mRNA extracted. The gene expression of each of these samples was analyzed using a pig generic 9K nylon microarray (AGENAE program; GEO accession number: GPL3729) spotted with 8931 clones derived from normalized cDNA banks from different pig tissues including testis and epididymis. Differentially expressed transcripts were obtained with moderated t-tests and F-tests and two data clustering algorithms based either on partitioning around medoÃ¯ds (top down PAM) or hierarchical clustering (bottom up HCL) were combined for class discovery and gene expression analysis. Tissue samples analysis defined seven transcriptomic units: testis, vas efferens and five epididymal transcriptomic units. Meanwhile transcripts formed only four clusters related to the tissues. We have then used a specific statistical method to sort out genes specifically overexpressed (markers) in testis, VE or in each of the five transcriptomic units of the epididymis (including VD). Among these markers some well-known epididymal genes were retrieved while some were new genes or genes not yet reported in these boar tissues. The specific regional expression of some of these genes was further validated by PCR and Q-PCR. We also searched for specific pathways and functions using available gene ontology information. Conclusions - This study fulfilled the gap between those done in rodents and human, and provides tools that will be useful for further studies on the biochemical processes responsible for the formation and maintain of the epididymal regionalization and the development of a fertile spermatozoa. Keywords: tissue type comparaison 96 samples - 12 tissue samples from 4 boars

Dataset Information

In vivo gene expression in granulosa cells during pig terminal follicular development.

Publications

In vivo gene expression in granulosa cells during pig terminal follicular development.

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