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Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

ABSTRACT: FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.

SUBMITTER: Bonnet A

PROVIDER: S-EPMC1533831 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

Bonnet A A Frappart P O PO Dehais P P Tosser-Klopp G G Hatey F F

Reproductive biology and endocrinology : RB&E 20060707

FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FS ...[more]

PMID: 16827936

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Project description:BACKGROUND:Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. RESULTS:We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. CONCLUSION:Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH. We focused our study on the transcripts we identified as the most interesting ones based on the BLAST results. Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found). These sequences will have to be better studied by a longer -or complete- sequencing to check their identity, and then by verifying the expression level of the transcripts in different parts of the worm.

Dataset Information

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

Publications

Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.

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