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Open Access Research article

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

Agnes Bonnet1*, Claudia Bevilacqua2, Francis Benne1, Loys Bodin3, Corinne Cotinot4, Laurence Liaubet1, Magali Sancristobal1, Julien Sarry1, Elena Terenina1, Patrice Martin2, Gwenola Tosser-Klopp1 and Beatrice Mandon-Pepin4

Author Affiliations

1 INRA, UMR444 Génétique Cellulaire, Auzeville, BP52627, F-31326 Castanet-Tolosan, France

2 INRA, UMR1313 Génétique Animale et Biologie Intégrative, Plateforme de Microgénomique expressionnelle ICE, F-78350 Jouy-en-Josas, France

3 INRA, UR631 Station d'Amélioration Génétique des Animaux BP52627, F-31326 Castanet-Tolosan, France

4 INRA, UMR1198 Biologie du Développement et de la Reproduction, F-78350 Jouy-en-Josas, France

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BMC Genomics 2011, 12:417  doi:10.1186/1471-2164-12-417

Published: 18 August 2011

Abstract

Background

Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.

The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.

Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.

Results

We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH).

A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.

Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.

Conclusions

The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.