title: Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by laser capture microdissection.
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.
authors.affiliation: INRA, UMR444 Génétique Cellulaire, Auzeville, Castanet-Tolosan, France. agnes.bonnet@toulouse.inra.fr
authors.forename: Agnes
authors.initials: A
authors.lastname: Bonnet
authors.forename: Claudia
authors.initials: C
authors.lastname: Bevilacqua
authors.forename: Francis
authors.initials: F
authors.lastname: Benne
authors.forename: Loys
authors.initials: L
authors.lastname: Bodin
authors.forename: Corinne
authors.initials: C
authors.lastname: Cotinot
authors.forename: Laurence
authors.initials: L
authors.lastname: Liaubet
authors.forename: Magali
authors.initials: M
authors.lastname: Sancristobal
authors.forename: Julien
authors.initials: J
authors.lastname: Sarry
authors.forename: Elena
authors.initials: E
authors.lastname: Terenina
authors.forename: Patrice
authors.initials: P
authors.lastname: Martin
authors.forename: Gwenola
authors.initials: G
authors.lastname: Tosser-Klopp
authors.forename: Beatrice
authors.initials: B
authors.lastname: Mandon-Pepin
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D002469:Cell Separation; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism*; D061346:Laser Capture Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012756:Sheep / Q000235:genetics* / Q000254:growth & development
chemical_list: D012313:RNA