title: Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density.
abstract: BACKGROUND
  In previous studies it has been shown that bovine granulosa cells (GC) cultured at a high plating density dramatically change their physiological and molecular characteristics, thus resembling an early stage of luteinization. During the present study, these specific effects on the GC transcriptome were comprehensively analysed to clarify the underlying mechanisms.
  
  
  METHODS
  GC were cultured in serum free medium with FSH and IGF-1 stimulation at different initial plating density. The estradiol and progesterone production was determined by radioimmunoassays and the gene expression profiles were analysed by mRNA microarray analysis after 9 days. The data were statistically analysed and the abundance of selected, differentially expressed transcripts was re-evaluated by qPCR. Bioinformatic pathway analysis of density affected transcripts was done using Ingenuity Pathway Analysis.
  
  
  RESULTS
  The data showed that at high plating density the expression of 1510 annotated genes, represented by 1575 transcript clusters, showed highly altered expression levels. Nearly two-thirds were up- and one third down-regulated. Within the top up-regulated genes VNN2, RGS2 and PTX3 could be identified, as well as HBA or LOXL2. Down-regulated genes included important key genes of folliculogenesis like CYP19A1 and FSHR. Ingenuity pathway analysis identified "AMPK signaling" as well as "cAMP-mediated signaling" as major pathways affected by the alteration of the expression profile. Main putative upstream regulators were TGFB1 and VEGF, thus indicating a connection with cell differentiation and angiogenesis. A detailed cluster analysis revealed one single cluster that was highly associated with the upstream regulator beta-estradiol. Within this cluster key genes of steroid biosynthesis were not included, but instead, other genes importantly involved in follicular development, like OXT and VEGFA as well as the three most down-regulated genes TXNIP, PAG11 and ARRDC4 were identified.
  
  
  CONCLUSIONS
  From these data we hypothesize that high density conditions induce a stage of differentiation in cultured GC that is similar to early post-LH conditions in vivo. Furthermore we hypothesize that specific cell-cell-interactions led to this differentiation including transformations necessary to promote angiogenesis.
authors.affiliation: Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196, Dummerstorf, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Baufeld
authors.affiliation: Institute for Immunology, University of Rostock, 18055, Rostock, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Koczan
authors.affiliation: Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196, Dummerstorf, Germany. vanselow@fbn-dummerstorf.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Vanselow
mesh_terms: D000818:Animals; D002417:Cattle; D002450:Cell Communication / Q000502:physiology*; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D005260:Female; D006107:Granulosa Cells / Q000502:physiology*; D040081:Protein Array Analysis / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D059467:Transcriptome / Q000502:physiology*
chemical_list: D012333:RNA, Messenger
keywords: Bovine; Cell density; Gene expression; Granulosa cells; Marker genes; Microarray; Signaling pathways