RESEARCH ARTICLE Molecular Reproduction & Development 80:166–182 (2013) In Vitro Maturation of Oocytes Alters Gene Expression and Signaling Pathways in Bovine Cumulus Cells MOHAMAD SALHAB, 1,2,3,4 SOPHIE DHORNE-POLLET, 5 SYLVAIN AUCLAIR, 1,2,3,4 CATHERINE GUYADER-JOLY, 6 DAPHNE ´ BRISARD, 1,2,3,4 ROZENN DALBIES-TRAN, 1,2,3,4 JOELLE DUPONT, 1,2,3,4 CLAIRE PONSART, 6 PASCAL MERMILLOD, 1,2,3,4 AND SVETLANA UZBEKOVA 1,2,3,4 * 1 INRA, UR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France 2 CNRS UMR7247, Nouzilly, France 3 Universit e Franc ¸ois Rabelais, Tours, France 4 IFCE, Nouzilly, France 5 INRA, UMR1313 G en etique Animale et Biologie Int egrative, Jouy-en-Josas, France 6 UNCEIA, Maisons-Alfort, France SUMMARY In vitro maturation (IVM) of immature oocytes is widely used in assisted reproduction technologies in cattle, and is increasingly used to treat human infertility. The devel- opment competence of IVM oocytes, however, is lower than preovulatory, in vivo- matured oocytes. During maturation, cumulus cells (CC) are metabolically coupled with an oocyte and support the acquisition of its developmental potential. Our objective was to identify genes and pathways that were affected by IVM in bovine CC. Microarray transcriptomic analysis of CC enclosing in vitro- or in vivo-mature oocytes revealed 472 differentially expressed genes, including 28% related to apoptosis, correlating with twofold higher cell death after IVM than in vivo, as detected by TUNEL. Genes overexpressed after IVM were significantly enriched in functions involved in cell movement, focal adhesion, extracellular matrix function, and TGF-beta signaling, whereas under-expressed genes were enriched in regulating gene expres- sion, energy metabolism, stress response, and MAP kinases pathway functions. Differential expression of 15 genes, including PAG11 (increased) and TXNIP (decreased), which were never detected in CC before, was validated by real-time RT-PCR. Moreover, protein quantification confirmed the lower abundance of glu- tathione S-transferase A1 and prostaglandin G/H synthase 2, and the higher abun- dance of hyaluronan synthase 2 and SMAD4, a member of TGF-beta pathway, in CC after IVM. Phosphorylation levels of SMAD2, MAPK3/1, and MAPK14, but not MAPK8, were higher after IVM that in vivo. In conclusion, IVM provokes the hyper-activation of TGF-beta and MAPK signaling components, modifies gene expression, leads to increased apoptosis in CC, and thus affects oocyte quality. Mol. Reprod. Dev. 80: 166–182, 2013. ß 2013 Wiley Periodicals, Inc. Received 19 October 2012; Accepted 17 December 2012 * Corresponding author: INRA, UR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France. E-mail: svetlana.uzbekova@ tours.inra.fr Mohamad Salhab and Sophie Dhorne-Pollet contributed equally to this work. Grant sponsors: French National Research Agency and Apisgen (OSCILE ANR-08-GENM-033 and OVOGENAE2 ANR-07-GANI-004 programs) Published online 30 January 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22148 Abbreviations: CC, cumulus cells; COC, cumulus–oocyte complex; ECM, extracellular matrix; IVM, in vitro maturation; IVF, in vitro fertilization. Genes: AKT, protein kinase B; CAPG, capping actin filament protein; CRHBP, cortico- tropin releasing hormone binding protein; ERK, extracellular signal-regulated kinase; FOS, v-fos FBJ murine osteosarcoma viral oncogene homolog; FSHR, FSH receptor; GSTA1, glutathione S-transferase alpha 1; HAS2, hyaluronan synthase 2; MAPK, mitogen-activated protein kinase; ME3, malic enzyme 3; MMP9, matrix metallopeptidase 9; NOG, Noggin; PAG11, pregnancy asso- ciated glycoprotein; PDE, phosphodiesterase; PTGS2, prostaglandin G/H synthase and cyclooxygenase 2; SDC2, syndecan; SERPINA5, serpin pepti- dase inhibitor, clade A member 5; TGF-beta, transforming growth factor beta; TMSB10, thymosin beta-10; TXNIP, thioredoxin interacting protein. ß 2013 WILEY PERIODICALS, INC.