Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes

• Published in: Domestic animal endocrinology Vol. 42; no. 1; pp. 50 - 62
• Main Authors: Nagyova, E., Scsukova, S., Nemcova, L., Mlynarcikova, A., Yi, Y.J., Sutovsky, M., Sutovsky, P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2012
• Subjects: Animals; Cell Adhesion Molecules - biosynthesis; Cumulus Cells - drug effects; Cumulus Cells - metabolism; Cysteine Proteinase Inhibitors - pharmacology; extracellular matrix; Extracellular Matrix - drug effects; Extracellular Matrix - metabolism; Female; fluorescent antibody technique; follicle-stimulating hormone; granulosa cells; HAS2; Leupeptins - pharmacology; luteinizing hormone; Oocyte-cumulus complex; Oocytes - drug effects; Oocytes - metabolism; preovulatory period; Progesterone; Progesterone - biosynthesis; Proteasome; Proteasome Endopeptidase Complex - metabolism; Proteasome Inhibitors; protein metabolism; proteins; proteolysis; Real-Time Polymerase Chain Reaction - veterinary; reverse transcriptase polymerase chain reaction; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; secretion; steroidogenesis; Swine; TNFAIP6; tumor necrosis factors; Ubiquitin; Ubiquitin; HAS2; Oocyte-cumulus complex; Progesterone; TNFAIP6; Proteasome
• ISSN: 0739-7240; 1879-0054
• DOI: 10.1016/j.domaniend.2011.09.003

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of
TNFAIP6 and
HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of
TNFAIP6 and
HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway–regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.