Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation.
Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.
Novak
Susan
S
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4.
Paradis
François
F
Savard
Christian
C
Tremblay
Karine
K
Sirard
Marc-André
MA
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System; D002678:Chimera; D019976:Cloning, Organism; D018920:Coculture Techniques; D003593:Cytoplasm / Q000378:metabolism*; D004527:Egg Proteins / Q000302:isolation & purification*; D005260:Female; D005786:Gene Expression Regulation; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D040901:Proteomics; D012333:RNA, Messenger / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D004527:Egg Proteins; D012333:RNA, Messenger