PARENT SESSION
SLIDE SESSION 16: FERTILIZATION
Chairs: William Breed, Nuch Tanphaichitr, Becky Sortini (Trainee)
Univ Ottawa-UCU Auditorium
1:30 PM-3:30 PM
436
PORCINE OOCYTE PROTEINS ARE ASSOCIATED WITH ISOLATED SOMATIC CELL NUCLEI IN A CELL-FREE INCUBATION SYSTEM.
Novak, Susan1, Savard, Christian1, Sirard, Marc-Andre1, 1
ABSTRACT- One of the major obstacles to be overcome for the success of cloning using nuclear transfer is the effective reprogramming of the differentiated cell nucleus by the oocyte cytoplasm. Our objective was to investigate which oocyte proteins are transported into the somatic cell nucleus, and thus may participate in reprogramming the cell nuclei. Porcine oocytes were collected and selected from follicles 2-6 mm in size from prepubertal gilt ovaries. Denuded GV stage and in vitro matured oocytes (n=200 each) were lysed in the presence of proteinase inhibitors by repeated freeze-thaw in liquid nitrogen. After centrifugation to remove cell debris, oocyte proteins were labelled using NHS-LC-Biotin (Pierce) that allows them to pass through cell membranes once labelled. Unreacted biotin was removed by repeated washing steps through an ultracentrifugation filter (10 000 MW cutoff), and the labelled proteins recovered in a collecting buffer. After 3 days of serum starvation, cultured porcine fibroblast nuclei were isolated using a Nuclei-Pure Prep kit (Sigma). The nuclei (300) were then incubated with labelled proteins (10ul) in the presence of an energy-regenerating system for 1.5 h at room temperature. The nuclei were washed twice with a storage buffer, and along with pre-incubation labelled oocyte proteins separated on SDS-PAGE gels, transferred onto membranes and immunodetected by Neutravidin-HRP (Pierce). This experiment was repeated 3 times, and results were consistent across replicates. Five labelled oocyte proteins from GV stage and in vitro matured oocytes were detected in the incubated nuclei, ranging in size from 30 to 129 kDa. Three of those proteins were consistently detected in both GV stage and in vitro matured oocytes. These preliminary results show that several oocyte proteins become associated with the nuclei of somatic cells, and may ultimately be responsible for the reprogramming process of somatic cells during nuclear transfer procedures. Project supported by CORPAQ.
KEY WORDS: pig, oocyte proteins, nuclear reprogramming