102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

Abstract

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins.