Current developments in IVF and animal cloning have resulted in increasing demand for large quantities of oocytes and ovarian follicles at specific stages of development. These medical and scientific needs may be met by developing an optimal culture system for preantral follicles. In this study, we investigated the growth of porcine preantral follicle cultures in different media and in the presence and absence of serum. Follicles were manually dissected from ovaries obtained from prepubertal gilts at a local slaughterhouse, and cultured for 3 days in M199 or NCSU23 medium supplemented with porcine FSH, transferrin, L-ascorbic acid and insulin. Follicle diameters were measured on day 1 and 3 of culture. In Experiment 1, the effect of supplementing culture medium with fetal calf serum (FCS) on porcine preantral follicle growth was examined. In the group of cultures supplemented with FCS, follicle diameter after 3 days of culture, survival rate and antrum formation rate in the FCS group were significantly higher than those of the control group. In Experiment 2, the effects of culture medium (M199 and NCSU23) on follicle growth were compared. Follicle diameters were increased in the M199 group, compared with those in NCSU23 (p<0.05), but we observed no significant differences in survival and antrum formation rates between cultures grown in the two media. In conclusion, supplementation of the culture medium with serum enhances preantral follicle growth and antrum formation, and M199 is superior to NUSU23 for porcine preantral follicle culture in vitro.

To examine the differential expression of proteins during the cycling (70～80% confluences) and G0/G1 (full confluences) phases in porcine fetal fibroblast cells, we used a global proteomics approach by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. Cycling cell were harvested at approximately 70% to 80% confluent state while cells in G0/G1 phase were recovered after maintenance of a confluent state for 48 hr. Cellular proteins with isoelectric points ranging between 3.0～10.0, were analyzed by 2‐DE with 2 replicates of each sample. A total of approximately 700 spots were detected by 2‐D gels stained with Coomassie brilliant blue. On comparing the cell samples obtained from the cycling and G0/G1 phases, a total of 13 spots were identified as differentially expressed proteins, of which 8 spots were up‐regulated in the cycling cell and 5 were up‐regulated in the G0/G1 phase. Differentially expressed proteins included K3 keratin, similar to serine protease 23 precursor, protein disulfide‐isomerase A3, microsomal protease ER‐60, alpha‐actinin‐2, and heat‐shock protein 90 beta. The identified proteins were grouped on the basis of their basic functions such as molecular binding, catabolic, cell growth, and transcription regulatory proteins. Our results show expression profiles of key proteins in porcine fetal fibroblast cells during different cell cycle status.

The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta, the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.