This study was carried out to evaluate the effects of glucose and sodium phosphate on in vitro development of porcine oocytes matured and fertilized in vitro. When the culture medium was supplemented with various concentrations of glucose, the higher proportions (23 and 26%) of oocytes developed to morular or blastocyst stages were at the concentrations of 2.78 and 5.56 mM than 0 (9%; P<0.05) and 11.12 mM (18%). In experiment to evaluate effect of sodium phosphate during in vitro development of porcine oocytes, a significantly (P<0.05) higher proportions of embryos developed to morular or blastocyst stages was obtained with sodium phosphateof 0.28 (25%) and 0.53 (27％) mM than 0 (15%), 1.05 (19%) and 2.10 (10%) mM. On the other hand, when oocytes were cultured in medium with (0.53 mM) sodium phosphate, the proportions of developed embryos were significantly (P<0.05) higher in medium without (29%) that than with (14%) 5.56 mM glucose. However, a higher proportion of embryos developed to morular or blastocyst stages were obtained in medium with (23%) that than without (8%) glucose (P<0.05). The minimum essential medium (MEM) added to the culture medium were higher regardless of presence of sodium phosphate and glucose on the development of embryos. Although sodium phosphate and glucose could support morular and blastocyst development to a limited extend (10∼24%), significantly higher proportion (36%) at morular or blastocyst stages was obtained by MEM adding in the medium with sodium phosphate and glucose. These results suggest that the early development of in vitro fertilized porcine oocytes can be maintained efficiently by glucose and sodium phosphate when they were cultured in medium with MEM.

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants (△24/83 and △38/83) and triple mutant (△24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.