cDNA cloning of bovine midkine and production of the recombinant protein, which affects in vitro maturation of bovine oocytes

In the present study, we cloned bovine midkine (bMK) cDNA by RT- and RACE-P CR, and determined its nucleotide sequence. The nucleotide and deduced amin o acid sequences of bMK showed a high degree of homology to those of mouse and human MK. Moreover, a large amount of recombinant bMK (rbMK) was produc ed using a baculovirus expression system and the protein was purified to ho mogeneity by heparin affinity chromatography. To examine whether treatment with rbMK during in vitro maturation (IVM) of bovine cumulusenclosed oocyte s affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine cumulus-enclosed oocytes obtained from slaughterh ouse-derived ovaries were cultured for 24 hr in IVM medium without (control ) or with various concentrations (50-400 ng/ml) of rbMK, followed by in vit ro fertilization (IVF) and culture. Although rbMK treatment during IVM did not affect the rates of nuclear maturation and postfertilization cleavage o f oocytes, rbMK at all experimental concentrations significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared to the control. Next, it was examined whether heparitinase (HTN) treatment of cumulus-enclosed oocytes would affect the enhancing activity of rbMK du ring IVM on the developmental competence of oocyte after IVF. The enhancing activity of rbMK was completely suppressed by HTN (1.0 mU/ml) treatment. T hese results indicate that the presence of rbMK during IVM of bovine cumulu s-enclosed oocytes can enhance their developmental competence to the blasto cyst stage after IVF and suggest that heparan sulfate chains on the cell su rface of cumulus cells and/or oocytes are required for bMK to exert its eff ect. (C) 2000 Wiley-Liss, Inc.