(W411) DEVELOPMENT OF MULTIDRUG RESISTANCE TYPE I P-GLYCOPROTEIN FUNCTION DURING IN VITRO MATURATION OF PORCINE OOCYTE.
Arai, Manabu1, Yamauchi, Nobuhiko1, Fukuda, Hiroaki1, Soh, Tomoki1, Hattori, Masa-aki1, 1 Kyushu University, Fukuoka, Japan
ABSTRACT- Multidrug transporter P-glycoprotein (P-gp) is an energy-dependent efflux pump that exports its substrates out of the cell. It is coded with the multidrug resistance type I (MDR1) gene in humans, which belongs to the ATP-binding cassette transporter superfamily. P-gp is considered to function as a defense mechanism against natural xenotoxics, as well as anticancer drugs in normal tissues. The present study was focused on the development of MDR1 gene-dependent defense in porcine oocytes, and performed to investigate the expression and function of MDR1 during in vitro oocyte maturation. The expression and function of this gene during in vitro oocyte maturation were analyzed by means of reverse transcription-polymerase chain reaction (RT-PCR) analysis and the rhodamine 6G efflux from oocytes as a marker of MDR1-type P-gp function. Cumulus-oocyte complexes were cultured for 0 h, 3 h, 28 h and 44 h in the maturation medium to obtain the germinal vesicle (GV), first metaphase and second metaphase (MII) stage oocytes. The P-gp function was assessed by means of the rhodamine 6G efflux from oocytes with P-gp inhibitors such as verapamil and PSC-833, estradiol or bisphenol A. RT-PCR analysis using primer sets based on the human cDNA sequence revealed the expression of MDR1 in the GV and MII stage oocytes, and its partial cDNA was very similar to that of human cDNA ranged from nucleotide number 2282 to 2783, with 87.6% homology in nucleotide. Verapamil, an inhibitor of calcium pump, and PSC-833 significantly inhibited the rhodamine 6G efflux from the MII stage oocytes, whereas estradiol and bisphenol A were ineffective. In the GV stage oocyte, however, all the reagents were ineffective in the rhodamine 6G efflux. The rhodamine 6G efflux from oocytes was detected at the MII stage more than at the GV stage. Thus, it was proved that MDR1 is expressed in porcine oocytes through the first meiosis stage, but that function of MDR1-type P-gp is poor at the GV stage. However, the MDR phenotype functions in the MII stage oocyte.
KEY WORDS: MDR type 1 gene, oocyte maturation, rhodamine 6G efflux, self-defense