Effects of cyclophosphamide on maturation and subsequent fertilizing capacity of pig oocytes in vitro.

This study examines the effects of cyclophosphamide, a widely used anti-cancer agent, on the maturation of pig oocytes and on their subsequent fertilizing capacity in vitro. Pig cumulus-oocyte complexes collected from prepubertal gilts were cultured in Waymouth MB 752/1 medium supplemented with sodium pyruvate (50 micrograms/ml), luteinizing hormone (0.5 microgram/ml), follicle-stimulating hormone (0.5 microgram/ml), and 17 beta-estradiol (1 microgram/ml) in the presence or absence of cyclophosphamide for 24 hr; they then were cultured without hormonal supplements in the presence or absence of cyclophosphamide for an additional 16-24 hr. The breakdown of germinal vesicle (GVBD) and changes in glutathione (GSH) content before in vitro fertilization were assessed. Oocytes containing one polar body and a metaphase plate were regarded as matured. Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was also examined. Treatment of oocytes with increasing concentrations (1-1000 micrograms/ml) of cyclophosphamide for 48 hr resulted in a dose-response inhibition of the rate of maturation, but had no effect on GVBD. Increasing duration (12-48 hr) of treatment with cyclophosphamide (100 micrograms/ml) led to a time-dependent inhibition of nuclear maturation, achieving statistical significance by 24 hr. The addition of cyclophosphamide (100 micrograms/ml) to maturation medium immediately after culture, 12 hr or 24 hr after culture also decreased the percentage of oocytes matured during a 48-h culture period. Exposure of oocytes to cyclophosphamide (100 micrograms/ml) for 40 hr did not prevent sperm penetration, not affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus at 8 hr after insemination. The concentration of GSH, an important factor for male pronuclear formation, in pig oocytes was determined by an enzymatic cycling assay. The concentration found was 8.15 +/- 1.19 mM per oocyte. Exposure of oocytes to cyclophosphamide (100 micrograms/ml) had no effect on GSH concentration. These results demonstrate that cyclophosphamide directly inhibits the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect, apparently, is not mediated through a decrease in the level of intracellular glutathione.