In vitro maturation of bitch oocytes: effect of sperm penetration.

In contrast to most mammals, bitches ovulate immature oocytes at the germinal vesicle stage. Spermatozoa can be present in the oviduct of bitches at ovulation. The aim of this study was to investigate the influence of premature fertilization on the resumption of meiosis. In addition, the relative behaviour of male and female chromatin was observed. Canine cumulus-oocyte complexes cultured in medium 199 supplemented with 20% fetal calf serum for 24 h were incubated in vitro in the presence or absence of fresh dog semen for 24 h, and then rinsed and cultured in the same medium for a further 24 h. Chromatin was stained by propidium iodide and all oocytes were examined under laser scanning confocal microscopy. Results show that sperm penetration can occur in vitro in immature oocytes and that this induces a resumption of meiosis: at 72 h, the percentage of oocytes at the germinal vesicle stage decreased significantly and the percentage of oocytes beyond metaphase I increased. However, premature fertilization was ineffective for 40% of oocytes, which remained at the germinal vesicle stage. Measurement of chromatin areas in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage, indicating that male and female chromatins are sensitive to the same cell cycle regulators.