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Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan 606
In a previous study, it was shown that nuclear maturation from the germinal vesicle stage to division of the first polar body in rat oocytes could be completed in a defined medium, but that the proportion of oocytes penetrated and cleaved was low (Niwa & Chang, 1975), perhaps because of some fault in the maturation process. The present study was to determine the number of oocytes penetrated and cleaved in vitro when they were cultured at various stages during maturation in vivo.
White rats of the Wistar strain were used. Immature females (21-25 days old) were induced to superovulate by a s.c. injection of 10 i.u. PMSG (Serotropin: Teikoku-Zoki Co.) 48 hr before an i.p. injection of 10 i.u. HCG (Puberogen: Sankyo Co.). Oocytes were recovered at various times after the HCG injection according to the procedures described
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Preantral follicles containing oocytes of 70–89.5 μm in diameter were isolated from pig ovaries and cultured in collagen gel for up to 16 days, in the presence of serum, FSH and oestradiol. Formation of follicular antra occurred as the culture proceeded. The oocytes had been enclosed by granulosa cells and contacts between the oocytes and processes of the enclosing cumulus cells were maintained over the culture period. After 16 days of culture, 30–40% of the oocytes were of normal appearance, and the diameters of about half of these oocytes were larger than 100 μm. When the oocytes grown in vitro were liberated from the follicles and cultured for a further 48 h in modified Krebs–Ringer bicarbonate solution, 6, 30 and 60% of the oocytes larger than 90, 100 and 110 μm underwent germinal vesicle breakdown, respectively. Progression to metaphase II was observed in 40% of oocytes that were over 110 μm in diameter, whereas no oocyte less than 90 μm in diameter resumed meiosis. The relationship between the size and meiotic competence of oocytes was similar for oocytes grown in vitro or in vivo. Oocytes grown and matured in vitro were penetrated by spermatozoa and formed a female pronucleus, but decondensation of the sperm head was incomplete. The results demonstrate for the first time that pig oocytes from preantral follicles can grow up to their final size, acquire meiotic competence, and be penetrated by spermatozoa in vitro.
