Growth and development of rat oocytes in vitro

Rat oocytes from preantral follicles have been shown to grow and acquire meiotic competence in vitro. Follicles were isolated by enzymatic digestion of ovaries from infant (10- or 11-day-old) Wistar rats. Follicles were cultured for up to 20 days in Minimum Essential Medium (MEM) supplemented with 2 mM hypoxanthine to maintain meiotic arrest. When cultures were begun, oocytes were in midgrowth phase (40-45 microns diameter), and were incapable of undergoing meiotic maturation when placed in hypoxanthine-free MEM. Oocytes grew and acquired meiotic competence during culture for 20 days attaining mean diameters of 62.6 +/- 0.6 microns and 61.1 +/- 0.6 microns in two experiments. Germinal vesicle breakdown (GVB) occurred in 60-70% of oocytes when transferred to MEM without hypoxanthine. Concomitant with oocyte growth and maturation were spontaneous increases in follicular production of progestins, androgens and estrogens. When oocytes grown and matured in this system were inseminated in vitro with epididymal sperm, 36.1% and 25.8% were penetrated by one or more sperm in two experiments. However, fertilization was not generally normal with multiple penetrations and abnormal numbers of pronuclei (one or three) being common, suggesting that in these oocytes cytoplasmic maturation was incomplete or abnormal. In the two experiments, normal fertilization (two pronuclei and one sperm tail in the vitellus) occurred in 34.6% and 47.1% of penetrated oocytes with development of these apparently normal zygotes to two-cell embryos being 66.7% and 62.5%, respectively.