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In vitro growth of oocyte–granulosa cell complexes isolated from cryopreserved ovine tissue

A culture system has been designed in which enzymatically isolated oocyte–granulosa cell complexes from fresh and frozen–thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte–granulosa complexes ranging from 100 to 240 µm in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 pm generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen–thawed tissue and measuring 190–240 µm on the day of isolation formed antral cavities in 25 ± 9% and 18 ± 6% (mean ± SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen–thawed oocyte–granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte–granulosa cell complexes that formed antral cavities (18 ± 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 ± 4%). All oocyte–granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 ± 2 µm on the day of isolation, to 131 ± 3 pm at the end of culture. These results show that oocyte–granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.