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

A culture system has been designed in which enzymatically isolated oocyte-g ranulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue c an be grown to antral size in vitro. Oocyte-granulosa complexes ranging fro m 100 to 240 mu m in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 mu m general ly 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 mu m on the day of isolation formed antral ca vities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. Th e effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. in cultures suppleme nted with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral caviti es (18 +/- 7%). However, antrum formation was significantly less frequent i n cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulo sa cell complexes maintained a three-dimensional structure throughout cultu re and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antr um formation in serum-free medium containing testosterone. Oocytes recovere d after 30 days of culture were viable and had increased in diameter from 7 8 +/- 2 mu m on the day of isolation, to 131 +/- 3 mu m at the end of cultu re. These results show that oocyte-granulosa cell complexes isolated from c ryopreserved ovarian tissue can be grown to antral size in vitro with simil ar efficiency to those isolated from fresh tissue.