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 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns 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 microns 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 microns on the day of isolation, to 131 +/- 3 microns 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.
Newton
H
H
Centre for Reproduction, Growth and Development, Leeds General Infirmary, UK.
Picton
H
H
Gosden
R G
RG
D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D004958:Estradiol / Q000096:biosynthesis; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D008954:Models, Biological; D009865:Oocytes; D011374:Progesterone / Q000096:biosynthesis; D012756:Sheep
D016895:Culture Media, Serum-Free; D011374:Progesterone; D004958:Estradiol