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

Abstract

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.

Human oocyte and ovarian tissue cryopreservation and its application

“…Studies in mice and sheep have shown that oocyte-granulose complexes isolated from the frozen-thawed ovarian tissue can grow to antral size [124,125]. The maturation of primordial follicles in vitro, which are capable of successful fertilization, has been demonstrated in a mouse model [126].…”

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

“…The vials were plunged directly into liquid nitrogen (−196 °C) and stored for up to 5 days before thawing. The freezing curve used in this study was chosen because most procedures currently used to cryopreserve oocytes [25], [26], [27], [28], [29] and [30] and ovarian tissue [10], [11], [12], [14], [15], [18], [24], [31], [32] and [33] stipulate a cooling rate of 0.3-0.5 °C/min from the seeding temperature (usually −5 to −9 °C) to a lower temperature, usually between −30 and −40 °C.…”

Carbohydrate metabolism by murine ovarian follicles and oocytes grown in vitro

“…The process of folliculogenesis involves many intricate and timely developmental events, which must be replicated in the laboratory. Avascular preantral follicles survive relatively well in vitro and culture of preantral follicles is possible in several species, including mouse (Nayudu & Osborn 1992, Boland et al 1993, Cortvrindt et al 1996, Rose et al 1999, sheep (Newton et al 1999, Picton et al 2003 and human (Roy & Treacy 1993, Picton & Gosden 2000. However, after long-term culture, oocyte viability is reduced and this is especially a problem in large animals and humans.…”