Dissociation and preservation of preantral follicles and immature oocytes from female dasyurid marsupials

Abstract

The mammalian ovary contains numerous immature preantral follicles that are not dependent on endocrine support, unlike the more mature hormone-dependent antral follicles. Preantral follicles can be enzymatically dissociated to yield immature oocytes that survive sub-zero preservation better as they lack a temperature-sensitive meiotic spindle. These techniques are highly applicable to gamete banking, which is an urgent requirement for Australian carnivorous marsupials as several species have rapidly declining populations and risk extinction. The present study developed protocols for the transport, dissociation, preservation and culture of granulosa cell-oocyte complexes (GOC) from the ovaries of dasyurid marsupials. High viability of GOC following enzymatic dissociation is reported and it was demonstrated that GOC are of significantly better quality following refrigerated storage for 24 h compared with storage at room temperature. Oocytes from primary follicles were not damaged by cold shock or the toxicity of vitrification media and following vitrification in liquid nitrogen 69.42+/-2.44% of oocytes were viable. However, the surrounding granulosa cells demonstrated significant damage post-thaw. These granulosa cells proliferated during a 48-h culture period resulting in significant improvements in GOC quality. The present study is a valuable step towards cryostorage of dasyurid gametes and represents fundamentally important methods by which we can contribute to the conservation of Australia's native predators.

Fertility preservation through gonadal cryopreservation

“…Cat [135,215,216] Cortical tissue [135,216] Isolated follicles [215] CSF Ultra-structural analysis [216] Xenotransplantation [135] IVC of isolated follicles for 1 week [215] Normal ultra-structure [216] Development of antral follicles [135] Follicular survival [215] No Horse [217] Cortical pieces Recovery of endocrine function [138,218] and antral follicle development [219] No Dasyurids [222] Isolated follicles Vitrification IVC and viability analysis…”

Genome Banking for Vertebrates Wildlife Conservation

“…Open systems were also used to vitrify granulosa-oocyte complexes (GOC) from primary follicles of marsupials. In two different studies the fat-tailed dunnart (Sminthopsis crassicaudata) (Czarny et al, 2009b) and the Tasmanian devil (Sarcophilus harrisii) (Czarny & Rodger, 2010) GOC were vitrified in self-made OPS. Post-warming viability was about 70% in both studies.…”

“…What unifies all these studies is the fact that only small number of oocytes were cryopreserved and only in vitro post thaw / warming evaluations were conducted. Immature oocytes seem to be less prone to damages caused by the chilling, freezing and thawing or warming procedures (Arav et al, 1996) and they, too, can be cryopreserved by slow freezing (Luvoni et al, 1997) or vitrification (Arav et al, 1993;Czarny et al, 2009b). Preantral oocytes can be preserved inside the follicle and about 10% seem to be physiologically active after thawing and one week of culture.…”

Mammalian reproduction out of cryopreserved cells and tissues: current state of the art and future options

“…Immature oocytes seem to be less prone to the damage caused by chilling, freezing and thawing, and they can be cryopreserved by controlled-rate freezing (Luvoni et al, 1997;Jewgenow et al, 1998;Jewgenow & Paris, 2006) or vitrification (Arav et al, 1993;Czarny et al, 2009). Preantral oocytes can be preserved inside the follicle and c. 10% seems to be physiologically active after thawing and 1 week of culture (Jewgenow et al, 1998;Nayudu et al, 2003).…”