In Vitro Growth of Preantral Follicles Isolated from Cryopreserved Ovine Ovarian Tissue

Abstract

In the present study, we compared the in vitro development of sheep preantral follicles obtained from unfrozen or frozen ovarian cortex. After thawing, follicles stored by a slow-freezing protocol with dimethyl sulfoxide (DMSO) or ethylene glycol (EG) were mechanically isolated and cultured for 10 days. After 1 day, approximately 50% and 34% of the DMSO and EG follicles, respectively, showed overt signs of degeneration, as confirmed by histological analysis. Follicles that survived thawing grew and formed antral-like cavities, without significant differences among experimental groups. However, the percentages of healthy oocyte-cumulus cell complexes (OCCs) retrieved from in vitro-grown follicles, as well as estradiol, were lower in DMSO than in EG or unfrozen follicles. Although cryopreservation did not cause appreciable differences in follicle morphological aspects, frozen OCCs showed lower metabolic cooperativity levels, as determined by [3H]uridine uptake. During culture, oocytes increased in diameter, but the percentage of germinal vesicle stage-arrested oocytes showing a rimmed chromatin configuration was significantly lower in the frozen groups. Our results indicate that cryopreserved sheep preantral follicles underwent growth in vitro but that freezing/thawing specifically affected gap junctional permeability and impaired the progression of regulative processes, such as the acquisition of a specific oocyte chromatin configuration. Moreover, because the cryoprotectant toxicity test excluded the occurrence of direct cellular damage, this method allowed us to discriminate the effects exerted by different cryoprotectants during the cryopreservation procedure on whole-follicular development.

Cryopreservation of swine ovarian tissue: Effect of different cryoprotectants on the structural preservation of preantral follicle oocytes

“…Cryopreservation of ovarian tissue proved to be effective for a wide range of species including sheep [3], [4], [10], [20] and [24], cattle [5], [16] and [21], goats [22] and [25] and humans [18] and [26].…”

Bio-banking: An Emerging Approach for Conservation of Fish Germplasm

“…Early studies on ovarian tissue cryopreservation were performed in mouse [37], and has been proven to be effective on other species such as sheep [37,38], goat [39] and pig. Ovarian tissue cryopreservation can be a viable alternative to cryopreservation of oocytes or embryos [40][41][42].…”

Preservação de fertilidade

“…De qualquer maneira não haveria a exposição ao risco de reimplantar células malignas. Outra possibilidade é o isolamento de folículos primordiais a partir do tecido congelado-descongelado, utilizando-se técnicas de digestão enzimática 37,38 ou por dissecção mecânica 39,40 , seguido de cultivo para maturação in vitro. As téc-nicas atualmente propostas para maturação in vitro (MIV) de folículos, e que vêm obtendo sucesso nos centros de reprodução assistida, partem de folículos imaturos, mas já em estádio de desenvolvimento mais avançado, ou de folículos antrais visualizados ao ultra-som e captados sem estimulação de ovulação prévia ou com baixas doses de gonadotrofi nas 41,42 .…”