Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes

Abstract

NBD-phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform. When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

“…Additionally, the in vitro fertilization rate was similar for frozen and fresh oocytes (70% versus 81%). In other studies, there was successful cryopreservation of oocytes of several species, with live births reported for mice [36], rabbits [37], and cows [1]. The success obtained in these experiments may be due to the cryopreservation protocols being more appropriate for the developmental stage of the oocytes and the preservation forms used (oocytes without cumulus cells), as well as to intrinsic characteristics of each species.…”

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle—stage mouse oocytes

“…However, the success rate of human oocyte cryopreservation is still low. Achievement of a clinically feasible technique requires further research to better understand and overcome cryopreservationassociated stresses and problems such as spindle disorganization (1-3), disruption of cytoskeletal elements (3,4), and increased polyploidy (5)(6)(7).…”

“…Studies using metaphase II (MII) human (10,11), mouse (1,12), cow (2), and rabbit (4) oocytes showed that cooling, cryoprotectants, or freezing and thawing cause depolymerization of the MII spindle microtubules with a consequence of chromosomal scattering. In addition, exposure of MII oocytes to cryoprotectants or a freeze-thaw cycle disrupts microfilament network and induces formation of several cytoplasmic microtubular asters or network (3,4). Although these alterations in microfilament and cytoplasmic microtubule organization of MII oocytes can be reversed by a subsequent incubation, changes in the spindle morphology are partially irreversible (3,4,12).…”

“…In addition, exposure of MII oocytes to cryoprotectants or a freeze-thaw cycle disrupts microfilament network and induces formation of several cytoplasmic microtubular asters or network (3,4). Although these alterations in microfilament and cytoplasmic microtubule organization of MII oocytes can be reversed by a subsequent incubation, changes in the spindle morphology are partially irreversible (3,4,12). The partially irreversible damage seemed to contribute to increased digyny after fertilization (3).…”

The potential for gamete recovery from non-domestic canids and felids

“…It has already been demonstrated that the cryopreservation of oocytes originating from antral follicles is successful in mice (Carrol and Gosden, 1993), rabbits (Vicent et al, 1989) and bovine (Fuku et al, 1992), followed by IVF after thaw, resulting in the birth of normal offspring in all of these species. In domestic felines, Luvoni and Pellizzari (2000) demonstrated that the mature oocyte could be cryopreserved and, soon after, fertilized in vitro with success.…”