Effects of Cryopreservation on the Meiotic Spindle, Cortical Granule Distribution and Development of Rabbit Oocytes

Abstract

Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation.

Live Birth from Slow-Frozen Rabbit Oocytes after In Vivo Fertilisation

“…While numerous reports of studies designed to investigate oocyte cryopreservation have been published in some species [64], few works have been performed in rabbit [10], [20]–[28] and only two recent works compared slow-freeze and vitrification methods [24], [26]. Moreover, live offspring were obtained only in one report, using the slow-freezing method [10].…”

“…To this end, different technologies for in vitro development of rabbit embryos have been assayed, such as in vitro fertilisation (IVF) [31], [32], intracytoplasmic sperm injection (ICSI) [23], [33]–[36], and parthenogenetic activation [24], [26], [37]–[39]. Although it seems possible, IVF has not been successful in rabbit and a repeatable IVF technique has not yet been developed, possibly due to the lack of an efficient in vitro capacitation system for rabbit spermatozoa linked to the poor permeability of sperm plasma membrane [40].…”

“…The success rate of rabbit ICSI is less than 4% [34], [35]. Some studies have employed parthenogenetic activation technique as an alternative tool to evaluate oocyte competence and in vitro development in rabbit [24], [26]. However, one substantial limitation to this technique is that it cannot obtain offspring.…”

Long-term cryopreservation of human oocytes does not increase embryonic aneuploidy

“…Given the widespread adoption of OC technology and its marked projected growth, improving our understanding of the potential risks to the oocyte and developing embryo is imperative. Animal and human studies have demonstrated slow cooling-and vitrification-induced damage to the meiotic spindle, and data suggest that cryoprotectants may induce chromatin contraction and potentially jeopardize chromosomal competence (10). However, studies suggest that metaphase spindles repolymerize, and chromatin properly realigns postcryopreservation, and we previously reported that 96% of oocytes demonstrating a visible spindle before freezing have one after thawing (23)(24)(25)(26).…”

“…The oocyte is particularly vulnerable to physical injury during the cryopreservation process, owing to its large volume and high water content, and cryopreservation causes alterations to the oocyte, including zona pellucida thickening and premature cortical granule exocytosis (4). Concerns have been raised regarding the risk of meiotic spindle disruption and aneuploidy in cryopreserved oocytes, and conflicting animal and human studies have demonstrated slow cooling-and vitrification-induced damage to the meiotic spindle (5)(6)(7)(8)(9)(10).…”