Vitrification of calf oocytes: Effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development

Abstract

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes

“…Some scientists believe that in most species such as bovine [23,24] and human [25], germinal vesicle (GV) oocytes are more amenable to vitrification than metaphase II (MII) oocytes. These authors thought that the specific structure of MII oocytes, such as the cortical granules and microtubular spindles, is the main cause of this cryosensitivity [26].…”

Vitrification of Domestic Cat Oocytes – Effect on Viability and Integrity of Subcellular Structures

“…1). Subcellular structures (microtubules, microfilaments and chromosomes) of all oocytes were separately attributed to one of the above mentioned patterns (Albarracı´n et al 2005;Luciano et al 2009). …”

Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells

“…In mammalian species, the factors involved in vitelline block are not well defined. However, it is well known that the vitrification procedure exerts a detrimental effect(s) on oocytes; for example, the most serious damage after vitrification is disorganization of spindles potentially caused by microtubule disassembly in vitrified oocytes [40]. Recently, paclitaxel treatment prior to vitrification has been shown to improve the developmental ability of mouse [41], human [42], bovine [43] and porcine [44] oocytes.…”