Microtubulin configuration and mitochondrial distribution after ultra‐rapid cooling of bovine oocytes

Abstract

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.

Impact of anisosmotic conditions on structural and functional integrity of cumulus–oocyte complexes at the germinal vesicle stage in the domestic cat

“…These oocytes were incubated for 15 min in 400 nM Mito-tracker green (Molecular Probes, Inc., Eugene, OR), washed in PBS for 10 min, and then mounted on a Teflon slide for evaluation of mitochondrial distribution by epifluorescence microscopy. Distributions were classified and recorded for each oocyte as normal (homogenous distribution throughout the cytoplasm) or abnormal (centered or heterogeneous distribution in the cytoplasm; Rho et al, 2002;Fulka et al, 2004).…”

“…Structure of the GV chromatin strongly influences successful nuclear and cytoplasmic maturation in vitro as demonstrated in mouse (Miyara et al, 2003), mare (Choi et al, 2006), and cow studies. Likewise, cytoplasmic mitochondrial distribution within the immature oocyte (also reflecting the microfilament organization) plays a critical role in driving later embryo development (Wang et al, 2000;Stojkovic et al, 2001;Rho et al, 2002;Van Blerkom et al, 2002;Velilla et al, 2006). Because GV chromatin structure and mitochondrial distribution may both be influenced by non uniform shrinking or swelling of an oocyte exposed to an anisosmotic solution, these two targets of interest were chosen as indicators of developmental competence.…”

“…Steps to cryopreservation can be detrimental to the functionality of cumulus-oocyte communications in mouse (RuppertLingham et al, 2003(RuppertLingham et al, , 2006, cow , and porcine oocytes (Wu et al, 2006). Interestingly, it has been demonstrated that cytochalasin B (CB) increases cytoskeletal flexibility and preserves the coupling between the oocyte and its cumulus cells, especially during times of hyperosmotic exposures (Le Gal et al, 1994;Isachenko et al, 1998;Rho et al, 2002). Therefore, in preparation for forthcoming cryopreservation trials, the objective of this study was to evaluate and characterize the impact of osmotic stress on structural and functional integrity of COC at the GV stage in the domestic cat.…”

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

“…Developmental arrest was observed more frequently in vitrified group than fresh control group ( Table 1), suggesting that oocyte activation by sperm penetration was suboptimal in the vitrified-warmed oocytes. Vitrification induced the reduction of maturation promoting factor (MPF) in ovine oocytes before fertilization [34], the damage of mitochondria in bovine oocytes [35], and of endoplasmic reticulum in mouse oocytes [36]. Post-warm oocytes may have been recovered by 2-h culture prior to IVF in the present study, but the harmful effect of remaining intracellular CPA (EG and/or DMSO) could not be estimated.…”

Cryo-survival, fertilization and early embryonic development of vitrified oocytes derived from mice of different reproductive age

“…A significant DNA fragmentation has been found in vitrified bovine oocytes [38]. It has been reported that the process of oocyte vitrification can induce profound modifications to mitochondria, cortical granules, microvilli, oolemma, smooth endoplasmic reticulum (SER) and cytoskeleton [39][40][41][42][43]. Therefore, taken together, it appears that the quality of blastocysts is detrimentally affected by both maternal reproductive age and vitrification/ warming procedures.…”