Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation

Abstract

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage.

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

“…These alterations are consistent with previous findings [11,36]. Interestingly, the cytoplasmic structure of the vitrified oocyte was not altered, as evidenced by location and morphology of cellular membrane, organelle, CG, lipid droplets, mitochondria and cytoplasmic vesicles, resembling structures in fresh oocytes.…”

“…Meiotic inhibition in vitro has been described to induce ultrastructural modifications in the bovine oocyte [11,17,18]. In the present work, meiotic arrest prior to IVM of fresh oocytes did not alter the structure of CC, which showed expansion and absence of morphological changes in organelles.…”

“…Oocytes matured for 0, 6 and 12 h had less ability to survive cryopreservation than 24 h IVM oocytes [15]. In bovine oocytes, active transcription and translation mostly occurs at GVBD and to a lesser extent at later stages of meiotic maturation [11,[39][40][41][42].…”

“…Studies on chilling sensitivity conducted on MII stage oocytes found that the main damage occurred due to meiotic spindle disorganization followed by microtubule depolymerization [11,26,43,44]. Cryopreservation of immature oocytes could provide a partial solution for damage inflicted to cytoskeleton by vitrification, as oocytes do not contain polymerized tubules at this stage.…”

“…This can be primarily explained by nuclear damage [10,11], including spindle disorganization and loss of clumping of microtubules [10,12,13], as well as other ultrastructural alterations seen in vitrified oocytes [14]. The nuclear stage at which oocytes are submitted to cryopreservation influences further ability of the oocyte to develop after in vitro fertilization (IVF) [15].…”