Recent progress in cryopreservation of bovine oocytes

Abstract

Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

Figure 1

Hypotheses regarding cryoinjuries in mammalian oocytes. (a) Premature cortical granule exocytosis causes the hardening of zona pellucida, leading to block of sperm penetration. (b) Disorganization of microtubules means depolymerization of tubulin proteins, leading to misassembly of meiotic spindles, and subsequently resulting in misalignment of chromosomes and inhibition of the second polar body extrusion. (c) Multiple aster formation, resulting from ooplasmic dysfunction to support MTOC, is a possible cause of low blastocyst yield.

Figure 2

Structures of chemicals used for improvement of cryosurvival of bovine oocytes and resulted in significantly higher blastocyst yield in vitro [84, 85]. (a) L-carnitine, (b) ROCK inhibitor Y-27632, (c) α-tocopherol.

Figure 3

Effect of ROCK inhibition during postwarm recovery culture on revivability of bovine mature oocytes [85]. (a) Proportion of vitrified-warmed bovine oocytes exhibiting the formation of no, single, or multiple sperm aster(s). The abnormal incidence of multiple aster formation was inhibited by the recovery culture with Y-27632. Immunostaining against α-tubulin (green) and nuclear staining with DAPI (blue) were performed at 10-hour post-insemination (hpi). (b) Accelerated timing of first cleavage in bovine oocytes vitrified-warmed and rescued with Y-27632. Asterisks indicate significant difference at P < 0.05.