Rescue of Vitrified-Warmed Bovine Oocytes with Rho-Associated Coiled-Coil Kinase Inhibitor1

Abstract

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

“…However, there are conflicting reports on the positive effect of L-carnitine on cryosurvival of bovine oocytes (Phongnimitr et al 2013). Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes (Hara et al 2012), can be inhibited by a short-term culture of the postwarming oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor, with a significantly higher blastocyst yield of 21% compared with 14% in untreated control (Hwang et al 2013).…”

Multiple aster formation is frequently observed in bovine oocytes retrieved from 1-day stored ovaries

“…Difference in multiple aster formation rate between fresh and stored ovary-derived oocytes was small despite of 210 the presence of significant difference (Fig. 2B) when compared to the difference described for vitrified-warmed oocytes with or without ROCK inhibitor treatment (Hwang et al 2013). Therefore, this may explain the failure of ROCK inhibition-mediated improvement in blastocyst yield from stored ovary-derived oocytes.…”

“…Therefore, treatment with the ROCK inhibitor may be useful to suppress the multiple aster formation by stabilizing astral structure. Hwang et al (2013) reported that ROCK inhibition-assisted suppression of multiple 205 aster formation in vitrified-warmed bovine oocytes led to a significant improvement of blastocyst yield from 14 to 21%. However in the present study, it may be reasonable to conclude that multiple aster formation is not directly involved in the developmental potential of bovine oocytes impaired by ovary storage (Table 2).…”

“…Hepes-buffered TCM-199 supplemented with 5% (v/v) FBS, 0.2 mM sodium pyruvate, and 50 µg/ml gentamycin sulfate (TCM-199/5%FBS) was used as the base medium for ROCK inhibition of stored ovary-derived oocytes prior to the IVF, as 100 reported previously (Hwang et al, 2013). In vitro fertilization and culture Spermatozoa were prepared from commercially available frozen semen of a Japanese Black bull.…”

“…We reported that impaired development of sperm aster in bovine oocytes was caused by a procedure for intracytoplasmic sperm injection (Hara et al, 2011), and that frequent multiple aster formation after in vitro fertilization (IVF) was induced by vitrification of in vitro-matured bovine oocytes (Hara et al, 2012), with 65 impaired migration and development of pronuclei prior to the first cleavage. Recently, we have reported that a short-term culture of vitrified-warmed bovine oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the incidence of multiple aster formation, resulting in an improvement of the blastocyst yield (Hwang et al, 2013). 70…”