Effects of Rho-associated protein kinase inhibitor on meiosis and embryo development of feline oocytes after vitrification

Abstract

For decades, gamete cryopreservation is markedly interesting method for assisted reproductive technologies (ARTs) in several species such as human, cat, dog, buffalo and goat. Vitrification is a denoting applied procedure for tissues and cells for long term storage. However, there are several disadvantages of all types of cryopreservation that potentially damages to cell so-called cryoinjuries. Cryoinjuries occur during cryopreservation procedure that damages cells or tissues and finally lead to cell death. Indeed, rho-associated coiled-coil protein kinase (ROCK) manipulates the cellular function and plays a pivotal role in cell apoptosis and death. In the present study, we examined the effects of ROCK inhibitor on oocyte maturation and developmental competence of vitrified-warmed feline oocytes. LIMK-1 and LIMK-2 were determined in cumulus cells and oocytes (experiment 1). The toxicity of the ROCK inhibitors at various concentrations ROCK inhibitor (0, 10, 20 and 40 µM) was tested in experiment 2. The experiment 3 examined the effect of vitrification on gene expression. The effects of ROCK inhibitor treatment on post-warming development of vitrified oocytes were examined in experiment 4. Our results showed the expressions of LIMK1 and LIMK2 in fresh oocytes and cumulus cells. Cumulus oocyte complexes (COCs) cultured with ROCK 10 µM had the statistical highest metaphase II (MII) rate (p<0.05) when compared to controls. However, high concentration of ROCK inhibitor (40 µM) negatively affected on meiotic competence and but not for developmental competence. The gene expression of LIMK1 after in vitro maturation of non-cryopreserved oocytes supplemented with 10 µM ROCK inhibitor for 12 hours significantly up regulated to higher levels than other groups (p<0.05). Incubation of vitrified-warmed COCs with 10 µM ROCK inhibitor significantly increased cleavage rate (36.13±3.76%) and tended to increase morula and blastocyst rates when compared to non-treated group (27.40±2.54%) (p<0.05). In conclusion, this study demonstrated that ROCK cascade presences in feline oocytes and cumulus cells. A chosen dose of 10 µM ROCK inhibitor is an appropriate dose for improving feline meiotic resumption rate, especially after cryopreservation. However, high dose of ROCK inhibitor adversely affected to meiotic resumption.