48 CARBOXYLATED ϵ-POLY-L-LYSINE (COOH-PLL) IMPROVES DEVELOPMENTAL ABILITY AND REDUCES MITOCHONDRIAL DAMAGE OF VITRIFIED PORCINE EMBRYOS AT THE PRONUCLEAR STAGE

Abstract

Although cryopreservation of pronuclear-stage embryos has increasing importance in the production of genetically modified animals via microinjection, it is well known that cryopreserved porcine embryos show low developmental ability. Cryopreservation causes damage to organelles such as mitochondria that are involved in metabolism and play important roles in embryonic development. To reduce the damage in embryo cryopreservation, we focused on carboxylated ε-poly-l-lysine (COOH-PLL), which has been recently developed as a new cryoprotective agent. Carboxylated ε-poly-l-lysine has antifreeze protein properties, such as inhibition of ice recrystallization (Matsumura and Hyon 2009). In this study, we examined the effect of COOH-PLL on development and mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Porcine follicular oocytes were matured (in vitro) and then fertilized (IVF) in vitro. At 10 h after IVF, the presumptive embryos were centrifuged to visualise the pronuclei. Embryos with 2 or 3 pronuclei (PN embryos) were used for vitrification. The PN embryos were exposed to equilibration solution for 10 min and then exposed to vitrification solution for 1 min before being plunged into LN on Cryotop®. Vitrification solution was phosphate buffered saline supplemented with 30% (vol/vol) ethylene glycol + 0.5 M sucrose + 20% (vol/vol) fetal calf serum + 0 or 20% (wt/vol) COOH-PLL (P0 or P20). Development to the blastocyst stage of the vitrified PN embryos was observed after in vitro culture for 158 h. Nonvitrified embryos were used as a control (fresh). Fresh and vitrified with P0 or P20 PN embryos were stained with MitoTracker® Red CMXRos. The embryos stained with MitoTracker® Red CMXRos were investigated by using a laser-scanning confocal microscope. Mitochondrial fluorescent intensity of embryos was evaluated by measuring the mean numbers of pixels in the fluorescent area (mean/area) using the NIH ImageJ software. The blastocyst rate of embryos vitrified with P20 (19.4%) was significantly (P < 0.05) higher than that of P0 (1.3%), and there were no significant differences in blastocyst rates between P20 and fresh group (28.4%; P > 0.05). The mitochondrial fluorescent intensity of embryos vitrified with P20 (2.8 mean/area) was significantly higher than that of P0 (1.5 mean/area), but the intensity of embryos vitrified with P20 was significantly lower than that of fresh (4.2 mean/area; P < 0.05). Our results suggest that supplementation of COOH-PLL with vitrification solution improved development and also reduced mitochondrial damage of vitrified porcine embryos at the pronuclear stage. Further studies are required to clarify the effect of COOH-PLL on various kinds of damage caused by cryopreservation such as other organelles, including endoplasmic reticulum damage, cytoskeleton damage, or DNA fragmentation of vitrified or warmed embryos in pigs.