The Role Of Rock Protein In The Development Of In-Vitro Fertilized Porcine Embryos

Pig is one of the important livestocks in our country, which is the closest animal to humans regarding their genomic and metabolic physiology. The investigation on the factors that are affecting the development of early in-vitro fertilized porcine embryos will provide the references for the improvement of survival rate of the in-vitro produced and cloned embryos. The development of porcine early embryos relies on the interaction of a variety factors. Rho-associated coiled-coil protein kinases (ROCKs) are downstream effectors of the Rho GTPases, which involved in oocyte maturation and embryonic development. Two isoforms of Rho kinases,ROCK1 and ROCK2,have been described in mammalian cells, which are playing important roles in cytoskeleton, gene transcription, secretion and apoptosis.This study focused on the role of ROCK1 and ROCK2 in the development of porcine in-vitro produced embryos and its impact on the quality of the blastocyst. Firstly, the distribution of ROCK1 and ROCK2 in oocytes and porcine in-vitro fertilized early embryos was analyzed by quantative real-time PCR and immunofluorescence. The results showed that ROCK1 and ROCK2 both expressed in porcine oocytes and in vitro fertilized preimplantation embryos, ROCK mRNA increased significantly from the two-cells stage to eight-cell stage in porcine embryos, began to decrease at the morula stages and hardly expressed at blastocyst stage. ROCK1 was stronger in nucleus of GV stage oocytes and 2-4 cell embryos, and began to shift to cytoplasm from 8 cell stage. ROCK2 was detected at the cell membrane in GV and M I stage oocytes, and in the cytoplasm of the blastocysts.To further investigate the role of ROCK1 and ROCK2 in the development of porcine in-vitro produced embryos, Y27632, a specific inhibitor of ROCKs, and ROCK activator lysophosphatidic acid (LPA) was added to the culture medium. The results showed that the cleavage and blastocyst rate decreased along the incretion of the Y27632 and some of the blastocysts were abnormal. When the concentration of Y27632 increased to 15μM, the blastocysts could not form. But the blastocysts formation was increased when 10μM LPA added with Y27632. Furthermore, the LPA alone improved the development of the embryos. ROCK inhibitor and activator also affected the cell numbers in the blastocysts. The total cell number was decreased when Y27632 was added, but the ICM/TE was increased, which resulted from the TE cell number reduction and ICM cell number incretion. The blastocysts could not form, when Y27632 added at day 2-3 of the embryo cleavage and at day 4-5 of the embryo development, which indicated the ROCK protein played important role in the blastocyst densification. Our data indicated that ROCK protein is essential for the porcine in-vitro produced embryos developed to the blastocyst stage, and ROCK1 and ROCK2 may exert different biological functions during this period.