Studies on Embryo Development Following Intra Cytoplasmic Sperm Injection and Morphogenetic Analysis of Primordial Germ Cell Migration in Aves

Abstract

Primordial germ cells (PGCs) migrate across the embryo to the gonads where they differentiate and function. Both of morphogenetic and actively through are used for their movement. We have examined the spatial and temporal action of PGCs. As results, PGCs migrated passively toward the anterior region from the preliminary location. However, PGCs and somatic cells are shown different migration speed when they reached the anterior region. PGCs demonstrated markedly faster migration than somatic cells. The results reveal that chicken PGCs use sequential passive and active migration toward the germinal crescent, and only PGCs migrated to germinal crescent. Cryopreservation of avian semen for preserving the avian genetic resource has been studied for more than 80 years, and there have been many technical difficulties due to the complexity of avian reproductive system. We found that the mixture shown the highest fertility composes of 8% glycerol with 3% DMA in prefreezing diluents. However the mixture was a harmful effect with the addition of cryoprotectant especially with glycerol. Our results show that the sperm preserved in the mixture composed of 6% glycerol containing 5% DMA had 5% less motility. Thus we used that mixture and the sperm preserved in the mixture produced fertilized eggs. Additionally, we tested incubation time for glycerol removal by monitoring under scanning electron microscopy (SEM). We found that complete removal of glycerol requires at least 30 min incubation time. We have investigated the ability of cryopreserved/thawed quail sperm by intracytoplasmic injection to the unfertilized ovum. An injected egg was incubated with egg shell surrogate system. We have used both PLC zeta (PLCζ) and inositol 1,4,5-trisphosphate (IP3). Embryo development ratio increased significantly compared to fresh sperm only (90% vs. 13%). Avian species surrogate egg shell system has been adapted in many different experimental fields. However, the viability needs to be improved and the system should be more simplified for commercial uses. We have established the quail egg surrogate system for embryo developmental study. The system has produced high percentage of hatchability in both thick albumin capsulated and non-capsulated egg, 78% and 60% respectively. Furthermore, we have succeeded in higher hatchability in single cell stage of embryo incubation. This study could provide deeper understanding of germ cell movement mechanism in early embryo developmental stage. And, we tested for ICSI by producing chick from cryopreserved sperm for avian genetic resource conservation. We have established knowledge in avian embryo development to understand embryo fertilization and developmental stratagem for atmosphere of ex ovo culture system used by surrogate egg shell system complementation. This study could bring deeper understanding in avian culture system with germ cell migration and possibilities of hatching chick by ICSI and surrogate egg shell system for conservation of genetic resource in the future.