Kyung Soo Kang

Conserved expression pattern of chicken DAZL in primordial germ cells and germ-line cells

The autosomal gene deleted in azoospermia-like (DAZL), which was identified as a member of the deleted in azoospermia (DAZ) family, is homologous to the Drosophila gene BOULE. The authors investigated the sequence similarities of chicken DAZL (cDAZL) with several invertebrate and vertebrate DAZL proteins using CLUSTAL X. A comparison of the primary sequence of cDAZL with other DAZL proteins indicated significant similarities: 70-82% with reptiles, 63-68% with mammals, 51-67% with amphibians, and 42-49% with fishes. The conserved expression pattern of cDAZL was examined by reverse transcription-PCR, quantitative real-time PCR, and in situ hybridization during primordial germ cell (PGC) settlement in the gonads and germ-line development. Among several tissues examined on embryonic day E6.5, DAZL expression was detected specifically in male and female gonads. Quantitative real-time PCR and in situ hybridization revealed strong cDAZL expression in PGCs. When the PGCs differentiated into germ cells, cDAZL expression was slightly decreased; however, expression was continuously detected in germ-line cells until the adult stage. We inferred that cDAZL expression was conserved in PGCs and during germ-line differentiation until the adult stage, making them a valuable molecular marker for studies of PGC differentiation and germ-line development in chickens.

Current genomic editing approaches in avian transgenesis.

The chicken was domesticated from Red Jungle Fowl over 8000years ago and became one of the major food sources worldwide. At present, the poultry industry is one of the largest industrial animal stocks in the world, and its economic scale is expanding significantly with increasing consumption. Additionally, since Aristotle used chicken eggs as a model to provide remarkable insights into how life begins, chickens have been used as invaluable and powerful experimental materials for studying embryo development, immune systems, biomedical processes, and hormonal regulation. Combined with advancements in efficient transgenic technology, avian models have become even more important than would have been expected.

Molecular and biological aspects of early germ cell development in interspecies hybrids between chickens and pheasants

Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications.

Spatial and Temporal Action of Chicken Primordial Germ Cells during Initial Migration

In most animals, primordial germ cells (PGCs) originate from an extragonadal region and migrate across the embryo to the gonads, where they differentiate and function. During their migration, PGCs move passively by morphogenetic movement of the embryo or move actively through signaling molecules. To uncover the underlying mechanism of first-phase PGC migration toward the germinal crescent in chickens, we investigated the spatial and temporal action of PGCs during primitive streak formation. Exogenously transplanted PGCs migrated toward the anterior region of the embryo and the embryonic gonads when they were transplanted into the subgerminal cavity, but not into the posterior marginal zone, in Eyal-Giladi and Kochav stage X embryos. These results indicate that for passive migration toward the anterior region the initial location of PGCs should be the central region. Notably, although PGCs and DF-1 cells migrated passively toward the anterior region, only PGCs migrated to the germinal crescent, where endogenous PGCs mainly reside, by active movement. In a live-imaging experiment with green fluorescence protein-expressing transgenic embryos, exogenous PGCs demonstrated markedly faster migration when they reached the anterior one-third of the embryo, while somatic cells showed epiblast movement with constant speed. Also, migrating PGCs exhibited successive contraction and expansion indicating their active migration. Our results suggest that chicken PGCs use sequential passive and active forces to migrate toward the germinal crescent.

The distribution of neuron-specific gene family member 1 in brain and germ cells: Implications for the regulation of germ-line development by brain

Vesicular acidification at early endosomes dissociates endocytosed receptor‐ligand complexes. The ligands, receptors, or both are then directed to late endosomes for degradation or recycled back to the plasma membrane. Of neuron‐specific gene (NSG) family members, early endosomal protein neuron‐specific gene family member 1 (NSG1) is the most important in receptor recycling. In this study, we characterized chicken NSG1 (cNSG1). We found several functional sites related to endocytotic machinery in cNSG1 that were highly conserved with most other vertebrate NSG1 proteins. We examined the tissue and duration specificity and the temporal and spatial patterns of cNSG1 expression. cNSG1 expression was preferentially located in all regions of the brain, neuroendocrine glands, and spinal cord. Unexpectedly, cNSG1 expression was strongly detected during male and female germ‐line development. Expression of NSG1 in two apparently unrelated cell types such as neurons and germ cells suggests NSG1 roles in neurons and germ‐cells chemotaxis and endocytotic machinery. Developmental Dynamics 240:850–861, 2011.

Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70-120min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved-thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

Comparative Study on Various Growth Performances of Korean Oge and White Leghorn Chickens

Genetic resource conservation of indigenous species is important to conserve terrestrial, aquatic and avian species throughout the world. The native Korean Oge (KO) chickens are important and protected indigenous avian species in Korea. This KO breed is very popular in Korea because of its external appearances of blackish color. The characterization of morphology and growth performances of KO were performed since 1980s, however the information is still not sufficient for breed selection. In this study, we compared various growth performances including body growth, tibia length, shank length, chest width, chest grith and length of tail feather between male and female chickens of KO and widely known White Leghorn (WL) breeds at 1 week, 2 weeks, 5 weeks, 10 weeks, and 24 weeks. We observed differences on various growth performances at different aged groups between KO and WL chickens. This study may help for the selection of chicken breeds based on age, body growth and meat production.

Production of Interspecific Germline Chimeras via Embryo Replacement

ABSTRACT In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

Normal Development and Hatchability of Korean Oge Chickens in White Leghorn Surrogate Eggshells

The avian embryos have been used as a good model to study embryonic development. Due to its unique development in the eggshell, avian embryos can be cultured and hatch in the surrogate eggshell system. In this study, we examined the viability, normal development and hatchability of Korean Oge (KO) chicken embryos in White Leghorn (WL) surrogate eggshells. Donor KO embryos at 3-day and 4-day-old were transferred into recipient WL eggshells, incubated for further 18 days at 37.5℃ with 70% of humidity until hatching. The viability of 3-day-old KO embryos at 7, 14 and 21 day in surrogate eggshell were 70.0%, 43.8% and 23.1%, respectively. In contrast, the viability of 4-day-old KO embryos at 7, 14 and 21 day in surrogate eggshells were 87.1%, 55.6% and 36.0%, respectively. The hatchability of KO embryos transferred into surrogate eggshells at 3-day-old was 23.1%, whereas embryos transferred at 4-day-old was 36.0%. Furthermore, the development of all viable embryos from 3-day group and 4-day group were normal. Our results suggested that culture of KO embryos in WL surrogate eggshells is highly possible, and transfer of donor embryos at 4-day-old may yield higher percentage of hatchability. This study may provide potential knowledge for the conservation of wild and endangered birds through surrogate system.