• Molecules and Cells
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• 제25권3호
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• pp.358-367
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• 2008

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.

• Asian-Australasian Journal of Animal Sciences
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• 제8권6호
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• pp.557-562
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• 1995

In this study, characteristics of chick primordial germ cells (PGCs), which is the founder cell of the germline, and gonadal development of the chick embryo between 12hrs and 6 day of incubation were investigated by transverse serial sections of chick embryos under the light microscopic observation. In embryo stage 20 (3 day of incubation), there are a lot of PGCs at the mesenchym, which were moving to the thickened epithelium (gonadal ridge). The PGCs arrive at both right and left gonad primordial in equal number prior to stage 24 (4 day of incubation), but in the following stages, the distribution of the PGCs became asymmetrical. More PGCs colonized the left than the right gonad, but the reason for the unequal distribution of PGCs is uncertain. The PGCs have mostly settled in the gonadal ridge (GR) at 6 day embryo. This study was conducted to investigate characteristics of the PGC migration and gonadal formation and observe the best condition for PGC isolation, culture and to attempt the possibility of the production for transgenic germline chimeras with manipulated PGCs.

• Asian-Australasian Journal of Animal Sciences
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• 제13권11호
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• pp.1514-1517
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• 2000

In chicken, exogenous genes introduced into germinal crescent region (GCR) of the early developmental stage, where primordial germ cells (PGCs) were concentrated, were successfully transferred to the gonads via PGCs. The foreign genes were also confirmed to be successfully incorporated into F1 and F2 generations. We tried to incorporate the exogenous genes into PGCs by lipofection, then the DNA mixture was injected into GCR at stage 3-5 or 9-11 of embryonic development (Hamburger and Hamilton, 1951). The manipulated eggs were incubated, and hatched chicks were reared until sexual maturation. F1 generation was obtained from the DNA-treated chicken (DNA-chicken) mated with normal birds. Furthermore, F2 generation was also obtained from the F1 chicken mated with normal birds. The transfer of introduced foreign genes were confirmed by marker gene detection methods and PCR analysis in the hatched chicks, F1 and F2 generations. However, in our experiments, DNA-chickens showed abnormal characteristics such as low egg production rate, abnormal appearance and decreased number of spermatozoa. In the case of F1 chicken, low egg production and the deterioration of sperm capacity for insemination in male chicken were observed.

• 한국가축번식학회지
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• 제21권3호
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• pp.311-319
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• 1997

This study was carried out to investigate the ultrastructural changes of spermatozoa obtained from 20 of 3-year-old male chum salmon(Oncorhynchus keta) collected and analysed in middle October in 1995. The ultrastructural changes of gonad of fingerlings were examined to describe the sex differentiation of this species. The results obtained in this study were as follows : In spermatozoa, the nucleus is dense and homogeneous. Two spheroidal mitochondria(about 350nm long) are situated in parallel between the nucleus and the axoneme. Spermatozoa mitochondria are assembled into an organized sheath surrounding the outer dense fibres and axoneme of the flagellar midpiece. The sheath flagellum is situated beneath the base of the sperm head. The primordial germ cells of 6.8~7.2${\mu}{\textrm}{m}$ in size, which were buried under fibrous mesenchymal tissue between gut duct and notochord of larva with a total length of 2.4cm at 50 days after hatching. In juvenile of 10.5cm in total length at 70 days after hatching, the gonad was occupied by bundles of oogonia. The dense drumstick bodies(large arrows) are observed in the nuclei of the primordial gonad and surrounding tissue cells of fingerling at 70 days after hatching. The oval Barr bodies(asterisk) are observed in the nuclei of the primordial germ cells under the mitosis(2n). Note the large mitochondria, ribosomes and rough endoplasmic reticulum in the cytoplasm. Accordingly, the fingerlings at 70 days after hatching are identified as the female(xx). In result, the gonadal sex differentiation begins from the 70 days after hatching in chum salmon.

• 한국발생생물학회지:발생과생식
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• 제10권3호
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• pp.193-196
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• 2006

이 연구는 태생경골어인 쏨뱅이의 번식생태학적 기초자료를 제공하고자 체내 자어와 출산 후의 시원생식세포의 출현, 원시 생식소와 난소 분화과정을 조직학적으로 조사하였다. 약 $10\;{\mu}m$의 시원생식 세포는 체내 자어의 초기 소화관과 중심신관 사이 복막에 위치하였다. 출산 후 31일째부터 생식원세포가 생식원기에 도달하였으며, 체세포들이 분열 증식하여 원시생식소를 형성하였고, 출산 후 49일째에 난소강이 형성되기 시작하였으며, 출산 후 60일째에 생식원기의 양쪽 끝에서부터 난소강 형성이 관찰되었다. 출산 후 79일째에는 난소강 안쪽을 따라 생식세포들이 분열 증식하기 시작하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제15권9호
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• pp.1227-1231
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• 2002

A powerful tool for chicken transgenesis could be established by employing a germline chimera production through primordial germ cell transplantation. This study was conducted to examine whether foreign gene-transfected gonadal primordial germ cells (gPGCs) have a migration activity into the gonad after transfer to recipient embryos. In Experiment 1, gPGCs of Korean Ogol Chicken were retrieved from 5.5-day-old embryos and subsequently transferred to the dorsal aorta of 2.5-day-old White Leghorn embryos after being labeled with PKH26 fluorescent dye. To confirm migration activity after transplantation, recipient embryos were sacrificed and examined on 3 days after transfer. Sex determination was concomitantly undertaken to examine whether sex of recipient embryos could affect the migration activity of gPGCs. All of embryonic gonads examined showed positive signals with PKH26 fluorescence and W-chromosome specific band by polymerase chain reaction (PCR) was detected in male embryos when gPGCs with ZW chromosome were transferred to recipient embryos. In Experiment 2, retrieved gPGCs were transfected with LacZ gene-containing cytomegalovirus promoter ($pCMV{\beta}$) by electroporation and subsequently transferred to recipient embryos. LacZ gene expression was identified in the gonads of 6 or 10-day-old recipient embryos and hatched-chicks. A total of 20 embryos and 12 hatched-chicks were examined and 11 of them (10 embryos and one hatched chicken; 11/32=34.4%) expressed $\beta$-galactosidase, a marker substance of LacZ gene. The results of this study demonstrated that foreign gene-transfected gPGCs can migrate and settle down into the gonad after being transferred into the blood vessel of the recipient embryos. This established technique will contribute to developing a peer biotechnology for transgenic chicken.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2002년도 가을 학술발표논문집
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• pp.98-99
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• 2002

Collection of large number of gonadal Primordial germ cells(gPGCs) is a prerequisite factor for improving germline transmission efficacy in the aves, In this study a magnetic-activated cell sorter(MACS) was applied for improving retrieval efficacy of quail gPGCs and the migration capacity of MACS-separated gPGCs was further examined after being transplanted to recipient embryos. We also induced germline chimeras by transfer of MACS-separated quail gPGCs at the efficiency of 17.4% on average.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2001년도 제18차 정기총회 및 학술발표 PROCEEDINGS
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• pp.69-70
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• 2001

Lectins have great potential as to determine the alternation of the distribution of cell surface carbohydrates during cellular development and differentiation. Here, we investigated the presence and distribution of cell surface carbohydrates on chicken primordial germ cells (PGCs) during the migration and gonadal stages using a variety of lectins. A total of six FITC-labelled lectins from several specificity classes were used: ConA (glucose/mannose), WGA (N-acetylglucosamine), STA (N-acetylglucosamine), DBA (N-acetylgalactosamine/galactose), UEA-I (fucose) and PHA-E (oilgosaccharide). As a results, PGC-specific binding was observed in STA. PGCs of migration stage (2.5- and 5.5-day embyos) were STA-positive whereas PGCs of 10-day embryonic gonad were not. The results suggest that N-acetylglucosamine residuse are present specifically in migrating chicken PGCs and changes during development.

• Asian-Australasian Journal of Animal Sciences
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• 제13권5호
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• Molecules and Cells
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• 제38권10호
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• pp.895-903
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• 2015

Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development.