• Korean Journal of Ichthyology
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• v.6 no.2
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• pp.237-243
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• 1994

This work was conducted to study sex differentiation in the black sea bream, Acanthopagrus schlegeli (Bleeker), using a histological method for the appearance of primordial germ cell, formation of primitive gonads, differentiation of female and male from newly hatched larva to the ovotestis stage of fish. The 3~4 primordial germ cells of $6.8{\sim}7.2\;{\mu}m$ in size, which were buried under fibrous mesenchymal tissue between gut duct and notochord of pre-larva with a total length (T.L.) of 2.4 mm at 3 days after hatching. The proto-gonial cells were located in the epithelium of the coelom attached with pigment cells of juvenile with 6.4 mm in T.L. at 21 days after hatching. In juvenile of 20.8 mm in T.L. at 59 days after hatching, the proto-gonial cells were migrated to the retro-peritoneum through the lineshaped primitive gonad composed of fibrous mesenchymal tissue. In juvenile of 7.8 em in T.L. at 186 days after hatching, the mitotic division of proto-gonial cell appeared in the lineshaped primitive gonad having many eosinophilic granule cells and abundant fibrous connective tissue. In juvenile of 9.5 em in T.L. at 254 days after hatching, the gonad was occupied by abundant fibrous connective tissue, bundles of spermatocyte and spermatid. In juvenile of 10.5 cm in T.L. at 13 months after hatching, the gonad was divided into cortical layer and medullary layer. The former was composed of bundles of a few spermatocytes and proto-gonial cells, the latter was filled with the fibrous mesenchymal tissue and a few proto-gonial cells. In juvenile of 14.7 em in T.L. at 16 months after hatching, the gonad was separated into ovarian part and testicular part by the fibrous connective tissue. The ovarian part is consisted of ovarian cavity and oocytes of perinucleolus stage. The testicular part was occupied by spermatogonia in the cyst.

• BMB Reports
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• v.43 no.6
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• pp.438-444
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• 2010

Dnd (dead end) gene encodes an RNA binding protein and is specifically expressed in primordial germ cells (PGCs) as a vertebrate-specific component of the germ plasma throughout embryogenesis. By utilizing a technique of specific nucleic acids associated with proteins (SNAAP), 13 potential target mRNAs of zebrafish Dnd (ZDnd) protein were identified from 8-cell embryo, and 8 target mRNAs have been confirmed using an RT-PCR analysis. Of the target mRNAs, the present study is focused on the regulation of geminin, which is an inhibitor of DNA replication. Using electrophoretic mobility shift assay (EMSA), we demonstrated that ZDND protein bound the 67-nucleotide region from 864 to 931 in the 3'UTR of geminin mRNA, a sequence containing 60.29% of uridine. Results from a dual-luciferase assay in HEK293 cells showed that ZDND increases the translation of geminin. Taken together, the identification of target mRNA for ZDnd will be helpful to further explore the biological function of Dnd in zebrafish germ-line development as well as in cancer cells.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• Journal of Aquaculture
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• v.16 no.4
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• pp.240-244
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• 2003

The primitive gonad development in Paralichthys olivaceus was classified into (ⅰ) primordial germ cell (PGC), (ⅱ) genital ridge type (GRT), (ⅲ) primitive gonad type I (PGT I) and (ⅳ) primitive gonad type II stages. In the control group, PGC was recognizable on the 3rd days after hatching, and the primodial gonad after 24th days, while it was around the 22nd day after hatching in the group exposed to PCBs 3.0 $\mu\textrm{g}$/L for 30 days. Likewise, the progress of kidney development was recognized in four stages; unitubular type of mesonephric duct (UTMD), the branched mesonephric duct (BMD), convoluted tubule formation (CTF) and glomerulus appearing (GA) stage. It was structurally completed between the 25th and 30th day after hatching in two groups. There was no significant difference (p>0.05) in the time scale of development of gonad, and kidney between control and the PCBs - exposed group.

• Molecules and Cells
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• v.38 no.9
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• pp.743-749
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• 2015

Production of genome-edited animals using germline-competent cells and genetic modification tools has provided opportunities for investigation of biological mechanisms in various organisms. The recently reported programmed genome editing technology that can induce gene modification at a target locus in an efficient and precise manner facilitates establishment of animal models. In this regard, the demand for genome-edited avian species, which are some of the most suitable model animals due to their unique embryonic development, has also increased. Furthermore, germline chimera production through longterm culture of chicken primordial germ cells (PGCs) has facilitated research on production of genome-edited chickens. Thus, use of avian germline modification is promising for development of novel avian models for research of disease control and various biological mechanisms. Here, we discuss recent progress in genome modification technology in avian species and its applications and future strategies.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.197-205
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• 2013

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Journal of Aquaculture
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• v.13 no.3
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• pp.215-222
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• 2000

The primordial germ cell of the sweet fish was recognized from the 2-day old fry (tl : 0.66 cm), when it began to protrude into peritoneal cavity between meaonephric duct and gut. The primordial gonad, with the formation of genital ridge, developed on the 30-day old fry. Ovarian differentiation was identified by the presence of ovarian cavity and meiotic oocytes from the 90-day old fry (tl : 3.42 cm). Testicular differentiation was identified by the presence of spermatogonial cells with efferent duct from the 100-day old fry (11 : 4.50 cm). Hence the sweet fish belongs to the differentiated type of gonochoristic teleost.

• Korean Journal of Ichthyology
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• v.13 no.4
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• pp.248-253
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• 2001

An histological study was conducted to determine the initial treatment time and treatment duration in the use of sex-reversal hormones in relation to gonadal development and sexual differentiation in the bagrid catfish, Leiocassis ussuriensis. The primordial germ cell, which could be recognized from one-day-old fry, began to protrude into the peritoneal cavity between the mesonephric duct and the gut. The primordial gonad with a genital ridge was developed at 5~10 days after hatching. Sex differentiation of the ovary was identified by the ovarian cavity and meiotic oocytes from 20-day-old larvae. Testicular differentiation was also identified by spermatogonial cells from 20-day-old larvae. It may therefore be concluded that this species belongs to the differentiated type of gonochoristic teleost.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.55-63
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• 1995

The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.

• BMB Reports
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• v.44 no.3
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• pp.147-157
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• 2011

The meiotic process from the primordial stage to zygote in female germ cells is mainly adjusted by post-transcriptional regulation of pre-existing maternal mRNA and post-translational modification of proteins. Several key proteins such as the cell cycle regulator, Cdk1/cyclin B, are post-translationally modified for precise control of meiotic progression. The second messenger (cAMP), kinases (PKA, Akt, MAPK, Aurora A, CaMK II, etc), phosphatases (Cdc25, Cdc14), and other proteins (G-protein coupled receptor, phosphodiesterase) are directly or indirectly involved in this process. Many proteins, such as CPEB, maskin, eIF4E, eIF4G, 4E-BP, and 4E-T, post-transcriptionally regulate mRNA via binding to the cap structure at the 5' end of mRNA or its 3' untranslated region (UTR) to generate a closed-loop structure. The 3' UTR of the transcript is also implicated in post-transcriptional regulation through an association with proteins such as CPEB, CPSF, GLD-2, PARN, and Dazl to modulate poly(A) tail length. RNA interfering is a new regulatory mechanism of the amount of mRNA in the mouse oocyte. This review summarizes information about post-transcriptional and post-translational regulation during mouse oocyte meiotic maturation.