• Journal of Animal Reproduction and Biotechnology
• /
• v.39 no.1
• /
• pp.19-30
• /
• 2024

Background: Although an understanding of the proliferation and differentiation of fish female germline stem cells (GSCs) is very important, an appropriate threedimensional (3D) research model to study them is not well established. As a part of the development of stable 3D culture system for fish female GSCs, we conducted this study to establish a 3D aggregate culture system of ovarian cells in marine medaka, Oryzias dancena. Methods: Ovarian cells were separated by Percoll density gradient centrifugation and two different cell populations were cultured in suspension to form ovarian cell aggregates to find suitable cell populations for its formation. Ovarian cell aggregates formed from different cell populations were evaluated by histology and gene expression analyses. To evaluate the media supplements, ovarian cell aggregate culture was performed under different media conditions, and the morphology, viability, size, gene expression, histology, and E2 secretion of ovarian cell aggregates were analyzed. Results: Ovarian cell aggregates were able to be formed well under specific culture conditions that used ultra-low attachment 96 well plate, complete mESM2, and the cell populations from top to 50% layers after separation of ovarian cells. Moreover, they were able to maintain minimal ovarian function such as germ cell maintenance and E2 synthesis for a short period. Conclusions: We established basic conditions for the culture of O. dancena ovarian cell aggregates. Additional efforts will be required to further optimize the culture conditions so that the ovarian cell aggregates can retain the improved ovarian functions for a longer period of time.

• Fisheries and Aquatic Sciences
• /
• v.18 no.2
• /
• pp.183-193
• /
• 2015

Several transgenic marine medaka Oryzias dancena strains harboring a green fluorescent protein (GFP) reporter construct regulated by an endogenous ${\beta}$-actin promoter were established and their expression characteristics in relation to transgene copy numbers were examined in 21 transgene genotypes. Most of the transgenic strains displayed transgene insertion patterns typical of microinjection-mediated introduction of foreign DNA into fish embryos, characterized by the random integration of multiple transgene copies (ranging from 1 - 282 copies per cell), often accompanied by the formation of concatemer(s), as assessed by genomic Southern blot hybridization analysis and qPCR. Transgenic strains showed ubiquitous and continued temporal and spatial expression patterns of the transgenic GFP during most of their life cycle, from the embryonic stage to adulthood, enabling assessment of the expression pattern of the endogenous ${\beta}$-actin gene. However, a comparative evaluation of transgene copy numbers and expression levels showed that copy number-dependent expression, the stability of the ubiquitous distribution and expression efficiency per transgene copy varied among the transgenic strains. Fluorescence expression levels were positively correlated with absolute transgene copy numbers, whereas the expression efficiency per transgene copy was inversely related to the number of transgene integrant copies. Data from this study will guide the selection of potentially desirable transgenic strains with ubiquitous expression of a fluorescent transgene, not only in this marine medaka species but also in other related model fish species.

• Journal of Fisheries and Marine Sciences Education
• /
• v.26 no.2
• /
• pp.270-283
• /
• 2014

본 연구는 온도 변화에 따른 해산송사리, Oryzias dancena의 마취 효과 및 생리적 스트레스 반응을 평가하고, 고온 및 저온에서 최적의 마취 수온을 구명하였다. 해산송사리에서 고온 실험($36{\sim}42^{\circ}C$)과 저온 실험($4{\sim}10^{\circ}C$)을 통해 마취 효과를 조사하였으며, 각 마취수온에서 전어체의 cortisol과 glucose를 측정하였다. 각 수온 마취 실험 후, 전개체는 모두 생존하였으며, 해산송사리의 마취시간은 고온 실험에서 고온 일수록, 저온 실험에서는 저온 일수록 유의적으로 빨랐다(P<0.05). 회복 시간은 유의하게 고온 실험에서 수온이 감소할수록, 저온 실험에서는 수온이 증가할수록 유의적으로 느렸다(P<0.05). 아가미 운동수는, 고온 실험에서 수온이 증가함에 따라 빨랐으며, 저온 실험에서는 수온이 감소할때 빨랐다(P<0.05). 수온이 $38^{\circ}C$ 나 $8^{\circ}C$ 일때의 마취 조건에서 실험직후 전어체 cortisol이 최대치로, 실험후 6시간까지 점진적으로 감소한 반면, 전어체 glucose는 실험후 1시간에 최고치를 보이고 실험후 2시간까지는 감소하였다. 기존의 마취제를 쓰지 않은 본 연구로 인해, 해산송사리의 여타 연구에서 샘플 및 사용자가 보다 더 안전하고 용이한 샘플 취급이 가능할 것으로 사료된다.

• Journal of Embryo Transfer
• /
• v.33 no.4
• /
• pp.343-348
• /
• 2018

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including $28^{\circ}C$, $31^{\circ}C$, $34^{\circ}C$, and $37^{\circ}C$ and 2 basal media including Dulbecco's modified eagle's medium (DMEM) and Leibovitz's L-15 medium (L15). With the exception of a case cultured in L15 at $31^{\circ}C$, the rate of primary cell adherence reached 100% when the blastomeres were cultured over $31^{\circ}C$. The period for primary adherence was significantly shorter in the groups cultured in $34^{\circ}C$ and $37^{\circ}C$ than in the ones in $28^{\circ}C$ and $31^{\circ}C$. The proportion of subculture was significantly high in the group cultured in DMEM at $31^{\circ}C$ compared to the other groups. Collectively, we demonstrated that the culture in DMEM at $31^{\circ}C$ was effective to primary culture of the blastomeres derived from blastula embryos.

• Journal of fish pathology
• /
• v.24 no.2
• /
• pp.131-139
• /
• 2011

Bisphenol A (BPA) is mainly used in the production of epoxy resins and polycarbonate plastics, which is a known endocrine disruptor and acutely toxic to aquatic organisms. In this study, estrogenic effect of BPA was investigated on hybrid between Oryzias dancena and O. javanicus (ODJ). ODJ were exposed to BPA of various concentrations (eg. 2.5 mg/L, 5.0 mg/L and 10.0 mg/L) for 56 days. The growth rate, abnormality and the ratio of female and male were observed in test group and control group. As a result, the growth was $14.7{\pm}2.0$ mm in total length (TL) in 2.5 mg/L, $13.7{\pm}2.5$ mm in 5.0 mg/L, $12.8{\pm}2.5$ mm in 10.0 mg/L in test group while it was $18.0{\pm}1.2$ mm in TL in control group which was not treated with bisphenol A. The result showed that the growth decreased as the concentration of BPA increased. The abnormality rate was 13.6% in control group, 65.4% in 2.5 mg/L, 81.3% in 5.0 mg/L and 98.1 % in 10.0 mg/L which showed increase in abnormality as an increase of BPA concentration. As a result of analyzing ratio of sex in the test group and control group, 6.0% was examined to be interspecific in controls, 76.9% in 2.5 mg/L and 100.0% in 5.0 mg/L and 10.0 mg/L. In conclusion, these results suggest that BPA has estrogenic effect on ODJ.