• Journal of Veterinary Science
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• 제23권2호
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• pp.35.1-35.13
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• 2022

Background: The testis has been reported to be a naturally O2-deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells. Objectives: The objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs. Methods: Gradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry. Results: Results revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated (p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased (p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased (p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs. Conclusion: We demonstrate that DMOG can promote the spermatogenesis activity of SSCs.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.170.1-170.1
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• 2006

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2005년도 창립 30주년 및 춘계학술대회
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• pp.55-66
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• 2005

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.305-312
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• 2009

단분화성 정원줄기세포의 장기간 체외배양 중에 확립되는 다분화능 정원줄기세포는 배아줄기세포와 유사한 특성을 가져 3배엽성 세포로 체외분화가 가능하며 기형종을 형성할 수 있다. 본 연구에서는 선행 연구를 통해 outbred 생쥐(ICR strain)로부터 확립된 다분화능 정원줄기세포의 형질전환 가능성을 확인하며, 배아 내로 주입하여 유전적 키메라를 형성하는 효율을 배아줄기세포와의 비교를 통하여 검증하고자 하였다. 다분화능 정원줄기세포를 넣은 배아로부터 태어난 산자는 총 47마리(4.8%)가 태어나, 67마리(11.7%)가 태어난 배아줄기세포군에 비해 그 효율이 낮았다(P<0.05). 그러나 산자들 중의 키메라 생쥐의 비율은 다분화능 정원줄기세포 군으로 부터 3마리(6.4%)가 태어나 배아줄기세포 군으로부터 태어난 5마리(7.5%)와 유사하였다(P>0.05). 태어난 유전자변형 생쥐의 장기를 확인한 결과, 췌장, 심장, 뇌, 근육, 위, 피부, 정소에 GFP가 발현되는 것을 확인하였다. 또한 배아의 근육, 위, 뼈 등에서 anti-GFP 항체의 발현을 확인하였다. 이상의 결과를 종합하면 outbred 생쥐로부터 확립된 다분화능 정원줄기세포가 inbred 생쥐로부터 확립된 배아줄기세포와 마찬가지로 키메라 생쥐를 생산할 수 있는 전분화능을 가짐을 확인하였고, 새로운 유전자변형 동물의 생산을 위한 매개체로서의 가능성을 가진 것으로 여겨진다.

• Reproductive and Developmental Biology
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• 제38권2호
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• pp.85-91
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• 2014

Despite many researches related with in-vitro culture of porcine spematogonial stem cells (SSCs), adherent culture system widely used has shown a limitation in the maintenance of porcine SSC self-renewal. Therefore, in order to overcome this obstacle, suspension culture, which is known to have numerous advantage over adherent culture, was applied to the culture of porcine SSCs. Porcine SSCs retrieved from neonatal testes were suspension-cultured for 5 days or 20 days, and characteristics of suspension-cultured porcine SSCs including proliferation, alkaline phosphatase (AP) activity, and self-renewal-specific gene expression were investigated and compared with those of adherent-cultured porcine SSCs. As the results, the suspension-cultured porcine SSCs showed entirely non-proliferative and significantly higher rate of AP-positive cells and expression of self-renewal-specific genes than the adherent-cultured porcine SSCs. In addition, long-term culture of porcine SSCs in suspension condition induced significant decrease in the yield of AP staining-positive cells on post-day 10 of culture. These results showed that suspension culture was inappropriate to culture porcine SSCs, because the culture of porcine SSCs in suspension condition didn't stimulate proliferation and maintain AP activity of porcine SSCs, regardless of culture periods.

• Journal of Veterinary Science
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• 제21권1호
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• pp.13.1-13.14
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• 2020

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3+ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2006년도 제23차 정기총회 및 학술발표회
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• pp.43-58
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• 2006

As a bioreactor, bird has proved to be most efficient system for producing useful therapeutic proteins. More than half of the egg white protein content derives from the ovalbumin gene with four other proteins(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels of 50 milligrams or greater. And the naturally sterile egg also contains egg white protein at high concentration allowing for a long shelf life of recombinant protein without loss in activity. In spite of these advantages, transgenic procedures for the bird have lagged far behind because of its complex process of fertilized egg and developmental differences. Recently, a system to transplant mouse testis cells from a fertile donor male to the seminiferous tubules of an infertile recipient male has been developed. Spermatogenesis is generated from transplanted cells, and recipients are capable of transmitting the donor haplotype to progeny. After transplantation, primitive donor spermatogonia migrate to the basement membrane of recipient seminiferous tubules and begin proliferating. Eventually, these cells establish stable colonies with a characteristic appearance, which expands and produces differentiating germ cells, including mature spermatozoa. Thus, the transplanted cells self-renew and produce progeny that differentiate into fully functional spermatozoa. In this study, to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos, the spermatogonial stem cell techniques were applied. This system consisted of isolation and in vitro-culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.171-177
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• 2009

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• 한국발생생물학회지:발생과생식
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• 제24권4호
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• pp.249-261
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• 2020

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.