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

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

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

• Clinical and Experimental Reproductive Medicine
• /
• 제50권4호
• /
• pp.213-222
• /
• 2023

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

• Toxicological Research
• /
• 제33권2호
• /
• pp.107-118
• /
• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

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

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
• /
• pp.4-4
• /
• 2002

Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. Spontaneous apoptosis of germ cells was observed in the testis of adult mice and experimentally induced busulfan treated mice increased this apoptosis to such an extent that there was a decrease in the weight of the testis. (omitted)

• 한국수정란이식학회지
• /
• 제32권4호
• /
• pp.343-351
• /
• 2017

Porcine spermatogonial stem cells (SSCs) prefer three-dimensional (3D) culture systems to 2D ones for the maintenance of self-renewal. Of the many 3D culture systems, agar-based hydrogels are candidates for supporting porcine SSC self-renewal, and there are various types of agar powder that can be used. In this study, we sought to identify an agar-based 3D hydrogel system that exhibited strong efficacy in the maintenance of porcine SSC self-renewal. First, 3D hydrogels with different mechanics were prepared with various concentrations of Bacto agar, lysogeny broth (LB) agar, and agarose powder, and the 3D hydrogel with the strongest alkaline phosphatase (AP) activity and greatest increase in colony size was identified for the different types of agar powder. Second, among the porcine SSCs cultured in the different 3D hydrogels, we analyzed the colony formation, morphology, and size; AP activity; and transcription and translation of porcine SSC-related genes, and these were compared to determine the optimal 3D hydrogel system for the maintenance of porcine SSC self-renewal. We found that 0.6% (w/v) Bacto agar-, 1% (w/v) LB agar-, and 0.2% (w/v) agarose-based 3D hydrogels showed the strongest maintenance of AP activity and the most pronounced increase in colony size in the culture of porcine SSCs. Moreover, among these hydrogels, the strongest transcription and translation of porcine SSC-related genes and largest colony size were detected in porcine SSCs cultured in the 0.2% (w/v) agarose-based 3D hydrogel, whereas there were no significant differences in colony formation and morphology. These results demonstrate that the 0.2% (w/v) agarose-based 3D hydrogel can be effectively used for the maintenance of porcine SSC self-renewal.

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

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.21-21
• /
• 2003

Male germ cell apoptosis has been extensively explored in rodent. In contrast, very little is known about their susceptibility to apoptosis stimuli of developing germ cell stages at the time when germ cell depletion after busulfan treatment occurs. Furthermore, it is still unanswered how spermatogonial stem cells are resistant to busulfan treatment. We examined the change of gene expression in detail using cDNA microarray analysis of mouse testis treated with busulfan. A subtoxic dose of busulfan (40mg/kg of body weight) transiently increased 228 mRNA levels among of the 8000 genes analyzed. TagMan analysis confirmed that the mRNA levels such as defensive protein, support protein, enzymatic protein, transport protein, and hormonal protein were rapidly increased. These results were re-confirmed by real-time PCR analysis. However, the expression levels of these genes induced by busulfan treatment were significantly reduced in control testis, indicating that both of male germ cells and somatic cells after busulfan treatment induces self-defense mechanism for protection of testicular cell death. Among them, we conclude that defense proteins play a key role in testis injury induced by busulfan.