• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.4-4
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• 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)

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.21-21
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• 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.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.54-61
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• 2023

Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro. This basic method of germ cell generation might be helpful in the prospective applications of this technology.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.213-218
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• 2015

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.2
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• pp.75-81
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• 2018

Objective: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. Methods: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at $36^{\circ}C$ for 3 more weeks. Results: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. Conclusion: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.

• Animal cells and systems
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• v.2 no.4
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• pp.495-500
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• 1998

D-type G1 cyclins are known to be crucial for the progression of mitotic cell cycle in mammals. Although many studies have been performed to elucidate the roles of D-type cyclins, it is largely unknown whether D-type cyclins are directly involved in the regulation of meiotic germ cell development. In the present study, we examined the expression patterns of D-type cyclins (cyclin D1 and D3) during male germ cell development by northern blot and in situ Hybridization analyses. In the adult testes, we detected a 4.2 kb cyclin D1 mRNA and two different sizes (2.3 kb and 1.8 kb) of cyclinD3 mRNAs. The short form of the cyclin D3 transcript was testis-specific. Along with the testicular development, expression of cyclin D3 mRNA was increased whereas cyclin D1 mRNA was gradually decreased. in situ hybridization study also revealed that the expression of cyclin D3 was restricted to the postmeiotic germ cells. Furthermore, the 2.3 kb transcript was highly expressed in the round spermatids and decreased in the elongated spermatids/residual bodies, while the 1.8 kb transcript was expressed in elongated spermatids/residual bodies more abundantly. Sucrose-gradient separation of polysomal RNA fractions demonstrated that some portions of the 2.3 kb transcript are translationally active, while the 1.8 kb transcript is likely to be inactive. Taken together, the present data suggest a functional importance of cyclin D3 expression in the differentiated postmeiotic male germ cells.

• Animal cells and systems
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• v.8 no.1
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• pp.65-70
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• 2004

Rbm is a male infertility gene located in the AZFb region of the Y chromosome. Expression pattern of Rbm indicates that Rbm is critical for early phase of male germ cell development. It shares strong structural homology with hnRNP G, suggesting a function as an RNA processing factor. In order to gain a clue on the molecular mechanisms of Rbm on male germ cell development, we examined interactions of Rbm with selected proteins in yeast. The results revealed specific interactions between Rbm, hnRNP K and Tra2${\alpha}$. These results suggest that hnRNP K and Tra2${\alpha}$ may be functional partners of Rbm in male germ cells. We propose a model in which hnRNP K may playa role as a platform for Rbm and Tra2${\alpha}$.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.43-52
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• 1996

This study was carried out to establish the in vitro short-term culture system of developing male germ cells by purifing germ cells of various stages. The decapulated testicular cells were incubated with collagenase (lmg/ml) and try psin (2.5mg/ml) in HBSS. After separating male germ cell, the separated germ cells were stained with heamatoxylin/eosin and determined developing stages under light microscopy. The purity of pachtene spermatocytes a and round spermatid were 85%, respectively. Yield of total male germ cells was highly variable between individuals, with a mean value of 3.5 to 4.5 ${\times}$ 10$^7$ cells/testis. Viability of the cell was over 97% after separation. In DMEM medium, the optimal cell number for culture is approximately 1 x 10$^5$ cells/dish, but low cell den-sities than 1 ${\times}$ 10$^5$ cell/dish showed a decreased cell viability. Furthermore, about :36.8% of pac-hytene cells was successfully cultured for 6 days and some of cells were developed to secondary spermatids and round spermatids. Therefore, our data suggested that this culture conditions will be utilize as a feasible tools to produce tran-sgenic livestock using techniques such as intrac-ytoplasmic injection and cell fusion.

• Journal of Radiation Protection and Research
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• v.10 no.1
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• pp.29-40
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• 1985

The objectives of present study is to investigate genetic damage of radiation in mammalian male germ cell and. to establish available screening method for determining genetic hazard by radiation. Several methods were employed to measure the genetic damage of radiation as follows: Sperm head counts, frequency occurrence of sperm with abnormal head shape, fertility, activity of LDH-X, and the induction of unscheduled DNA synthesis (U.D.S.) in male mouse were performed with the passing of time after irradiation by making use of the sequence of event that occurs during spermatogenesis. Sperm head counts and activity of LDH-X in testes were gradually reduced by increased radiation dose and with the passing of the time after irradiation. Frequency occurrence of sperm with abnormal head shape, sterile period, and the induction of unscheduled DNA synthesis were increased by increased radiation dose. It is suggested that since germ cell is a direct reflection of genetic complement, the use of male germ cell is rapid and convenient method for measuring genetic damage by radiation.

• Herbal Formula Science
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• v.29 no.1
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• pp.1-8
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• 2021

Objectives : The purpose of this study was to investigate the antioxidant activity of water extract of Ojayeonjonghwan (OYH) in GC-1 germ cell lines. Methods : DPPH radical scavenging activity and cell viability assays in GC-1 germ cell lines were performed. In addition, the protective effects of OYH against hydrogen peroxide-induced oxidative stress in GC-1 germ cell lines were examined by measuring cell viability after H2O2 treatmet. The formation of ROS and the antioxidant enzymes activity such as SOD and catalase were measured in the same condition. Results : OYH scavenged DPPH radical dose-dependent manner and the IC50 was 63.79 ㎍/ml. OYH showed no cytotoxicity at concentration of 1, 10, 100 ㎍/ml. The hydrogen peroxide-induced cytotoxicity of GC-1 germ cell lines was protected to 53.66% by OYH at concentration of 10 ㎍/ml. OYH effectively inhibited ROS production in GC-1 germ cell lines. Mn SOD and catalase protein expression were significantly increased in GC-1 germ cell lines, but Cu/Zn SOD protein expression was not significantly changed. Conclusions : In conclusion, OYH has antioxidant activities against hydrogen peroxide-induced oxidative stress in GC-1 germ cell lines.