• Development and Reproduction
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• v.8 no.1
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• pp.27-33
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• 2004

To investigate the mechanism of germ cell death in postnatal stage of mouse, the involvement of apoptotic executioners, caspase-3 and caspase-activated DNase(CAD), and apoptotic initiators, Bax Fas and Fas ligand, in the germ cell death has been studied. Immune-labels of active caspase-3 and CAD were located in TUNEL-positive, apoptotic, oocytes as well as normal oocytes of primary or secondary follicles. CAD immune-labels were also detected in the nucleus of TUNEL-positive oocytes. Most of oocytes showing positive immune-labeling of active caspase-3 or CAD had vacuoles in their cytoplasm, which is the morphological characteristic of oocyte during folliclar atresia. Bax immune-stains were detected in the atretic oocytes which showed the vacuole in their cytoplasm. Positive immune-labels for Fas ligand was localized in TUNEL-positive or atretic oocytes. Presence of immunoreactivity of active caspase-3 and CAD in TUNEL-positive germ cells implicate that active raspase-3 and CAD might play a role in germ cell apoptosis during early development of mouse ovarian follicle. Immunohistochemical localization of Bax and Fas ligand in TUNEL-positive oocytes suggests that these might be the most plausible modulator of oocyte apoptosis.

• The Korean Journal of Zoology
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• v.27 no.1
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• pp.13-24
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• 1984

The fertilized eggs of Rana dybowskii were irradiated with UV (254 nm wave length) on the vegetal hemisphere to investigate the effects on the primordial germ cells (PGCs) and axis formation. The investigations were carried out in two ways; namely time course and UV dose. Up to 1,600 $ergs/mm^2$ of UV dose, irradiated at 60 min. after fertilization, there was no effect on the PGC number. However, the number of PGC comparing with that of unirradiated control was decreased more than 40%. As the amount of irradiation was increased, the number of PGC was inversely declined. The maximal dose of irradiation which eliminates PGC completely without inducing any axis abnormality was 4,800 $ergs/mm^2$. If the eggs were irradiated earlier with this amount the severer effect could be obtained. Thus the UV effect on the PGC number was most effective when irradiated by 60 min. post fertilization. Thereasfter stage. At UV doses over 9,600 $erge/mm^2$ other effects start to appear; namely abnormalities of nerual tube and axis formation. Therefore, comparative study on the UV sensitivity of PGC and axis formation was carried out. It was revealed that UV effect on the axis was drastically decreased at the time of $0.7\\sim0.8$ between fertilization and 1st cleavage, while the germ plasm was sensitive to UV until 4 cell stage.

• Advances in Traditional Medicine
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• v.8 no.2
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• pp.111-124
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• 2008

In the present study, an attempt has been made to assess whether the effect of benzene extract of Ocimum sanctum leaves on the ultrastructural changes in the epithelial cells of the cauda epididymis, its subsequent recovery in the seminiferous epithelium and fertility of male albino rats. Wistar strain male albino rats were orally administered benzene extract of 250 mg/kg body weight of O. sanctum leaves followed by subsequent recovery maintaining suitable controls for 48 days. Results indicate decrease in the weights of testis, epididymis and seminal vesicles. Other accessory organs were not affected. Total count, cell and nuclei diameters of germ cells and Leydig cells were reduced. Cauda epididymis exhibited significant reduction in epithelial height and nuclei diameter of epithelial cells. Cells showed vacuolization with exhibit of signs of degeneration. Ultra study revealed that, in general, the cauda epididymis was affected and in particular, the principal, clear and basal cells were highly disturbed. Further, there was decrease in the size of lipid droplets, mitochondria, Golgi complex, endoplasmic reticulum and accumulation of lysosomal bodies. Fertility performance test showed no implantation in female rats mated with O. sanctum treated rats. Moreover, their recovery after withdrawal of treatment was observed suggesting that the effect of the treatment is transient and reversible. A recovery period resulted in normal spermatogenesis and fertility, suggesting reversible antispermatogenic and antifertility effects of the plant.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• Reproductive and Developmental Biology
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• v.39 no.2
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• pp.43-47
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• 2015

Sirtuin proteins are evolutionary conserved Sir2-related $NAD^+$-dependent deacetylases and regulate many of cellular processes such as metabolism, inflammation, transcription, and aging. Sirtuin contains activity of either ADP-ribosyltransferase or deacetyltranfease and their activity is dependent on the localization in cells. However, the expression pattern of Sirtuins has not been well studied. To examine the expression levels of Sirtuins, RT-PCR was performed using total RNAs from various tissues including liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Sirtuins were highly expressed in most of tissues including the testis. Immunostaining assay showed that Sirt1 and Sirt6 were mainly located in the nucleus of germ cells, spermatocytes, and spermatids in the seminiferous tubules, whereas Sirt2 and Sirt5 were exclusively present in the cytoplasm of germ cells and spermatocytes. Our results indicate that Sirtuins may function as regulators of spermatogenesis and their activities might be dependent on their location in the seminiferous tubules.

• Applied Microscopy
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• v.15 no.1
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• pp.1-12
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• 1985

In this study, the pathway and date of migrating Primordial germ cells (PGCs) were observed light microscopically and ultrastructural changes of them during migration were observed by electron microscopic examination. For these purpose, alkaline phosphatase reactions were used for identifying the PGCs and acid phosphatase reactions were used for observing their degenerating activities. Also, effects of actinomycin D on the migration of PGCs were examined. According to these results, at the 9th gestation day, PGCs were observed in the endodermal cells of yolk sac, at the 11th gestation day, they were seen in the hindgut and then entered into the dorsal mesentery by the 13th gestation day. At the 14th gestation day, they were located in the genital ridges. When PGCs were located in the hindgut and genital ridges, the positive reactions of alkaline phosphatase were dominated, but acid phosphatase reactions were limited in all stage except they were in dorsal mesentery. However, these reactions were lessened in case of actinomycin D treatment. By electron microscopic examination, PGCs had pseudopodia, tail process, trailing cytoplasm and nuage as the ultrastructural characteristics. In addition, these morphological features were damaged by actinomycin D treatment.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.47-47
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• 2001

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally,？ 2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p〈0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of ？2-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.72-78
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• 2001

It is possible ot prepare protoplasts of the zygomycete fungus, Phycomyces blakesleeanus, by digesting the cell wall of spore germlings with commercially available chitinase and chitosanase. However, the cells without any cell walls immediately form large aggregates, and thus, it is difficult to isolate the individually separated protoplasts. Inherent problem with the formation of aggregates in preparing protoplasts could be solved by the use of bovine serum albumin (BSA). As a result, we were able to prepare a large number of single protoplsts quickly and easily. We took time-lapse photomicrographs of the formation of protoplasts, and found that there were certain regions of the cell wall of spore germlings that were sensitive to chitinase and chitosanase, although the cell wall of the original spores is known to be insensitive to these enzymes. There are two kinds of cell walls on a spore germling; one with a bound wheat germ agglutinin (WGA), and the other a bound concanavalin A (ConA). Furthermore, only cells with walls which had bound WGA were able to regenerate, while those with walls with bound ConA were not able to regenerate.

• Parasites, Hosts and Diseases
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• v.25 no.2
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• pp.154-158
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• 1987

As a series of systematic classification of paramphistomes, the worms in the rumen and reticulum were collected on 214 Korean cattle slaughtered at Jeonju abattoir from January, 1986 to April, 1987 and were classified by means of morphology. Afterwards, the karyotype of Paramphistomum cervi(Zeder, 1790) was detected by means of modified air.drying method from germ cells of the worms. The results were summarized as follows: 1. In the chromosome number of 254 p. cervi, the haploid cell was n:9 and the diploid 2n=18. The meiotic divisions were observed frequently; 1,924 haploid and 32 diploid cells were reliable. Nine pairs of mitotic chromosomes were homologous in the metaphase stage, and the chromosomes were composed of five medium-sized metacentrics (m) , subtelocentrics(st) or submetacentrics(sm) and four small-siRed subtelocentrics(st) or submetacentrics(sm). Meiotic metaphase was composed of five medium and four small chromosomes in size. 2. As a series of C-banding method, C-band was showed in centromeric region from all of the haploid germ cells. Whereas chromosome No. 3 and 5 included heterochromatin on the tip region, chromosome No. 4 on the distal region and No. 6 proximal region. And chromosomes No. 2 and 8 showed a remarkable C-band distinguished from other chromosomes.

• Molecules and Cells
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• v.23 no.1
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• pp.49-56
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• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.