• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.48-48
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• 2003

본 실험은 spermatogonia 단계에 발현하는 유전자를 찾기 위하여 suppression subtractive hybridization를 수행하였다. 기존에 mouse에서는 spermatogonia 특이적인 유전자들이 밝혀져 있기 때문에 pig에 특이적인 유전자를 찾기 위하여 pig 250days testis와 pig 60days testis를 재료로 하여 실험하였다. SSH를 통하여 254days testis에 특이적으로 발현되는 후보유전자를 7개 찾았고 25days testis와 60days testis 의 Northern blot을 통하여 25days에 과발현하고 60days에 발현의 양이 대폭 줄어드는 spermatogonia 유전자로 생각되는 후보유전자 2개를 선택하여 pig tissue northern blot, genomic DNA southern blot, RT-PCR 그리고 In-situ hybridization을 수행하였다. Tissue northern blot과 RT-PCR을 통하여 후보자 1번은 간과 폐, 난소, 정소에서 발현하고, 후보유전자 15번은 난소와 정소에서만 특이적으로 발현함을 알았다. DNA sequence analysis와 NCBI Blast search를 통하여 후보자 1번은 다른 종에서 밝혀진 유전자였고 후보유전자 15번은 어느 종에서도 밝혀지지 않은 새로운 유전자였다. Degenerated primer를 통하여 후보자 1번의 pig full sequence를 밝히고 NCBI에 등록하였다. 그리고 In-situ hybridization을 통하여 후보유전자득이 20일째 testis의 Leydic cell에서 많이 발현되고 adult testis에서는 발현이 감소하는 결과를 얻었다. 이것으로 보아 위의 두 후보유전자는 spermatogonia에 직접 관련된 유전자이기 보다는 spermatogonia의 발달에 영향을 주는 leydic cell 특이발현을 가진 유전자로 사료되어진다.

• Korean Journal of Veterinary Research
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• v.26 no.2
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• pp.201-210
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• 1986

The cycle of the seminiferous epithelia in the testis of mature Korean native cattle was divided into twelve stages, using criteria the morphological changes in the acrosomic system and the nuclei of developing spematids and germ cells. The results were summarized as follows; 1. The minimum number of tripe A spermatogonia were the average of 1.8 in both at stages I and VI, while maximum numbers were the average of 4.2 at stage XII. Some type A spermatogonia divided at stage XII to produce the type intermediate(IN) spermatogonia at following stage I. The intermediate type spermatogonia divided at stage IV to produce the type B spermatogonia at stage V. 2. The type B spermatogonia divided at stage VII to produce the preleptotene primary spermatocytes at stage XII. The pachytene primary spermatocytes divided at stage XI to produce the secondary spermatocytes at stage VII. The secondary spermatocytes observed at stag XII divided to give rise to the round spermatids at following stage I. Each numbers of the first spermatocytes and of spermatids were almost constant, respectively, through the cycle of the seminiferous epitherium. 3. The relative frequencies of each stage among stages I to XII of the cycle of the seminiferous epithelia were 6.1, 3.7, 5.2, 7.8, 2.2. 3.3, 13.8, 18.4, 11.8, 7.2, 18.1% and 2.4%, respectively.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.35-41
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• 2005

This study was carried out for development of effective preservation on animal genetic resources. Spermatogonia cells are the germline stem cells and they can be restored to adult animal with proliferation and differentiation intentionally. When the spermatogonia cells were purified from seminiferous tubules and were cultured at $32^{\circ}C$, the cells were actively proliferated. The culture medium consisted of TCM199 plus $10\%$ FCS and coculture with Sertoli cells supported cultivation of spermatogonia cells. By passing 40 days of incubation, spermatogonia cells formed the germline colony or shape of ES-like colony or reconstruction of pseudo-seminifcrous tubule shape. At 40 days, the cultured cells were no sign for differentiation to spermatocyte or spermatid. The experiment of induced differentiation of this cells is needed.

• Korean Journal of Veterinary Research
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• v.32 no.3
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• pp.281-293
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• 1992

Classification of the cycle of seminiferous epithelia into 12 stages by the morphological changes in acrosomal system and evaluation of the relative frequency of stages and the cell association were histologically performed in the mature Korean native Jin-do dogs. The results were summarized as follows; 1. The minimum number of type A spermatogonia averaged 1.01 at stages I, while maximum number averaged 2.47 at stages XII. Some type A spermatogonia divided at stage XII to produce the type intermediate(IN) spermatogonia at the following stage I. The type IN spermatogonia divided at stage IV to produce the type B spermatogonia at stage V. 2. The type B spermatogonia divided at stage VI to produce the preleptotene primary spermatocytes at stage VII. The secondary spermatocytes observed at stage XII. The secondary spermatocytes observed at stage XII divided to give rise to the round spermatids at the following stage I. The numbers of the first spermatocytes and spermatids were almost constant, respectively, through all the cycles of seminiferous epithelium. 3. The acrosomal vesicle was invaginated to occupy one third to half of spermatid nucleus at the cap phase, which was different from that of rodent and ruminant spermatid nuclei. 4. The relative frequencies of stages I to XII of seminiferous epithelia cycle were 10.34, 4.84, 5.03, 8.22, 10.86, 6.63, 6.42, 18.88, 10.17, 6.18, 7.62% and 4.81%, respectively.

• Development and Reproduction
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• v.19 no.1
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• pp.1-10
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• 2015

The purpose of the present study was to examine the seminiferous epithelium cycle of Bombina orientalis using a light microscope. The cycle was divided into a total of 10 stages, according to the morphological characteristics of the cells. The spermatogenetic cells included primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatid and sperm. At stage I, the primary spermatogonia was located closer to basal lamina of the seminiferous tubule without spermatocyst formations. Especially at the stage II, the secondary spermatogonia were located in the spermatocyst. The primary and secondary spermatocytes were found from stages III to VI. The secondary spermatocytes were smaller in size than the primary spermatocytes, but they had thicker nucleoplasm and smaller nuclei. The round-shaped, early sperm cells were formed in stage VII, and further divided at stage VIII to have more concentrated nucleoplasm before division to matured sperm cells. At stage X, the matured sperm cells emerged from the spermatocyst. Considering the above results, this study presented the special characteristics in the generation and type of sperm formation. The germ cell formation occurred in various stages, like the perspectives of Franca et al (1999), ultimately, providing taxonomically useful information.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.23-36
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• 1993

To investigate the cycle and relative frequences and the fine structure of seminiferous epithelia in mature Jindo dogs, histologic study was performed. The results obtained were summarized as follows; 1. Type A spermatogonia appeared approximately 1.6 times as many at stage II as compared to stage I while type In spermatogonia appeared small amount in stage III, IV and V. type B spermatogonia were found during the stage VI to VIII, though not detectable during stage I to V. The type B spermatogonia divided at stage VII to produce the preleptotene primary spermatocytes at stage VIII. The number of primary spermatocytes of the leptotene phase markedly increased during stage I to II, and the primary spermatocytes of the pachytene phase were shown the least in number at stage IV. The secondary spermatocytes could be seen only at stage IV. 2. The relative frequencies of each stage from stages I to VIII of the cycle of seminiferous epithelia were 31.6, 11.9, 10.0, 3.2, 8.2, 10.1, 11.7 and 13.2% respectively. 3. On electron microscopic observations, acrosomal vesicle of spermatids appeared larger though the bulk of germ cells were the morphologically same as those of the other animal species. Thread line structures light microscopically observed in the cytoplasm of Sertoli cell were the longitudinal orientation of mitochondria.

• Radiation Oncology Journal
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• v.8 no.1
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• pp.17-27
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• 1990

The effects of both hyperthermia alone and X-ray irradiation combined with hyperthermia on rat testis have been investigated. The histological changes were observed on 15 and 30 days after treatment. There was no histological change of rat testis by hyperthermia alone. The earliest change by X-ray irradiation was the degeneration of the spermatogonia of the seminiferous tubule, which was appeared in 2 Gy group. Necrosis of the spermatogonia was severe in 6 Gy group and complete atrophy was developed in 8 Gy group. With increased dose of radiation, the degree of changes of tubules was increased. In combined group of X-ray irradiation and hyperthermia, the histological change of the seminiferous tubule was more severe than X-ray alone group. Necrosis and atrophy of the spermatogonia were appeared in 2 Gy and complete atrophy of spermatogonia was seen in 6 Gy group. Thermal enhancement ratio (calculated at the complete atrophy of the spermatogonia) was 1.3 in this experiment. There was no difference in observation time inverval between 15 and 30 days after each treatment in all groups.

• Animal cells and systems
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• v.8 no.3
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• pp.197-203
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• 2004

CD30 is a member of tumor necrosis factor receptor (TNFR) superfamily and has pleiotropic functions including cell activation, proliferation, differentiation, and death, depending on cell types and stage of differentiation. Although CD30 expression has been described mainly in hematopoietic tissues, several types of nonhematopoietic tumors including embryonic carcinoma and germ-cell tumors express CD30. We examined CD30 distribution in the testis and epididymis from wild type and CD30-deficient mice. In the testis, spermatogonia, spermatocytes and Sertoli cells expressed CD30, but not in spermatids. Spermatogonia and spermatocytes near the basement membrane strongly reacted to anti-CD30. In the epididymis, CD30 expression was exclusively observed in luminal epithelia and some interstitial cells. Taken together, these results show a spatio-temporal regulation of CD30 expression in mouse testis and epididymis and suggest a possible role of CD30 in spermatogonia and spermatocytes.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.387-396
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• 2005

The aims of the study were to establish a short-term screening test for detecting testicular toxicity of chemicals in rats and to determine whether a 2-week administration period is sufficient to detect testicular toxicity of 2-bromopropane (2-BP) as an example. Male Sprague-Dawley rats were subcutaneously administered with 1000 mg/kg/day of 2-BP or its vehicle for 2 weeks. Ten male rats each were sacrificed on days 3, 7 and 14 after the initiation of treatment. Parameters of testicular toxicity included genital organ weights, testicular sperm head counts, epididymal sperm counts, motility and morphology, and qualitative and quantitative histopathologic examinations. The early histopathological changes observed on day 3 of treatment included degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, and decreased number of spermatogonia in stages II and V. On day 7 of treatment, atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, multinuclear giant cells, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in stages VII and XII. On day 14 after treatment, a significant decrease in the weights of testes and seminal vesicles was found. Atrophy of seminiferous tubules, exfoliation of germ cells, degeneration of spermatogonia and spermatocytes, mature spermatid retention, vacuolization of Sertoli cells, decreased number of spermatogonia in stages II and V, and decreased number of spermatocytes in all spermatogenic stages were also observed. In addition, a slight non-significant decrease in testicular sperm head counts, daily sperm production rate and epididymal sperm counts was found. The results showed that 2 weeks of treatment is sufficient to detect the adverse effects of 2-BP on male reproductive organs. It is considered that the short-term testicular toxicity study established in this study can be a useful tool for screening the testicular toxic potential of new drug candidates in rats.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.63-65
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• 2002

Identification of spermatogonial stem cell-specific surface molecules is important in understanding the molecular mechanisms underlying the maintenance and differentiation of these cells. We have found that spermatogonia from busulfan treated mice expressed an autoantigen that distinguishes between undifferentiated and differentiated spermatogonia. Four to six weeks after busulfan treatment, germ cells located in the basal compartment of seminiferous epithelium show isotype-specific IgG deposits that form due to autoimmunity. Before busulfan treatment, the level of testicular IgG was very low but IgG levels began to increase after week 4 and peaked at week 6. When cells from the busulfan treated testis were analyzed using laser scanning cytomeoy (LSC), the frequency of cells positive for IgG deposits, 6-integrin, and 1-integrin were 16.5${\pm}$3.8%, 11.8${\pm}$2.6%, and 9.0${\pm}$ 1.4%, respectively. Immunofluorescent staining suggested that most, if not all of the cells with IgG-deposits isolated from a laminin-coated dish, were also positive for a spermatogonial stem cell marker \ulcorner6-integrins as well as for a germ cell-specific marker TRA 98. We determined serum and intratesticular IgG levels and the soundness of seminiferous tubule basement membrane from busulfan treated mice using electron microscopy, in order to study the mechanism responsible for IgG deposits in spermatogonia. We found that the basement membranes of seminiferous tubules from busulfan treated mice were severely impaired when compared to those of normal adult, neonates and w/wv mice. Furthermore, new blood cells were observed in the surface of the damaged basement membrane along the seminiferous tubules. These results suggest that the IgG in spermatogonial stem cells accumulates from circulating blood through the impaired basement membranes induced by busulfan treatment. Taken together, our study suggests that IgG can be used as a new marker for undifferentiated spermatogonia cells.