• Journal of Embryo Transfer
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• v.17 no.2
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• pp.137-143
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• 2002

This study was conducted to evaluate the effect of sugar kinds and combination of various sugars, and straw size and pre-freezing time on motility of post-thaw spermatozoa in canine. In general, the extender was supplemented with fructose or glucose for semen freezing. The extender used in .this study was Tris-citric acid extender (Tris-buffer) supplemented with 20％ Egg-yolk, 8％ glycerol, 1％ Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer. were combined such as control (fructose, xylose, trehalose), two combinations (Fru＋Tre, Fru＋Xy1, Tre＋Xy1) and three combinations (Fru＋Tre＋Xy1). The motility after CASA analysis in Fru＋Tre＋Xy1 was higher than those in fructose, trehalose, xylose, Fru＋Tre, Fru＋Xy1, Tre＋Xy1 (69 vs. 58, 61, 50, 65, 20, 54). However, the progressive motility after CASA analysis in Fru＋Tre group was higher than those in fructose, trehalose, xylose, Fru＋Xy1, Tre＋Xy1, Fru＋Tre＋Xy1 (59％ vs. 47, 55, 42, 13, 49, 44％). The survival rate of post-thaw spermatozoa in 0.25 $m\ell$ straw for 10 min pre-freezing was significantly higher than those in 0.25 $m\ell$ straw for 10 min, 0.25 and 0.5 $m\ell$ for 5 min (80＋0.0 vs. 65＋7, 68＋16, 58＋8％; P＜0.05). The results indicated that the progressive motility of post-thaw spermatozoa in 70 mM Fru ＋Tre (two combination) following CASA analysis and 0.25 ml straw for 10 min pre-freezing time could be better for freezing of semen in canine.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.91-96
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• 2003

The aim of this study was conducted to evaluate the effect of sugar kinds and combination of various sugars on acrosome damage of post-thaw spermatozoa in canine. The extender used in this study was Tris-citric acid extender (Tris-buffer) supplemented with 20% Egg-yolk, 8% glycerol, 1% Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer were combined such as control (fructose, xylose, trehalose), two combinations (Fru+Tre, Fru+Xyl, Tre+Xyl) and three combinations (Fru+Tre+Xyl). The acrosome damage rate of post-thaw spermatozoa in Eosin B & Fast Green stain in Fruc+Tre was higher than those in fructose, trehalose, xylose, Fruc+Xyl, Tre+Xyl, Fruc+Tre+Xyl (83.0$\pm$5.6 vs. 82.3$\pm$3.1%, 81.7$\pm$2.1%, 72.0$\pm$2.0%; 80.3$\pm$4.5%, 76.7$\pm$3.8%, 81.0$\pm$5.6). The motility after CASA analysis in Fru+Tre was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose(79$\pm$6 vs 75$\pm$3, 74$\pm$8, 71$\pm$11, 70$\pm$4, 66$\pm$15, 63$\pm$ 12%). However, the progressive motility after CASA analysis in Fru+Tre group was higher than those in Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose (67$\pm$7, 64$\pm$3, 62$\pm$6, 61$\pm$8, 60$\pm$2, 57$\pm$13, 53$\pm$10%). The results indicated that the acrosome damage & progressive motility of post-thaw spermatozoa in 70 mM Fruc+Tre (two combination) following Eosin B & Fast Green stain and CASA analysis.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Journal of Preventive Medicine and Public Health
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• v.38 no.3
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• pp.330-336
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• 2005

Objectives : The aim of this study was to investigate the effects of bisphenol A (BPA), an estrogen-like environmental endocrine disrupter, on the placental function and reproduction in rats. The mRNA levels of the placental prolactin-growth hormone(PRL-GH) gene family, placental trophoblast cell frequency and reproductive data were analyzed. Methods : The pregnancies of F344 Fisher rats ($160g{\pm}20g$) were detected by the presence of the copulatory plug or sperm in the vaginal smear, which marked Day 0 of pregnancy. Pregnant rats were divided into three groups. The control group was intraperitoneally injected with a sesame oil vehicle. The two remaining groups were injected with 50 or 500 mg/kg B.W/day of BPA, resuspended in sesame oil, on either days 7 to 11 or 16 to 20 of pregnancy, with the rats sacrificed on either day 11 or 20, respectively. The mRNA levels of PRL-GH and Pit-1a and b isotype genes were analyzed by Northern blot hybridization and reverse transcription-polymerase chain reaction. The hormone concentrations were analyzed by radioimmunoassay, and the frequency of the placental trophoblast cells observed by a histochemical study. Reproductive data, such as the placental weight and litter size, were surveyed on day 20. The fetal weight was surveyed for 4 weeks after birth. A statistical analysis was carried out using the SAS program (version 8.1). Results : The mRNA levels of the PRL-GH gene family, such as placental lactogen I, Iv and II, prolactin like protein A, C and Cv, and decidual prolactin-related protein were significantly reduced due to BPA exposure. The mRNA levels of the Pit-1a and b isotype genes, which induce the expression of the PRL-GH gene family in the rat placenta, were also reduced due to BPA exposure. The PL-Iv and PL-II concentrations were reduced in the BPA exposed group. During the middle to last stage of pregnancy (Days 11-20), a high dose of BPA exposure reduced the frequency of spongiotrophoblast cells, which are responsible for the secretion of the PRL-GH hormones. Reproductive data, such as the placental and fetal weights and the litter size, were reduced, but that of the pregnancy period was extended in the BPA exposed compared to the control group. Conclusions : BPA disrupts the placental functions in rats, which leads to reproductive disorders.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.165-174
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• 1999

Boar semen can be frozen successfully. However, there is a large variability in the extent of damage boar semen samples experiences during cryopreservation. This experiment was undertaken to find out factors that affect a post-thaw viability of boar spermatozoa. For this purpose, cryodiluents(BF5, LEY, Soejima and M-Soejima), cryoprotectants(glycerol. ethylene glycol, and propylene glycol), pre-freezing method(dryice-pellet, dryice-straw and L$N_2$vapour-st-raw) and total time required for freezing(2. 5, and 7 h) were compared as a factors. To investigate quality of semen during freezing process, motility(%), normal apical ridges(%, NAR), and proportion of living sperm(%) by flow cytometic analysis were assessed after collection, cooled, pre-frozen and post-thawing. Post-thaw motility of semen diluted with M-Soejima was 52.0%, respectively. When heparin, caffeine or heparin＋caffeine was added to 2nd cryodiluent of M-Soejima during freezing process, the highest motility after thawing was shown at the addition of caffeine (2mM), with 61.7$\pm$2.9% of motility. M-Soejima with heparin or caffeine was significantly higher than that of controI(p＜0.05). The result using glycerol(Gly), ethylene glycol(EG), propylene glycol(PG), and their mixture (Gly＋EG and Gly＋PG) as cryoprotectants, the highest motility was shown at the mixture treatment with Gly plus PG. However, the highest proportion of live spermatozoa was shown at Gly＋EG, there was no significantly difference among treatments(p＞0.05). When semen was pre-frozen with three manners(dryice-pellet, dryice-straw, and L$N_2$ vapor-straw), motility(%) of post-thaw spermatozoa was the highest in the L$N_2$ vapor-straw pre-freezing method of M-Soejima cryodiluent with 57.5% of motility, For a simple, economical and timesaving approach to freezing boar semen, total time required for freezing were 2, 5, and 7 hours, post-thaw motility were 43.8, 45.0 and 38.8%, NAR were 19.5, 22.7 and 28.5%, and viability were 20.8, 19.9 and 22.1%, respectively. This data suggests that boar semen diluted with M-Soejima cryodiluent contained caffeine, using mixture of glycerol and propylene glycol or ethylene glycol as cryoprotectants, frozen with 2 hours, can be taken better motility, NAR, and proportion of live spermatozoa.

• Development and Reproduction
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• v.6 no.2
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• pp.97-104
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• 2002

In most mammals, mature oocyte-cumulus complexer(OCCs) ovulate into the oviduct where fertilization by sperm takes place. However, the complex that fail to fertilize eventually undergoes degeneration while they reside in the oviduct. Yet there is no blown mechanism how both oocyte and cumulus cells degenerate. Using human follicular fluid (hFF), bovine oviductal tissue extract (BOX) and mouse OCC, the present study aimed to find how the oviduct influence the viability of the oocyte and cumulus cells in vitro. There was no difference of oocyte maturation rate between the control and BOX-treated groups. However, there was a significant difference in the survival of cumulus cells between two groups. Cumulus cells cultured in the presence of hFF alone underwent initially expansion and then they formed monolayer in the culture dish. Even after 72 hr, they proliferated well and showed fibroblast-like morphology. Cumulus cells cultured in the presence of both hFF and BOX also expanded after 24 hr, however, after 72 hr culture, they eventually detached and degenerated. Cumulus cells cultured in the BOX alone gave a similar drastic result. When the cumulus cells cultured in the presence of BOX were stained with DAPI, their nuclei showed partial condensation and fragmentation. After detailed analysis of these cells by TUNEL assay, many nuclei of them exhibited well stained spots indicating the signs of apoptosis. Based upon these observations, it is suggested that BOX might possess a factor that leads mouse cumulus cells to undergo apoptosis in vitro.

• Journal of Preventive Medicine and Public Health
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• v.37 no.2
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• pp.157-165
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• 2004

Objectives : This study aimed to investigate the toxic effects of chromium (VI) on the placental function and reproduction in rats. For the study, the placental prolactin-growth hormone (PRL-GH) gene expression, placental trophoblast cell differentiation and reproductive data were analyzed. Methods : The pregnancies of F344 Fisher rats were checked by the presence of a copulatory plug or sperm in the vaginal smear, which was defined as day 0 of the pregnancy. Pregnant rats were divided into the three groups. The control group was given tap water (chromium level < 0.001 ppm) and the remaining groups were given 250 or 750 ppm of chromium (VI) [as potassium dichromate], from day 7 to 19 of the pregnancy. Rats were sacrificed at days 11 and 20 of pregnancy. The mRNA levels of PRL-GH and Pit-1a and b isotype genes were analyzed by Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR). The hormonal concentration was analyzed by radioimmunoassay, and the differentiation of placental trophoblast cells were observed by histochemical studies. Reproductive data, such as placental and fetal weights, pregnancy period, and litter size, were surveyed at day 20 of pregnancy and after birth. A statistical analysis was carried out using the SAS program (version 8.1). Results : The mRNA levels of the prolactin-growth hormone (PRL-GH) family of genes were dose dependently reduced by chromium exposure. The mRNA levels of Pit-1a and b isotype genes that induce the expression of the PRL-GH family of genes were also reduced by chromium exposure. The PRL-GH hormonal concentration in the rat placenta, fetus and maternal blood were decreased by chromium exposure. In the middle stage of pregnancy (day 11), a high dose of chromium suppressed the differentiation of spongiotrophoblast cells that secret the PRLGH hormones. In the last stage of pregnancy (day 20), a high dose of chromium induced apoptosis of placental cells. Reproductive data, such as placental and fetal weights, litter size, were reduced, but the pregnancy period was extended in the group exposed to chromium compared with the controls. Conclusion : Chromium (VI) disrupts the ordered functions of the placenta, which leads to reproductive disorders in rats.

• Korean Journal of Poultry Science
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• v.39 no.4
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• pp.291-295
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• 2012

The use of methylacetamide (MA) as a cryoprotective agent for freezing Korean Native Black rooster Ogye semen was examined with artificial insemination. The diluted Ogye semen with HS-1 was subjected for 2 step dilution method of cryopreservation in which the final concentration of MA was adjusted to 7.5%. The sperm viability after thawing was reduced from $95.17{\pm}0.93%$ to $55.93{\pm}1.38%$ which was confirmed by live-death analysis based on Fluorescence-Activated Cell Sorting (FACS). The rates of fertilized eggs with fresh or frozen-thawed semen were reduced from $94.98{\pm}3.93%$ to $66.36{\pm}8.43%$ at day 7 with significant difference. However, the hatching rates of experiments at day 21 did not shown difference between $92.64{\pm}2.33%$ and $90.45{\pm}8.05%$ (P<0.05). With these results, the utilization of MA for freezing of Ogye spermatozoa could affect on viability of frozen-thawed semen but not on the fertility of lain eggs and hatchability of fertilized eggs and also provide possible tools of freezing for poultry genetic resource conservation.

• Toxicological Research
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• v.33 no.2
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• pp.107-118
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• 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.