• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.86-86
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. (omitted)

• 한국수정란이식학회지
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• 제25권2호
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• pp.111-116
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• 2010

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/$cm^2$, 1h) and $H_2O_2$ (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to $H_2O_2$. However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.33-40
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• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.69-69
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• 2002

Transgenic animals production tools have been valuable for research and purpose. The current methods of gene transfer, microinjection and nuclear transfer, which are widely used in transgenic animal production, but all most methods has only had limited success in production of larger species. Here, we report the possibility of a sperm-mediated gene transfer method in porcine embryos. Oocytes were collected from ovaries harvested at a local slaughterhouse were matured in 500${mu}ell$ drops of TCM-199 under mineral oil at 38.5$^{\circ}C$ in a humidified atmosphere of 5%CO2 in air. After 42-43h of in vitro maturation oocytes were denuded. for sperm injection into the cytoplasm of the porcine oocytes, sperm suspension in NIM medium are subjected extraction with TritonX-100 before mixing with a green fluorescent gene (GFP). Sperm with Tritonx-100 were prepared by adding TritonX-100 to a final volume of 0.05% in the sperm suspension and mixing by trituration for 60s before two wishes in NIM medium at 2$^{\circ}C$. A(ter wishing, sperm were mixed with TritonX-100 at $25^{\circ}C$ followed by washes at 2$^{\circ}C$. Sperm were resuspended in ice cold NIM to a final volume of 400${mu}ell$ and 2-20ng/${mu}ell$ DNA were triturated on ice for 60s. All microinjection was performed in HEPES-buffered CZB medium at room temperature within 2h. After culture in NCSU-23 for 72h, percent of porcine embryos transfected GFP gene are 20.7%(6/29) in 20ng/${mu}ell$ sperm-DNA mixed group and other groups were 3.7 %(2/54)and 4.7%(3/67). These data suggests that sperm-mediated gene transfer method should be used to the production tool of transgenic pig efficiently.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.56-56
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• 2001

The exogenous gene transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently used to produce transgenic mice and pigs. Sperm-mediated DNA transfer has the potential to markedly simplify the generation of transgenic animals. This method may serve as an alternative to the pronucleus injection of DNA for the production of transgenic pigs. Therefore, in this study, we investigated the expression of transgene after co-injection of spermatozoon or sperm head with green fluorescent protein (GFP) gene into in vitro matured porcine oocytes. Spermatozoon and sperm head, that was obtained by sonication, were treated with 0.03% Triton X-100 to remove the membrane. They were preincubated with linearized pEGFP-N1 for 1 min, and then embryos cultured NCSU23 medium for 2.5 days after co-injected of sperm and DNA. We monitored expression of GFP in embryos under epifluorescent microscope. The remove of sperm membrane did not alter the developmental competence of embryos after ICSI. At 7 days following injection, the rates of blastocysts following injection of intact sperm (15.0%), and of sperm with disrupted membrane (14.2%) were higher than that following IVF (10.0%). Porcine oocytes injected with sperm which co-cultured with DNA concentration of 1, 0.1, and 0.01 ng were 60, 65.7 and 75% and 18.5, 37.4 and 22.2% for rates of cleavage and GFP expression, respectively. In vitro matured porcine oocytes injected with sperm and isolated sperm head resulted in 69 and 59.7% of cleavage rates, respectively The rates of embryo GFP expressed did not significantly different between sperm (20.4%) and sperm head (20.0%) injection. The transgenic embryos with the clusters of positive blastomeres were observed under fluorescent microscope. Most of embryos expressed GFP gene showed mosaicism. They showed GFP expression at 1/4, 2/4 and 3/4 of blastomeres at the 4-cell stage. Among these 4-cell embryos, the expression rate of 1/4 blastomere group (54.6%) was higher than the other groups (15.3-30.7%). These results indicate that membrane disrupted sperm could attach with exogenous DNA, and that this procedure may be useful to introduce foreign gene into porcine oocytes. Therefore, our data suggest that the ICSI car be a useful tool to efficiently produce transgenic pig as well as other mammals.

• Toxicological Research
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• 제21권2호
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• pp.129-134
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• 2005

Styrene is a commercially important chemical used mainly in the production of plastics. A toxic effect exerted by styrene exposure may cause infertility, congenital anomalies or death in offspring. Treatment with styrene for 0, 50, 100, and 500 mg/kg for 5 days in Sprague-Dawley rats significantly decreased sperm motilities and sperm counts while sperm abnormalities were significantly increased. To determine the relationship between changes in sperm motilities and roles of reactive oxygen species (ROS), we determined the effect of styrene on ROS production and mRNA expression of antioxidant enzymes in rats. ROS production was enhanced by styrene treatment in a dose-dependent manner. The mRNA expression of catalase and superoxide dismutase (SOD) 2 was strongly suppressed by styrene treatment although SOD1 or glutathione peroxidase (GPX) 4 expressions were not significantly changed. Taken together, these results indicate that styrene may cause toxic effect in rat sperm cells by enhancing oxidative stresses.

• Clinical and Experimental Reproductive Medicine
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• 제45권3호
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• pp.101-109
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• 2018

In an age when a small quantity of sperm can lead to pregnancy through in vitro fertilization or intracytoplasmic sperm injection, selecting healthy sperm is important. Sperm DNA fragmentation (SDF) is known to be higher in infertile men. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the alkaline comet test are SDF tests that directly measure DNA damage and have shown closer correlations with assisted reproduction results than indirect tools such as the sperm chromatin structure assay or the sperm chromatic dispersion test. It is difficult; however, to endorse a single test as the best test overall; instead, it is best to select a testing method based on each patient's clinical condition and goals. In a couple struggling with infertility, if the male partner has a high level of SDF, he should aim to decrease SDF through lifestyle modifications, antioxidant treatment, and ensuring an appropriate duration of abstinence, and physicians need to treat the underlying diseases of such patients. If sperm DNA damage continues despite the patient's and physician's efforts, other methods, such as micromanipulation-based sperm selection or testicular sperm extraction, should be used to select healthy sperm with nuclear DNA integrity.

• 농업과학연구
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• 제47권4호
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• pp.979-985
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• 2020

A wide range of techniques have been developed to separate X or Y- chromosome-bearing sperm. In particular, bovine semen sex-sorted by using flow cytometry based on differences in the amount of DNA between X and Y chromosome bearing sperm is used in dairy farms. The first piglets were produced using sex-sorted sperm 30 years ago. However, sexed sperm have not been commercially available in pigs because the flow cytometry technique is not capable of sorting the high number of sperm required for porcine artificial insemination (AI), and the prolonged exposure to an electrical filed might damage to the DNA in sperm. The purpose of this study was to evaluate a boar sperm sorting method based on magnetic nanoparticles. A flow cytometer assay verified the efficacy of the magnetic nanoparticles (> 90% of sex-sorted sperm). In addition, a duplex polymerase chain reaction (PCR) assay using sex chromosome specific genes including SRY (sex-determining region Y; male), ZFY (zinc finger protein Y-linked; male), and ZFX (zinc finger protein X-linked; female) showed that in vitro fertilized porcine embryos by X and Y-chromosome bearing sperm were 100% female (40/40) and 72% female (35/48), respectively, at 8-cell or morula stages, suggesting that the sex-sorted sperm were fertile. In conclusion, our findings suggest that the sex-sorted method based on magnetic nanoparticles can be utilized for porcine sex-sorted AI.

• 한국수정란이식학회지
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• 제17권1호
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• pp.45-53
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• 2002

본 연구는 sperm-mediated gene transfer를 이용하여 ICSI에 의한 형질전환동물 생산의 기초자료로서 활용하기 위해, ICSI에 사용될 정자의 조건과, 그에 따라 적합한 돼지 정자와 외래유전자의 전처리 및 ICSI를 통한 수정을 및 후기 배로의 발달율과 외래유전자의 발현 여부를 조사하여 다음과 같은 결과를 얻었다. ICSI에 이용될 정자의 조건에 따라 정소상체미부정자, 사출정자 및 동결정자를 이용하여 ICSI후 수정율은 각각 72.3%, 64.1% 및 74.1%로 나타나 유의적인 차이를 나타내지 않았으며, 또한 후기배로의 발달율에 있어서도 각각 17.6%, 18.7% 및 15.0%로 나타나 각 처리군간의 유의적인 차이는 나타나지 않았다. 그리고, ICSI후 전기적 자극을 실시한 군과 실시하지 않은 군에서 난자의 활성화에 따른 수정율은 대조구로 이용한 shame injection과 전기적 활성화를 실시하지 않은 군에서 각각 47.1%와 46.3%로 나타나 전기적 활성화를 실시한 군의 79.6%에 비해 유의적인 차이를 나타내었다. 후기배로의 발달율에 있어서도 전기적 활성화를 실시한 군에서는 24.1%로 나타나 전기 적 활성화를 실시하지 않은 군에서의 14.4%와 유의적인 차이를 나타내었다. 그리고 대조구로 이용한 shame injection에 있어서 후기 배로의 발달율은 2.5%로 낮은 결과를 나타내었다. 또한, 정자와 pcDNA LacZ유전자의 처리시 electroporation 방법을 실시하여 ICSI후 난자를 각각 전기적 활성화를 실시한 군과 실시하지 않은 군에 있어서 유전자 발현율은 각각 30.2%와 24.2% 나타났으나, 두 처리군간에 유의적인 차이는 나타나지 않았다. 그러나, 두 군에서 pcDNA LacZ 유전자는 모두 mosaic 발헌 양상을 보였다. 이상의 실험 결과들을 종합해 보면, ICSI에 사용될 수 있는 돼지의 정자는 정소상체미부정자, 사출정자 및 동결정자 모두가 이용 가능하며, ICSI후 추가 전기적 자극에 의한 난자의 활성화가 수정율과 후기 배로의 발달율을 향상시킬 수 있음을 시사하였다. 따라서, 돼지에 있어서 정자의 외래유전자 도입에 대한 정확하고 실용적인 방법은 보고되고 있지는 않은 상태로, 정자와 외래유전자의 처리법을 향상시키기 위하여 다양한 방법으로 많은 연구가 요구된다.

• Mycobiology
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• 제36권4호
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• pp.255-259
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• 2008

The effect of aflatoxin-contaminated corn on albino mice was investigated using the sperm morphology assay. Blood parameter levels including; total white blood cells (WBC), total red blood cells (RBC), packed cell volume (PCV), serum bilirubin (SB) and fasting blood sugar (FBS) were also determined in the tested mice. Test mice were exposed to aflatoxin-contaminated corn (contamination level of 100 ppb) for $1{\sim}4$ weeks while aflatoxin-free corn and cyclophosphamide were used as negative and positive controls, respectively. Sperm cells showed varieties of morphological abnormality when assessed after 5 weeks. The percentage frequencies of the negative and positive controls were 18.8% and 48.87%, respectively, while the percentage abnormalities for the 1, 2, 3 and 4 weeks exposures were 41.38%, 48.17%, 57.13% and 61.67%, respectively. PCV, WBC, total bilirubin and glucose level values of mice in all concentrations were higher and statistically significant as compared to the negative control values using Dunnett's test. Therefore, abnormal sperm cell induction is concentrationdependent such that continuous consumption of aflatoxin-contaminated corn is capable of negatively affecting spermatogenesis by inducing or increasing the frequency of morphologically abnormal sperm cells produced.