개 동결 정자의 apoptosis, oxidative stress, DNA integrity에 관한 연구

김수희 전북대학교 대학원 2009 국내박사

The aims of the present study were designed to investigate impact of freezing-thawing process on apoptosis, oxidative stress and DNA integrity in addition to sperm conventional parameters and to compare the effects of sperm separation techniques (Glass wool [GW] filtration and Percoll density gradient [PDG] centrifugation) on removal of cryo-damage in canine sperm. And also, this study was conducted to determine correlations between sperm conventional parameters (progressive motility, viability and abnormality) and advanced parameters (membrane integrity, apoptosis, ROS production and DNA integrity), and correlations among advanced parameters. Five healthy and sexually mature dogs were used in this study, and semen was obtained by digital manipulation, cryopreserved and thawed. After thawing of cryopreserved semen, sperm conventional parameters such as sperm motility, viability and morphological defects were evaluated for comparison with fresh semen. Sperm membrane integrity (HOS test and 6-CFDA/PI fluorescent test), apoptosis (Annexin V [AN]/PI assay), intracellular H₂O₂ level (DCFDA/PI assay) and DNA integrity (SCSA) were also evaluated before and after freezing-thawing process. And thawed semen was treated by sperm separation techniques using glass wool and Percoll to identify the effect of them on removal of cryo-damaged sperm, and was also evaluated like the preceding before and after treatment. In addition, H₂O₂ stimulation test and recovery rate were evaluated in treated samples. Freezing-thawing process decreased motility, viability, normal morphology and membrane integrity in canine sperm as previously reported (p < 0.05). This process also decreased AN-/PI- sperm, and increased apoptotic index, intracellular H₂O₂ level of viable sperm and DNA fragmentation (p < 0.05). In the correlations among parameters, progressive motility and viability positively correlated with AN-/PI-, CFDA+/PI- and HOST responsive sperm, while negatively correlated with apoptotic index and intracellular H₂O₂ level of viable sperm. Abnormal morphology positively correlated with AN+/PI+ sperm and apoptotic index, while negatively correlated with CFDA+/PI- and HOST responsive sperm. The sperm samples treated by GW filtration after thawing showed significant increase in sperm motility, viability, normal morphology, membrane integrity and AN-/PI- sperm compared with whole samples (before filtration) (p < 0.05). In basal ROS level of viable sperm fraction, the GW filtrated samples showed significant decrease in the percentage of sperm with high intracellular H₂O₂ level and MFI value compared with whole samples, whereas PDG centrifuged samples just showed significant decrease in MFI value compared with whole samples (p < 0.05). When H₂O₂ stimulation was given in two separation groups, PDG centrifuged viable spermatozoa showed significantly higher increasing rate in intracellular H₂O₂ level compared with GW filtrated viable spermatozoa (p < 0.05). DNA fragmentation was no significant difference among whole and the two sperm treatments (p > 0.05). The recovery rate of progressive motile, viable, CFDA+/PI-, AN-/PI- and DCF-/PI- sperm was higher in GW filtrated samples than PDG centrifuged samples (p < 0.05). In the correlations among parameters, progressive motility and viability positively correlated with CFDA+/PI- and HOST responsive sperm, while negatively correlated with AN-/PI+ sperm. In particular, viability also negatively correlated with the percentage of sperm with high intracellular H₂O₂ level of viable sperm fraction. Abnormal morphology did not show correlations with all advanced parameters. In the correlations among advanced parameters, increase in apoptotic index positively correlated with increase in intracellular H₂O₂ level of viable sperm, but not correlated with the others. Increase in intracellular H₂O₂ level of viable sperm positively correlated with increase in DFI, while negatively correlated with increase in HOST responsive sperm. In conclusion, the freezing-thawing procedure significantly affects membrane integrity, apoptosis, ROS production and DNA integrity as well as conventional parameters in canine semen. It is therefore recommended that these parameters be used as an additional parameter for the assessment of sperm quality after freezing-thawing in canine semen. And these cryo-damages can be improved by treatment of GW filtration after freezing-thawing. It is considered that GW filtration may rather lower the risk of selecting sperm with abnormal function for ART. 이 연구는 개의 정액에서 동결이 일반적인 정자성상 뿐만 아니라 apoptosis, 산화적 스트레스, DNA integrity에 미치는 영향을 조사하고자 수행되었으며 동결·융해 후 glass wool (GW) 여과와 Percoll density gradient (PDG) 원심분리법과 같은 정자처리기법이 동결 손상된 정자의 제거에 이용될 수 있는지 알아보고자 수행되었다. 또한 도출된 결과를 이용하여 일반적인 정자성상과 정자 세포의 apoptosis, 산화적 스트레스, DNA integrity와의 상관관계를 규명함으로써 정자에 대한 기존의 일반 성상 검사를 포함하여 정자 핵 및 정자막의 손상과 대사기능의 변화를 평가하여 그 결과 생존성과 수정능력이 높은 정자를 선별하는 기초자료 및 선별방법을 제시하고자 하였다. 실험동물로서 5 마리의 성견 (Beagle 4 두, 슈나우저 1 두)을 사용하여 수지법으로 정액을 채취하였고 채취한 정액은 Sweden 배지를 이용하여 동결하였다. 정자의 동결전과 동결후를 비교하기 위하여 정자 운동성, 생존성, 기형율과 같은 일반적인 정자성상 검사를 수행하였고 정자 세포막의 온전성을 조사하기 위하여 HOS test와 6-CFDA/PI fluorescent test를, apoptosis를 조사하기 위하여 Annexin V (AN)/PI assay를, 세포 내 H₂O₂ 수준 조사를 위하여 DCFDA/PI assay를, 그리고 DNA 온전성을 조사하기 위하여 SCSA와 같은 검사들을 수행하여 평가하였다. 또한 융해된 정액은 생존성과 정상형태율이 높은 정자를 얻기 위한 방법으로 glass wool과 Percoll 처리 후 회수하였으며 회수된 정자들은 상기와 동일한 방법으로 평가되었다. 이 연구의 결과에서 개 정자의 동결-융해 과정은 정자의 운동성, 생존성 및 세포막의 온전성을 감소시켰으며 기형율을 증가시킨 것으로 확인되었다 (p < 0.05). 또한 동결 및 융해 후 동결 전 희석정액에 비해 AN-/PI- 정자의 감소가 나타났고, 정자의 apoptotic index와 살아있는 정자의 세포 내 H₂O₂ 수준 및 DNA fragmentation의 증가가 나타났다 (p < 0.05). 평가인자들의 상관관계 분석 결과, 개 정자의 전진 운동성과 생존성은 AN-/PI-, CFDA+/PI- 그리고 HOST 반응 정자들과 positive 상관관계를 보였으나, apoptotic index와 살아있는 정자 중 높은 H₂O₂ 수준을 가진 정자율과는 negative 상관관계를 보였다. 기형율은 AN+/PI+ 정자, apoptotic index와는 positive 상관관계, CFDA+/PI- 및 HOST 반응 정자들과는 negative 상관관계를 나타내었다. 융해 후 GW 여과에 의해 처리된 정자는 처리하지 않은 정자보다 운동성, 생존성, 형태학적 정상 정자율, 세포막 온전성 그리고 AN-/PI- 정자율에서 증가를 나타냄으로써 이 방법을 통해 더 양호한 정자를 선별할 수 있음이 인정되었다 (p < 0.05). 세포 내 H₂O₂ 수준 검사 결과, H₂O₂로 자극하지 않은 경우, GW 여과된 정자는 대조군에 비해 살아있는 정자에서 높은 수준의 H₂O₂를 가진 정자율 및 Mean Fluorescence Intensity (MFI)에서 감소를 보였으며 (p < 0.05), PDG 원심분리된 정자도 대조군에 비해 MFI에서 감소를 보였다 (p < 0.05). GW 및 Percoll 처리군을 H₂O₂로 자극하였을 경우, PDG 원심분리된 정자는 GW 여과된 정자에 비해 높은 H₂O₂ 증가율을 보였다 (p < 0.05). 그러나 GW 및 PDG에 의해 처리된 정자의 DNA fragmentation율은 대조군의 DNA fragmentation율과 유의적인 차이는 인정되지 않았다. 정자 처리 후 전진 운동성, 생존성, CFDA+/PI-, AN-/PI- 및 DCF-/PI- 정자의 회수율 비교에서, GW 여과군은 PDG 원심분리군보다 정자수의 높은 회수율을 보임으로써, GW 여과군은 PDG 원심분리군에 비해 전진 운동성, 생존성, 세포막 온전성, apoptosis, ROS 평가에서 더 양호하며 더 많은 수의 정자를 회수할 수 있음이 인정되었다 (p < 0.05). 이상의 결과에서 평가인자들의 상관관계 분석 결과, 개 정자의 전진 운동성과 생존성은 CFDA+/PI- 및 HOST 반응 정자들과 positive 상관관계를 보였으나, AN-/PI+ 정자와는 negative 상관관계를 나타내었다. 또한 생존성은 살아있는 정자 중 높은 수준의 H₂O₂를 가진 정자율과도 negative 상관관계를 나타내었다. Apoptotic index의 증가율은 살아있는 정자의 H₂O₂ 증가율과 positive 상관관계를 보였으며, 살아있는 정자의 H₂O₂ 증가율은 DNA fragmentation index (DFI) 증가율과 positive 상관관계를, HOST 반응 정자 증가율과는 negative 상관관계를 나타내었다. 이 연구의 결론으로서, 동결-융해 과정은 개 정액의 일반적인 정자 성상뿐만 아니라 정자 세포막 온전성, apoptosis, ROS 생성 및 DNA 온전성에 영향을 미치는 것으로 나타났다. 따라서, 정자의 핵 상태 및 대사기능을 판정하는 방법들은 동결·융해 후 정액의 질을 평가하고 정자의 생존성과 수정능력을 판정하는데 보다 정확한 방법이 될 것으로 판단된다. 또한 이러한 정자의 동결 손상은 동결-융해 후 GW 여과 처리에 의해 향상될 수 있으며 이는 체외수정 및 ICSI와 같은 번식공학에 적용시 보다 생존성과 수정능력이 높은 정자를 선택할 수 있을 것으로 생각된다. 본 연구는 개의 동결정자의 핵 상태와 대사기능의 변화를 조사한 최초의 보고로서 향후 적절한 동결 프로그램의 개발을 위한 기초자료가 될 것이며, 개 동결정액의 수정능력을 향상시킬 수 있는 정자 처리방안을 구축하는데 효과적으로 활용될 수 있을 것으로 판단된다.

Studies on eenome editing in avian species for acquiring resistances against to avian leukosis virus and site-specific recombination

이홍조 서울대학교 대학원 2017 국내석사

Avian species has known as the most suitable model animals in the research fields due to their unique embryonic development. In particular, their reproductive characteristics, such as a relatively short reproductive cycle and laying over hundreds eggs annually, enhance their suitability for this research area. With rapid development of biotechnology, the demand for genome-edited avian species has been also increased. Especially, the recently reported programmed genome editing technology that can induce gene modification at target locus in an efficient and precise manner facilitates establishment of animal models. In this regards, we evaluated these up-to- date biotechnologies including piggyBac transposition, Flipase/Flipase recognition target (Flp/FRT) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) in avian species. Furthermore, we investigated feasibility of semen cryopreservation in chicken for efficient preservation of avian genetic resources including genome-edited avian species. The first study was performed to evaluate the feasibility of CRIPSR/Cas9 system in specific chicken gene, which relating to avian viral disease. Avian leukosis virus (ALV) is a retrovirus that causes tumors in avian species, and its vertical and horizontal transmission in poultry flocks results in enormous economic losses. Despite the discovery of specific host receptors, there have been few reports on the modulation of viral susceptibility via genetic modification. We therefore engineered acquired resistance to ALV subgroup B using CRISPR/Cas9-mediated genome editing technology in DF-1 chicken fibroblasts. Using this method, we efficiently modified the tumor virus locus B (tvb) gene, encoding the TVB receptor, which is essential for ALV subgroup B entry into host cells. By expanding individual DF-1 clones, we established that artificially generated premature stop codons in the cysteine-rich domain (CRD) of TVB receptor confer resistance to ALV subgroup B. Furthermore, we found that a cysteine residue (C80) of CRD2 plays a crucial role in ALV subgroup B entry. These results suggest that CRISPR/Cas9-mediated genome editing can be used to efficiently modify avian cells and establish novel chicken cell lines with resistance to viral infection. Using same strategy, we also targeted chicken Na+/H+ exchange 1 (chNHE1) gene, which is known as host receptor specifically binds to ALV subgroup J. After first appearance of ALV subgroup J in 1991, the virus causes enormous economic losses in poultry industry. There is no chicken that have resistant against to the virus in nature, the demands for develop resistant chicken lines has been emphasized. The host receptor and the critical amino acid have been identified in avian cells, however, there is no report about acquiring resistant against to the virus mediated by host genome modification. Therefore, in this study, we modified the chNHE1 gene using CRISPR/Cas9 system, and validated the susceptibility of the modified cell lines. As results, we could find that artificially generating premature stop codon on chNHE1 receptor causes absolute resistant to ALV subgroup J, and indel mutations containing W38 is critical. Targeted recombination on W38 regions revealed that deletions only containing W38 are most effective to acquire resistant against to the virus. These results suggest that CRISPR/Cas9-mediated genome replacement could be utilized to precise genome editing in chicken cells and be a efficient tool for developing avian viral disease resistant lines. For the next study, we evaluated recombinase-mediated gene cassette exchange (RMCE) in avian species. Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce RMCE, one of the site-specific recombination technologies, and confirmed RMCE in TG chicken–derived cells. As a result, we established TG chicken lines that have, FRT pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. Next, we investigated the feasibility of semen cryopreservation in chicken to efficiently preserve the avian genetic resources including genome-edited chicken. The importance of genetic resource preservation has been highlighted in the literature as means of maintaining genetic diversity. Among the various methods of preserving such resources, semen cryopreservation can be advantageous because it reduces the time of restoring genetic resources and is less technique-dependent. The Korean Ogye (KO) chicken is a Natural Monument and is recognized as an important genetic resource in Korea. However, successful cryopreservation methods for KO chickens have yet to be reported. Therefore, we completed cryopreservation methods in KO chickens using N-methylacetamide (MA) as a cryoprotectant. Also we performed additional experiments to identify whether fertility and hatchability are affected by long-term storage. Finally, we examined sperm viability in the cryopreserved semen. Our results suggest that the cryopreservation method using MA can be applied to KO chickens regardless of storage period and could be a useful tool for the preservation the endangered avian species. Based on the researches, we identified that the efficient genome editing system mediated by piggyBac transposition, Flp/FRT and CRISPR/Cas9 could be utilized in chicken system. The results suggest that these genome editing technologies can contribute to facilitate the studies on avian species and give us chances to establish novel model for research area as well as industry area. Furthermore, preservation system mediated by semen cryopreservation of this study also will contribute to establish novel chicken lines including genome-edited chicken.

Effect of trehalose addition to frozen canine semen extender : 개 동결희석제에 Trehalose 첨가가 미치는 영향

Kang-Sun Park 충남대학교 대학원 2012 국내석사

정액동결보존은 생식보조기술의 한 분야로, 우수한 품종의 유전자원 유지와 멸종위기 희귀 종 보존을 위해 이용되고 있다. 동결희석액에는 완충제인 트리스 (Tris)와 난황, 탈지유, 당류, 아미노산 등이 첨가되어 동결해동정액의 질적 향상을 위한 연구가 계속적으로 진행되어 오고 있다. 이전 연구결과에서 같은 분류의 당류라 하더라도, 분자구조가 다르기 때문에 각기 다른 작용을 한다고 알려져 있으며, 당 성분에 따라 동결희석액에 미치는 영향이 다양하다는 연구결과가 있다. Trehalose는 두 개의 단당류 결합한 비환원성 이당류로써, 동결희석액 내에 첨가가 될 경우 인지질 이중 층 막으로 구성된 정자의 외부 막과 결합하여 막 구조를 안정화시키고, 정자 내의 수분을 탈수화시켜, 동결 시 발생하는 얼음결정형성을 줄여 보다 높은 정자의 운동성과 생존성을 갖도록 도와주는 것으로 다른 포유류의 실험결과를 통해 알려져 왔다. 아직까지 개 정액동결보존 실험에서는 Trehalose첨가를 통한 실험이 많지 않기 때문에, 본 연구의 목적은 비글 종에서 채취한 정액을Trehalose농도 별로 제작된 동결희석제를 이용, 동결해동 한 개 정액을 CASA (Computer Assisted Sperm Analysis)를 이용하여 객관적으로 운동능력을 각 지표 별로 확인하고, 정자염색을 통해서 수정능력에 가장 큰 영향을 미치는 첨체효소의 손상여부를 확인하였다. 그리고 동결된 정액을 각기 다른 해동조건을 설정하여, 최적의 정액 해동 조건을 모색하고, 해동 후 보관 조건에 따른 정자의 변화를 알아보고자 하였다. 본 실험에서 대조 군은 단당류 Glucose가 첨가된 동결희석제를 사용하였고, 실험 군에서는Trehalose 5, 15, 25, 35 mM농도 별로 제작된 동결희석제를 사용하였다. 급속동결방식을 이용하여 진행한 실험에서 Trehalose 25mM에서 가장 높은 정자 활력도(Motility) 47.84%를 기록하였고, 다른 운동지표 또한 다른 실험 군에 비해 유의적으로 상승하였기 때문에 이 농도를 최적 농도조건으로 확인하고 이후 실험을 진행하였다. 해동 조건에 따라 정액에 미치는 영향에서는, 온도가 상승할수록 해동정액의 활력도 및 운동지표들이 상승을 하였지만, 반면 첨체손상이 증가하는 경향을 보였다. 해동 온도에 따라서 유의적인 차이는 보이지 않았지만, 정자와 난자의 수정에 있어서 운동능력보다는 첨체손상여부가 중요하기 때문에, 36 ℃에서 60초간 해동하는 것이 다른 해동온도조건에 비해 첨체손상 빈도가 적은 것을 확인하였다. 그리고 해동정액을 4가지 온도조건 4, 17, 25 그리고 36 ℃에서 보관실험을 실시하였을 때, 보관시간이 길어질수록 정자의 운동능력은 미약해지고, 정자의 기형발생 빈도와 첨체 손상율이 증가하였다. 유의적 차이는 없었지만, 17 ℃에서 해동 후 보관하는 것이 다른 보관조건보다는 좋은 결과를 나타내었음을 확인하였다. 또한 수술적인 인공수정방법을 통해 Trehalose가 정자의 수정능력에 미치는 영향을 확인한 결과, 정상적으로 수정란의 발생, 착상 그리고 임신유지가 되는 것을 초음파 진단으로 확인하였다. 결론적으로, 개 정액동결에 있어 이당류 Trehalose의 첨가가 단당류를 첨가한 동결희석액에 비해서 정자의 동결해동 후 운동능력 증진시켰으며, 첨체손상의 발생빈도 또한 감소시켜주는 것 보아, Trehalose는 정자의 동결해동에 의한 손상을 방지하고, 정자의 질적 향상에 긍정적인 효과를 미치는 것으로 확인하였다.

여러 가지 항산화제가 제주 한라마의 액상정액과 제주 흑우의 동결정액에 미치는 영향

신상민 전북대학교 일반대학원 2022 국내박사

Cooled storage and cryopreserved semen is usually associated with artificial insemination and genetic improvement programs in livestock species. However, the long-term storage and cryo-damage inducing the rapid degrade in spermatozoa quality limits the development of this technology. Oxidative stress is the result of an imbalance between the body's reactive oxygen species(ROS) and antioxidants, which can lead to sperm membrane damage, apoptosis and decreased motility. In order to prevent oxidative stress on ROS, the method of feeding antioxidants or adding them to semen diluents was used. The aim of this study was to investigate the effects of addition of various concentrations of astaxanthin(ASX), myo inositol(MYO) and pentoxifylline(PENT) on Halla Horse stallion sperm functions during cooled storage(4℃) up to 96~120 h . In addition, this study was conducted to investigate the effect of L-methionine intake on sperm motility improvement in 6 Jeju Black Cattle and 1 Jeju Black Cattle with asthenozoospermia. Semen collected from four Halla Horse stallions using artificial vaginal method was centrifuged at 600 g for 15 min. Spermatozoa were diluted in INRA 96 extender supplemented with different concentrations ASX(0, 5 and 10 uM), MYO(0, 1 and 2 mg/ml), PENT(0, 3 and 6 mM). The sperm samples were stored at 4℃ up to 96 h(MYO, PNET)∼120 h(ASX). Analysis was performed every 24 h. CASA was used to evaluate sperm motility and viability, and flowcytometry was used to determine membrane integrity and apoptosis. 6 Jeju black cattles were fed with L-methionine with 10 g/day during 6 weeks. 1 Jeju black cattle with asthenozoospermia was intake with L-methionine with 10 g/day for 30 days. Semen of Jeju black cattle were collected by artificial vaginal technique. Analysis was performed every 10 days interval(1 Jeju black cattle) and every 2 weeks interval(6 Jeju black cattle). CASA was used to evaluate sperm motility and viability, and flowcytometry was used to evaluate membrane integrity and apoptosis. There was no significant difference in sperm viability, plasma membrane integrity and acrosome membrane integrity between treatment groups(ASX 0, 5 and 10 uM) In the ASX addition experiment, there was no difference in sperm motility between concentrations, but a decrease in motility was observed at 96 h and 120 h in the control group(p<0.05). Also, the early apoptosis sperm ratio between ASX concentrations at all times was significantly higher in the control group, and the non-apoptosis sperm ratio was significantly higher in the ASX added group(p<0.05) and, in the control group, the early apoptosis sperm ratio at 96h and 120 h was significantly lower, and the non-apoptosis sperm ratio was significantly lower at 96 h(p<0.05.). The progressive motility of fresh spermatozoa(from 80.60% to 95.40%) and incresed after L-methionine feeding in 6 Jeju black cattle(P<0.05). The viability, progressive motility, plasma membrane integrity(PMI) and acrosome membrane integrity(AMI) of spermatozoa incresed after L-methionine ingestion(viability: from 57.89% to 74.50%, progressive motility: from 58.91% to 86.75%, PMI: from 70.20% to 77.45%, AMI: from 69.53% to 77.83%) in 1 Jeju black cattle with asthenozoospermia. It is useful for long-term cooled storage by reducing the induction of apoptosis in the addition of ASX in Halla horse stallion. In addition, It was confirmed that ingestion of L-methionine in Jeju black cattle increased sperm motility.

Development of basic assisted reproductive technologies for Selachimorpha

김상화 서울대학교 대학원 2022 국내박사

Shark is a generic term for fish species belonging to the Class Chondrichthyes Superorder Selachimorpha and is a representative animal group that has successfully survived to date among early vertebrates. As they appeared in the early stages of vertebrate formation and thus contain the history of evolution, sharks are important subject of evolutionary biological studies in various aspects such as immunology, reproductive biology, and cancer biology. In addition, sharks play an important role in ecological point of view because they are the apex predators of the marine food chain and are contributing to maintain a balanced and stable ecosystem. It has already been revealed through several previous studies that if the number of sharks become seriously reduced, the entire food chain of the relevant area can collapse. Sharks are thus also called as keystone species because of their high importance. The problem is that sharks are currently in critical danger of extinction. According to the IUCN Red List, about 37% of chondrocytes are classified into “vulnerable (VU),” “endangered (EN),” and “critically endangered (CR)” groups, facing serious extinction. Decline of population had been perceived since the 1970s, and the biggest contributing factor has been pointed out as fishing, led by the shark's fin industry. Accordingly, conservational efforts have been carried out worldwide to reduce shark fishing, but their endangered status has not been easily improved due to their uniquely slow breeding rate. Artificial intervention by human is indispensable for the conservation of critically endangered animal species. Assisted reproductive technology has already been developed and applied to conservational works for various endangered species such as white cranes, Przewalski’s horses, elephants, and northern white rhinos. However, in the case of sharks, access to the animals is not easy, so few studies have been conducted thus far. Therefore, in this paper, a series of studies on assisted reproductive technology were conducted to contribute to the shark conservation. First, basic imaging atlas was established, and then shark semen cryopreservation protocol, hormone induced ovulation protocol, and hormone induced semen sampling protocol were developed. 1. As imaging analysis techniques are the basis of veterinary approaches, this study established detailed imaging atlas in sharks using computed tomography (CT) and magnetic resonance imaging (MRI) methods for the first time. Whole-body CT and MRI scans were performed with three young banded houndsharks (Triakis scyllium) of around 1 m in total body length, and each individual was cryosectioned into transverse, sagittal, and dorsal planes to compare and analyze with the images from CT and MRI scans. Atlas was established by classifying various organs and tissues in detail. However, it was impossible to confirm the reproductive system as the study was conducted on immature individuals. 2. Experiments were conducted on five male banded houndsharks (Triakis scyllium). One hour after 0.2 mL/kg of Ovaprim® administration, the shark's abdomen was gently massaged and the secondary cloudy portion of semen was sampled through urogenital papilla. The composition of an activating extender capable of maximizing the motility of stationary spermatozoa was established, which was designated as SSAE-1. To establish a cryopreservation protocol optimized for banded houndshark semen, a total of 8 extender solutions, 3 extension ratios, 15 cryoprotectants, 4 equilibration periods, 3 cooling rates, and 3 thawing temperatures were tested. The optimized protocol (Kim’s protocol) was as follows: extender, filtered seawater; extension ratio, 1:3; cryoprotectant, egg yolk 10% + ethylene glycol 10%; equilibration period, 10 min; cooling rate, 3 cm, 3 min; thawing temperature, 30℃, 10 s. The resulting post-thaw spermatozoa motility was 2.03%. 3. Experiments were conducted on the ovoviviparous shark banded houndshark (Triakis scyllium) and the placental shark (Triaenodon obesus) to confirm the applicability of salmon-derived gonadotropin-releasing hormone analogue (sGnRHa, Ovaprim®) throughout shark species with various breeding strategies. Prior to the experiment, normal blood sex hormone concentrations of females and males were identified in each of the species, and used as the base line for further experimental analysis. In both species, it was confirmed that Ovaprim® successfully induced changes in concentration of estradiol, progesterone, and testosterone in the blood, follicular maturation and ovulation in females, and semen release in males. The optimized injection protocols of Ovaprim® for future application to artificial insemination were as follows: male banded houndshark: 0.2 mL/kg administration and semen sampling 1 hour after administration; female banded houndshark: 0.2 mL/kg first administration and 0.5 mL/kg second administration with 24 hours of gap time; male whitetip reef shark: 0.2 mL/kg administration and semen sampling right after administration; female whitetip reef shark: 0.2 mL/kg first administration and 0.2 mL/kg or 0.3 mL/kg second administration with 24 hours of gap time. 상어는 판새아강 상어상목에 속하는 어종들의 총칭으로, 초기 척추동물 중 현재까지 성공적으로 생존해 있는 대표적인 동물군이다. 척추동물 형성 초기에 나타나 진화의 역사를 고스란히 담고 있는 만큼, 상어는 면역체계, 번식 전략, 암 저항성 등 다양한 측면에서 중요한 진화생물학적 연구의 대상이다. 뿐만 아니라 상어는 생태학적으로도 중요한 역할을 하는데, 해양 생태계 먹이사슬의 최상위 포식자로서 해당 생태계가 균형 있고 안정적으로 유지될 수 있도록 기여하고 있기 때문이다. 상어 개체 수에 이상이 생기면 먹이사슬 전체가 무너질 수 있다는 점은 이미 여러 선행연구를 통해 밝혀진 바, 그 중요성이 높기 때문에 핵심종이라고도 불린다. 문제는 이들이 현재 심각한 멸종위기에 처해있다는 점이다. IUCN Red List에 따르면, 연골어류의 37%가량이 취약 (VU), 절멸 위기 (EN), 절멸 위급 (CR)군으로 분류되고 있어 심각한 멸종위기 상태를 마주하고 있다. 이러한 양상은 이미 1970년대부터 확인되어 왔으며, 그 가장 큰 요인으로는 샥스핀 산업을 필두로 하는 어업이 지적되어 왔다. 이에 전세계적으로 상어 어획량을 줄이고자 하는 환경운동이 수행되어 왔으나, 상어 특유의 느린 번식속도에 기인하여 이들의 멸종위기 상태가 쉽게 개선되지는 않고 있는 실정이다. 심각한 멸종위기 상태의 동물 종 보전을 위해서는 사람의 인위적인 개입이 필수불가결하다. 이미 재두루미, 몽고말, 코끼리, 북부흰코뿔소 등 다양한 멸종위기 동물종에서 종 구제 및 보호를 위하여 보조생식기술이 개발 적용되고 있다. 그러나 상어의 경우 동물 자체에 대한 접근이 쉽지 않아 사실상 연구 되어있는 바가 거의 없다. 이에 본 논문에서는 상어 종 보전에 기여하고자 일련의 연구들을 수행하였다. 우선적으로 기초적인 영상의학 아틀라스를 확립하였으며, 이후 보조생식기술 개발을 위하여 상어 정액 동결보존 프로토콜 및 호르몬 유도 배란 프로토콜, 호르몬 유도 정액 샘플링 프로토콜을 개발하였다. 1. 영상의학적 분석 기반 확립은 모든 수의학적 접근 방식의 기본이 되는 만큼, 본 연구는 최초로 컴퓨터 단층촬영 (CT) 및 자기공명영상진단 (MRI) 방식을 이용하여 상어에서의 세밀한 영상의학 아틀라스를 확립하였다. 체장 1 m 안팎의 어린 까치상어 (Triakis scyllium) 세 마리의 전신 CT 및 MRI 스캔을 수행했으며, 각 개체들을 냉동 후 transverse, sagittal, dorsal 단면으로 잘라 실제 단면 모습과 CT, MRI상의 단면을 비교분석 하였다. 다양한 장기 및 조직들을 세밀하게 구분하여 아틀라스를 확립하였다. 다만 미성숙 개체들을 대상으로 연구를 수행하였던 만큼 생식계통에 대한 확인은 불가하였다. 2. 까치상어 (Triakis scyllium) 수컷 다섯 마리를 대상으로 실험을 수행하였다. Ovaprim®을 0.2 mL/kg 투여 후 1시간 뒤, 상어의 복부를 부드럽게 마사지하여 urogenital papilla를 통해 나오는 정액의 secondary cloudy portion을 샘플링 하여 실험에 이용하였다. 정지상태 정자의 motility를 최대로 만들 수 있는 활성화 용액 조성을 확립하였으며 이를 SSAE-1로 명명하였다. 까치상어 정자에 최적화된 동결보존 프로토콜을 확립하기 위하여 총 8종의 extender solution, 3종류의 extension ratio, 15종의 cryoprotectants, 4종의 equilibration periods, 3종의 cooling rates, 3종의 thawing temperature를 테스트 하였다. 결과적으로 정립된 Kim’s protocol은 다음과 같다: extender, filtered seawater; extension ratio, 1:3; cryoprotectant, egg yolk 10% + ethylene glycol 10%; equilibration period, 10 min; cooling rate, 3 cm, 3 min; thawing temperature, 30℃, 10 s. 프로토콜을 이용했을 때 최종적으로 확인된 정자의 해동 후 운동능은 2.03%로 확인되었다. 3. 난태생 상어인 까치상어 (Triakis scyllium)와 태반성 태생 상어인 화이트팁 상어 (Triaenodon obesus)에서 실험을 수행하여 다양한 번식전략을 지닌 상어 종 전반에 걸쳐 연어 유래 gonadotropin-releasing hormone (GnRH, Ovaprim®) 적용 가능성을 확인하였다. 실험에 앞서 두 상어종에서 암컷과 수컷의 정상 혈중 성호르몬 농도를 각각 확인하여 추후 실험 분석의 base line으로 이용하였다. 두 종 모두에서 Ovaprim®이 혈중 에스트로겐, 프로게스테론, 테스토스테론의 농도 변화를 성공적으로 유도하였으며, 암컷에서는 난포 성숙 및 배란을, 수컷에서는 정액 사출을 유도함을 확인하였다. 추후 인공수정에 적용할 수 있도록 Ovaprim®의 농도 및 투여 주기를 최적화한 결과는 다음과 같았다: 까치상어 수컷: 0.2 mL/kg 투여 1시간 뒤 정액 샘플링; 까치상어 암컷: 0.2 mL/kg 투여 24시간 뒤 0.5 mL/kg 2차 투여; 화이트팁 상어 수컷: 0.2 mL/kg 투여 직후 정액 샘플링; 화이트팁 상어 암컷: 0.2 mL/kg 투여 24시간 뒤 0.2 mL/kg 또는 0.3 mL/kg 2차 투여.

Reproductive characteristics of cloned male cats from somatic cell nuclear transfer : 체세포핵이식에 의한 복제고양이의 번식학적 특성에 관한 연구

최유진 경상대학교 대학원 2009 국내석사

본 연구는 체세포 핵 이식에 의한 복제 고양이에 번식적 특성을 규명하기 위하여 자연교미를 통하여 산자를 생산하였고 성호르몬을 분석하였으며 정액을 채취하여 동결 융해후 생존율 및 운동성, 기형율, 체외수정율 등의 특성을 살펴보았다. 그 결과 복제고양이 A, B, C, D 가 각각 3마리, 2마리, 4마리, 5마리의 산자를 생산하였으며, 이를 형태학적으로 분석하여 복제고양이 A와 B에서 각각 1마리 2마리의 Odd eye 색을 가진 산자가 태어났고 나머지는 Brown 또는 Blue 색을 가진 산자가 태어났다. 또한 모든 산자에서 흰바탕에 검은색 반점을 가지고 있었으며 암수의 성비는 각각 7마리씩으로 1:1의 성비를 보였으며, 모든 산자에서 Microsatellite 분석을 통한 친자감별로 모두 복제고양이의 산자임을 확인하였다. 성호르몬의 분석결과 테스토스테론의 수치가 복제고양이 그룹과 대조구에서 유의적 차이를 보였으며 LH 와 FSH에서는 유의적 차이가 없었다. 정액의 특성을 파악하기 위하여 정액을 채취 후 동결융해 하였고 이를 융해하여 운동성, 생존율, 기형율, 체외 수정율을 조사하였다. 그 결과 cloned D 를 제외한 모든 개체에서 성공적으로 정액을 채취하였다. 먼저 정자의 농도는 cloned A 에서 512 ± 140 (Range 368 &#8211; 685 x 106/mL) 로 가장 높았고, cloned C 에서 335 ± 92 (Range 274 &#8211; 469 x 106/mL) 로, cloned B에서 459 ± 159 (Range 336 &#8211; 510 x 106/mL) 와 대조구에서 680 ± 452 (Range 360 &#8211; 479 x 106/mL)) 비해 낮은 분포를 보였다. 정액의 동결융해 후 특성분석은 computer associated sperm analysis 으로 분석하였다. 그 결과 운동성과 전진운동성은 cloned B에서 다른 복제고양이나 대조구 보다 낮았으며 curvilinear, straight line, and average path velocities 등은 유의적으로 높았으나, straightness 는 낮게 나타났다. 동결융해 후 정자의 수정율을 살펴보기 위하여 IVF를 실시하였는데 모두 성공적인 배 발달율을 보였다. 난자의 분할율은 A, 74.4%; B, 71.4%; C, 86.2%; 대조구, 82.0% 이고 배 발달율은 A, 34.4%; B, 26.7%; C, 48.0%; 대조구, 43.9% 로 복제고양이 사이의 통계학적 유의차는 없었다. 이상의 결과들로 보아 체세포 핵 이식에 의한 복제고양이에서 번식학적 이상이 발견되지 않았고 정상적인 성 성숙과 성장을 보였다. 따라서 체세포 핵 이식에 의한 동물복제는 종의 보존방법으로서의 활용뿐만 아니라 고양이과의 멸종 위기종의 동물복제에 의한 복원에 기여할 수 있을 것츠로 판단되며, 그와 관련된 다양한 연구에 활용 가능할 것으로 사료된다.

돼지 정액 동결보존 시 마이오이노시톨이 정액의 기능과 수태율에 미치는 영향

라나 오스만 전북대학교 일반대학원 2023 국내석사

마이오 이노시톨은 비타민 B의 파생물 중 하나다. 칼슘과 글루코스의 농도를 조절하고, 지질의 합성, 세포의 성장, 세포의 발생과 형태 형성에 필수적인 역할을 한다. 이 연구에서는 다양한 농도의 마이오 이노시톨이 돼지의 정액에 미치는 영향에 대해 연구했다. 희석하여 냉각한 정액을 BF5 동결보존제와 함께 동결보존한 후 대조군을 포함한 다양한 농도의 마이오 이노시톨 (0.5, 1, 1.5, 2mg/mL)을 투여한 총 5개의 그룹으로 나누었다. 최소 24시간 후 정액을 해동하고 운동성, 생존능, 첨체의 보전성, 카스페이스, 미토콘드리아막 전위, 유전자 발현, 수정능, 활성산소, 세포 사멸 등 정자 파라미터를 평가했다. 그 결과, 0.5mg/mL 그룹은 다른 그룹에 비해 총 운동성 (TM)과 진행성 운동성 (PM)이 현저히 높았다. 그러나, 급속 진행성 운동성 (RPM), 중간 진행성 운동성 (MPM), 곡선 속도 (VCL), 평균 경로 속도 (VAP), 직선 속도 (VSL), 직진성 (STR), 선형성 (LIN)은 모든 실험군에서 유의미한 차이가 없었다. 0.5 및 1mg/mL 그룹의 생존율은 다른 그룹보다 유의하게 높았다. 0.5mg/mL 마이오이노시톨 처리 그룹의 첨체 보존성은 다른 그룹에 비해 유의하게 개선된 반면, 카스페이스 활성이나 미토콘드리아막 전위에서는 모든 그룹 간에 차이가 없었다 (P>0.05). 0.5mg/mL 그룹에 대한 유전자 발현 분석 결과, 대조군에 비해 Bcl-2 관련 X 단백질 (BAX)과 활성산소 조절인자 (ROMO1)의 발현이 유의하게 감소하고 정자 미토콘드리아 관련 시스테인 풍부 단백질 (SMCP)이 증가했으며, NADPH 산화효소 5 (NOX5), 스페르민 산화효소 (SMOX), 세포 사멸 조절 인자 (BCL2)는 유의미한 변화가 없었다. 체외 수정 실험 결과, 0.5mg/mL 마이오이노시톨 그룹은 대조군에 비해 더 높은 분열률과 배반포형성률을 보였다. 세포 사멸과 ROS 수치는 대조군에 비해 모든 마이오이노시톨 그룹에서 감소했으며, 특히 1mg/mL 그룹에서 유의미한 감소가 나타났다. 결론적으로, 마이오이노시톨로 돼지 정액을 처리하면 운동성, 생존능, 첨체 보존성 등의 정액 파라미터가 개선되었고, 활성 산소, 세포 사멸 수준, 산화 상태 관련 유전자 발현이 감소한 반면, 체외 수정 및 배아 생산율이 개선되었다. Myo-inositol is one of the vitamin B derivatives. It plays essential roles in regulating calcium and glucose levels, lipid synthesis, cell growth, cytogenesis, and morphogenesis. This study investigated the effects of different concentrations of myo-inositol on boar semen. Diluted, cooled semen was cryopreserved in a BF5 extender and divided into five groups: control and different concentrations of myo-inositol (0.5, 1, 1.5, and 2 mg/mL). At least after 24 h, semen was thawed, and sperm parameters were evaluated: motility, viability, acrosome integrity, caspase, mitochondrial membrane potential, gene expression, fertilizing ability, ROS, and apoptosis. The results showed that total motility (TM) and progressive motility (PM) were significantly higher in the 0.5 mg/ml group compared to the other groups. However, rapid progressive motility (RPM) and medium progressive motility (MPM), curvilinear velocity (VCL), average path velocity (VAP), straight linear velocity (VSL), straightness (STR), and linearity (LIN) were not significantly different for all experimental groups. The viability of the 0.5 and 1 mg/mL groups was significantly higher than the other groups. Acrosome integrity in the 0.5 mg/mL myo-inositol-treated group revealed significant improvement over other groups, while there was no difference between all groups in caspase activity or mitochondrial membrane potential (P > 0.05). Gene expression analysis for the 0.5 mg/mL group demonstrated a significant decrease in expression of Bcl-2-associated X protein (BAX) and reactive oxygen species modulator (ROMO1), an increase in sperm mitochondria-associated cysteine-rich protein (SMCP), and no significant change in NADPH oxidase 5 (NOX5), spermine oxidase (SMOX), or apoptosis regulator (BCL2) compared to the control group. In vitro fertilization revealed that the 0.5 mg/mL myo-inositol group had higher cleavage and blastocyst rates compared with the control group. Apoptosis and ROS levels decreased in all myo-inositol groups compared with the control group, with a significant decrease in the 1 mg/mL group. In conclusion, treatment of boar semen with myo-inositol improved boar semen parameters, including improved motility, viability, acrosome integrity; decreased ROS, apoptosis levels, expression of oxidative state-related genes, improved in vitro fertilization and embryo production rates.

Studies on embryo development following intra cytoplasmic sperm injection and morphogenetic analysis of primordial germ cell migration in aves

강경수 서울대학교 대학원 2015 국내박사

Primordial germ cells (PGCs) migrate across the embryo to the gonads where they differentiate and function. Both of morphogenetic and actively through are used for their movement. We have examined the spatial and temporal action of PGCs. As results, PGCs migrated passively toward the anterior region from the preliminary location. However, PGCs and somatic cells are shown different migration speed when they reached the anterior region. PGCs demonstrated markedly faster migration than somatic cells. The results reveal that chicken PGCs use sequential passive and active migration toward the germinal crescent, and only PGCs migrated to germinal crescent. Cryopreservation of avian semen for preserving the avian genetic resource has been studied for more than 80 years, and there have been many technical difficulties due to the complexity of avian reproductive system. We found that the mixture shown the highest fertility composes of 8% glycerol with 3% DMA in prefreezing diluents. However the mixture was a harmful effect with the addition of cryoprotectant especially with glycerol. Our results show that the sperm preserved in the mixture composed of 6% glycerol containing 5% DMA had 5% less motility. Thus we used that mixture and the sperm preserved in the mixture produced fertilized eggs. Additionally, we tested incubation time for glycerol removal by monitoring under scanning electron microscopy (SEM). We found that complete removal of glycerol requires at least 30 min incubation time. We have investigated the ability of cryopreserved/thawed quail sperm by intracytoplasmic injection to the unfertilized ovum. An injected egg was incubated with egg shell surrogate system. We have used both PLC zeta (PLCζ) and inositol 1,4,5-trisphosphate (IP3). Embryo development ratio increased significantly compared to fresh sperm only (90% vs. 13%). Avian species surrogate egg shell system has been adapted in many different experimental fields. However, the viability needs to be improved and the system should be more simplified for commercial uses. We have established the quail egg surrogate system for embryo developmental study. The system has produced high percentage of hatchability in both thick albumin capsulated and non-capsulated egg, 78% and 60% respectively. Furthermore, we have succeeded in higher hatchability in single cell stage of embryo incubation. This study could provide deeper understanding of germ cell movement mechanism in early embryo developmental stage. And, we tested for ICSI by producing chick from cryopreserved sperm for avian genetic resource conservation. We have established knowledge in avian embryo development to understand embryo fertilization and developmental stratagem for atmosphere of ex ovo culture system used by surrogate egg shell system complementation. This study could bring deeper understanding in avian culture system with germ cell migration and possibilities of hatching chick by ICSI and surrogate egg shell system for conservation of genetic resource in the future.

닭 정액 동결 시 동결보호제에 따른 정액성상 및 수정률에 미치는 영향

최진석 경상대학교 대학원 2013 국내석사

The purpose of this study was to evaluate the spermatozoal viability, acrosome integrity, mitochondrial activity and fertility in the frozen-thawed fowl semen by different cryoprotective agents and to figure out their correlations with rate of fertilization. The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at 5℃. After equilibration for 30 minutes, diluted semen was used by mixing an equal volume of semen diluent containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration. Diluted semen was put in 0.5mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen at -196℃. Frozen semen was thawed in water bath at 5℃ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed as fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. Intravaginal artificial insemination was performed twice a week by injecting 0.2mL of thawed semen directly into the vagina within 2 min after thawing. As the result of freezing rooster's semen, when using DMF, survival rate was 52.1±5.52% and it was significantly higher than that of using DMA(46.94±5.06%) and MA(36.56±4.66%)(p<0.05), respectively. For the rates of acrosomal membrane and death of sperm, semen using MA was highest with 61.16±1.86% and then followed by samples with DMA(47.48±1.9%) and DMF(36.56±1.42%), respectively with significant differences(p<0.05). For rate of sperm survival with intact mitochondrial membrane, semen frozen by using DMF was highest with 52.68±1.07% and then followed by samples with DMA(44.46±1.04%) and MA(38.36±1.88%), respectively with significant differences(p<0.05). Samples with DMF and DMA showed similar rates of fertilization 67.85% and 68.35%, respectively while Sample with MA showed somewhat lower rate of fertilization 60.95% but there were no significant differences. In summarizing the results of this study, when freezing rooster semen by using 7% DMF as cryoprotective agent, there were no significant difference between rates of fertilization but this sample was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotective agent which have the lowest influences on cell membranes and acrosome integrity of sperm and could be used to freezing conservation method for poultry genetic resources.