개 동결 정자의 apoptosis, oxidative stress, DNA integrity에 관한 연구 = Studies on Apoptosis, Oxidative Stress, and DNA Integrity in Cryopreserved Canine Sperm

다국어 초록 (Multilingual Abstract)

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.

국문 초록 (Abstract)

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.

목차 (Table of Contents)

• List of Tables vii
• General Abstract 1
• Chapter 1. Literature Reviews on Fundamental Cryobiology of Spermatozoa 5
• 1.1. The importance and need of spermatozoa cryopreservation 7
• 1.2. Fundamental protocol of spermatozoa cryopreservation 8
• 1.2.1. Basic steps for cryopreservation 8
• 1.2.2. Various protocols for cryopreservation in mammalian species 9
• 1.3. Appreciation of the cryopreservation phenomenon 11
• 1.3.1. Mechanism of cryoinjury during cooling and warming processes 11
• 1.3.1.1. Cooling and cold shock 11
• 1.3.1.2. Freezing and cellular injury 12
• 1.3.1.3. Thawing 14
• 1.3.2. Cryoprotectant agents (CPAs) 14
• 1.4. Evaluation of sperm function for detection of cryoinjury 16
• 1.4.1. The classical methods 16
• 1.4.1.1. Evaluation of ejaculates 16
• 1.4.1.2. Motility 16
• 1.4.1.3. Sperm morphology and viability 17
• 1.4.1.4. Sperm membrane integrity 19
• 1.4.2. New methods 19
• 1.4.2.1. Sperm plasma membrane integrity 20
• 1.4.2.2. Acrosomal membrane integrity 20
• 1.4.2.3. Mitochondrial integrity 21
• 1.4.2.4. Sperm capacitation 22
• 1.4.2.5. DNA status of spermatozoa 23
• 1.5. Recent study related with cryoinjury 24
• 1.5.1. Oxidative damage 24
• 1.5.1.1. Evaluation on the oxidative damage 26
• 1.5.2. Ability of the sperm to sustain embryonic development 27
• 1.6. References of literature review 29
• Chapter 2. Effects of Cryopreservation on Apoptosis, Oxidative Stress and DNA Integrity in Canine Sperm 40
• Abstract 41
• 2.1. Introduction 43
• 2.2. Materials and Methods 49
• 2.2.1. Animals 49
• 2.2.2. Semen collection 49
• 2.2.3. Semen freezing and thawing process 50
• 2.2.3.1. Preparation of semen diluent and extenders 50
• 2.2.3.2. Semen dilution and freezing 51
• 2.2.3.3. Semen Thawing 52
• 2.2.4. Sperm evaluation 53
• 2.2.4.1. Sperm motility 53
• 2.2.4.2. Determination of sperm concentration 53
• 2.2.4.3. Sperm morphological defects 53
• 2.2.4.4. Sperm viability 54
• 2.2.4.5. Sperm plasma-membrane integrity 54
• - CFDA/PI fluorescence assay 54
• - Hypo-osmotic swelling test (HOST) 56
• 2.2.4.6. Sperm apoptosis 56
• 2.2.4.7. Measurement of sperm intracellular hydrogen peroxide level 57
• 2.2.4.8. Sperm chromatin structure assay (SCSA) 58
• - Measurement procedures 58
• - Data analysis 59
• 2.2.4.9. Flow cytometric analysis 60
• 2.2.5. Statistical analysis 61
• 2.3. Results 63
• 2.3.1. Sperm motility and viability in fresh and frozen-thawed semen 63
• 2.3.2. Sperm morphological defects in fresh and frozen-thawed semen 66
• 2.3.3. Sperm plasma-membrane integrity in fresh and frozen-thawed semen 70
• 2.3.4. Sperm population setting for flow cytometry analysis 74
• 2.3.5. Sperm apoptosis in fresh and frozen-thawed semen 76
• 2.3.6. Measurement of intracellular hydrogen peroxide level in fresh and frozen-thawed semen 79
• 2.3.7. Sperm chromatin structure assay (SCSA) in fresh and frozen-thawed semen 82
• 2.3.8. Correlations between conventional and advanced parameters 85
• 2.4. Discussion 88
• 2.5. Conclusion 100
• 2.6. References 101
• Chapter 3. Effects of GW and PDG Separation for Removal of Spermatozoa Damaged by Frozen-Thawed Process in Canine 114
• Abstract 115
• 3.1. Introduction 118
• 3.2. Materials and Methods 123
• 3.2.1. Sample preparation 123
• 3.2.2. Sperm separation procedures 123
• 3.2.3. Sperm evaluation 125
• 3.2.3.1. Sperm motility 125
• 3.2.3.2. Determination of sperm concentration 125
• 3.2.3.3. Sperm morphological defects 125
• 3.2.3.4. Sperm viability 125
• 3.2.3.5. Sperm plasma-membrane integrity 125
• 3.2.3.6. Sperm apoptosis 126
• 3.2.3.7. Measurement of sperm intracellular hydrogen peroxide level 126
• - Induction of artificial oxidative stress 126
• - Determination of hydrogen peroxide level 126
• 3.2.3.8. Sperm chromatin structure assay (SCSA) 127
• 3.2.3.9. Flow cytometric analysis 127
• 3.2.3.10. Determination of recovery rate 127
• 3.2.3.11. Determination of increasing rate 128
• 3.2.4. Statistical analysis 128
• 3.3. Results 130
• 3.3.1. Comparison of conventional sperm parameters before and after sperm treatments 130
• 3.3.2. Sperm plasma-membrane integrity before and after sperm treatments 134
• 3.3.3. Sperm apoptosis before and after sperm treatments 137
• 3.3.4. Basal intracellular hydrogen peroxide level before and after sperm treatments 139
• 3.3.5. Measurement of intracellular hydrogen peroxide level according to stimulation in GW filtration and PDG centrifugation samples 141
• 3.3.6. Sperm chromatin structure assay (SCSA) before and after sperm treatments 145
• 3.3.7. Comparison of recovery rate in GW filtration and PDG centrifugation samples 147
• 3.3.8. Correlations between conventional and advanced parameters 149
• 3.3.9. Correlations among advanced parameters 151
• 3.4. Discussion 153
• 3.5. Conclusion 165
• 3.6. References 166
• General conclusion 178
• General abstract in Korean (국문 초록) 180