真鲷(Pagrosomus major)胚胎冷冻保存损伤机理及冷冻精子遗传物质稳定性研究

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	真鲷(Pagrosomus major)胚胎冷冻保存损伤机理及冷冻精子遗传物质稳定性研究
其他题名	Studies on cryoinjury of embryo and DNA stability of sperm in red sea bream (Pagrosomus major)
	徐西长
学位类型	博士
	2007-06-10
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	真鲷胚胎真鲷精子冷冻损伤超低温保存抗冻剂冰晶遗传物质稳定性
摘要	本文对真鲷心跳期胚胎对5种常用渗透性抗冻剂（DMSO、甘油、甲醇、丙二醇、乙二醇）和3种非渗透性抗冻剂（PVP、PEG-8000、蔗糖）的耐受性进行了研究。渗透性抗冻剂分6个浓度梯度（5%；10%；15%；20%；25%；30%）和3个时间组（10min；30min；1h）。非渗透性抗冻剂中，PVP、PEG-8000分3个浓度梯度（5%、10%、15%）和2个时间组（10min、30min），蔗糖为4个浓度梯度（5%、10%、15%、20%）和2个时间组（10min、30min）。实验结果表明，在渗透性抗冻剂组中，浓度为5%的处理组的孵化率（>90%）与对照组差异均不显著，随着抗冻剂浓度增大及处理时间的延长，真鲷心跳期胚胎的孵化率显著下降（P<0.05），在最高浓度的最长处理时间中胚胎孵化率均降到了0。总体上，真鲷心跳期胚胎对五种渗透性抗冻剂的耐受性从小到大依次为：甲醇 < 甘油 < 乙二醇 < DMSO < 丙二醇。对影响胚胎孵化率的三个因素（抗冻剂、浓度、时间）进行的因素效应分析结果表明，三种因素对孵化率的影响显著（P<0.05），并且浓度效应 > 时间效应 > 抗冻剂效应。在非渗透性抗冻剂组中，蔗糖组胚胎孵化率未呈显著变化；PVP组随着浓度及时间的增大，孵化率显著下降（P<0.05）；PEG-8000组随着浓度增大孵化率显著下降（P<0.05），但在两个时间组间差异不显著。相同处理情况下PEG-8000对真鲷心跳期胚胎的毒性要小于PVP。因素效应分析比较结果表明仅时间效应不显著，且抗冻剂效应 > 浓度效应 > 时间效应。对所用各种抗冻剂进行了渗透压测量，实验中使用的渗透性抗冻剂（5%-30%）的渗透压值在959-7980mOsm/kg之间，均高于使用海水的渗透压值（919mOsm/kg）；使用的非渗透性抗冻剂的渗透压值在316-1040mOsm/kg之间，除20%蔗糖渗透压值（1040mOsm/kg）高于海水外，其他非渗透性抗冻剂的渗透压值均要低于海水。对孵化率与相应的溶液渗透压值进行相关回归分析结果表明，渗透性抗冻剂的渗透压与孵化率呈显著的负相关（P<0.05），而非渗透性抗冻剂的渗透压与孵化率相关不显著。渗透性抗冻剂组的回归分析结果表明，二次方程的曲线拟合度最高，得到的回归方程分别为：Y10min = -2×10-8X2 10min - 6×10-5 X 10min + 1.5635 （R2 = 0.713），Y30min= 5×10-8X2 30min－0.0007 X 30min + 2.097（R2 = 0.681），Y1h = 7×10-8X2 1h－0.0008 X 1h+ 2.0397（R2= 0.725）。在真鲷胚胎对抗冻剂耐受性实验的基础上，挑选五种抗冻剂--10%DMSO、5%甘油、10%甲醇、20%丙二醇、10%乙二醇，浸泡真鲷心跳期胚胎30min后，分别以超速（130℃/min）、快速（20℃/min）、慢速（3℃/min）的速度降温并使用低温显微镜进行观察，依次记录Toif（油球结冰）、Teif（胚胎外部结冰）、Tiif（胚胎内部结冰）等结冰点，Toif值在-9~-23℃之间；Teif值在-21~-35℃之间；Tiif值在-21~-52℃之间。结冰顺序为先油球结冰，然后胚胎外部结冰随之内部马上瞬间变黑形成内部冰晶。随着降温速度的提高，各结冰温度值显著下降。各抗冻剂之间的Teif及Tiif值不同，Toif值之间没有显著差异。对两种玻璃化冷冻方法进行模拟观察，发现胚胎冰晶形成的顺序与非玻璃化过程不同--先内部结冰然后逐渐蔓延至外部形成外部冰晶，而且模拟玻璃化的内部结冰温度Tiif值（-52.56℃）显著（P<0.05）低于使用低浓度的同种抗冻剂超速降温组的Tiif值（-40.11℃）。在快速及慢速降温组中，20%丙二醇组的Tiif要显著的低于其他组（P<0.05）；在超速降温中，甲醇组的Tiif值要显著的低于其他组（P<0.05）。在Tiif小于30℃的实验组中获得形态完整胚胎的比例平均仅有30.77%；在Tiif大于30℃的实验组中获得形态完整胚胎的平均比例高达70.37%，模拟玻璃化组达到100%。各抗冻剂之间，复温后胚胎形态完整率10%甲醇组最高（77.78%）；其次依次为10%乙二醇（66.67%）、20%丙二醇（55.56%）和10%DMSO（55.56%）；5%甘油组最低（11.11%）；推测甲醇的对胚胎的渗透效果要好于其他组。综上推测：使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。我们对低温保存的真鲷精子核DNA损伤进行了研究以期为下一步胚胎遗传物质稳定性研究提供参考依据。研究方法为单细胞凝胶电泳（SCGE），针对研究对象，在实验过程中对传统的碱性单细胞凝胶电泳在铺胶方法、电泳条件等进行了改进。对精子细胞进行预处理，在碱性电泳液中使核DNA双链解链变性后电泳，EB染色lOmin后，在荧光显微镜下观察，每次随机观察50个左右的核DNA。结果表明，对荧光显微镜下观察到的精子核按彗尾长度及荧光强度划分等级，出现损伤的精子核DNA的损伤程度主要为轻度损伤和中度损伤，很少见有完全损伤的真鲷精子核。经5%、10%、18%、20%、25%、30%DMSO冷冻保存后的精子彗星率分别为33.47％ ± 8.95％; 35.91％ ± 19.44％; 48.95％ ± 8.90％; 43.33％ ± 11.19％; 55.80％ ± 38.94％。鲜精彗星率为31.43 ％ ± 2.68％。对比真鲷冷冻精液与新鲜精液的精子DNA的损伤状况，表明仅用30％ DMSO冷冻精子DNA损伤状况与鲜精差异显著(P<0.05)。综上所述，渗透性抗冻剂对胚胎的毒性与其渗透压值呈显著的负相关关系。丙二醇对真鲷心跳期胚胎毒性最小，甲醇较其他抗冻剂能更好的渗透入胚胎；玻璃化方法能显著降低Tiif值并能更好的保持超低温保存后胚胎的形态完整性，因此，使用丙二醇、甲醇作为抗冻剂以及玻璃化冷冻保存方法对真鲷心跳期胚胎超低温保存也许较为合适。常规使用的用于超低温保存真鲷精子的DMSO（浓度<15%）不会对精子核物质稳定性造成明显影响。由于胚胎较精子结构要复杂许多，对于真鲷胚胎损伤机理的研究还有大量工作可以开展。
其他摘要	The application of cryopreservation technology to the preservation of fish embryos could play a major role in seed production, genetic management of broodstock, and conservation of aquatic resources. Although investigators have examined fish cryobiology for over 50 years, successful cryopreservation of fish embryos has not been achieved. Red sea bream (Pagrosomus major) has a high commercial value in Chinese aquaculture production. Some attempts at red sea bream’s embryo cryopreservation have been carried out, but successful cryopreservation is still far from being achieved. In order to achive successful cryopreservation, several parameters（toxicity of cryoprotectants; membrane permeability; intraembryonic ice formation and so on） should be taken into account in the design of a cryopreservation protocol. In this paper, the cryoprotectant and osmotic tolerance , intraembryonic ice formation of embryos in red sea bream (Pagrosomus major) were analyzed expeimentally. The effect of sperm cryopreservation on sperm DNA stability in red sea bream was also investigated. Heart-beating stage embryos were incubated in five permeable cryoprotectants -- dimethyl sulfoxide (DMSO), glycerol (GLY), methanol (MeOH), 1,2-propanediol (PG), ethylene glycol (EG), in concentrations ranging from 5% to 30% for 10min, 30min and 1h. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. PG was best tolerated by the embryos. In group of non-permeable cryoprotectant--5%, 10% and 15% polyvinyl pyrrolidone (PVP) and PEG-8000, 5%, 10%, 15%, 20% sucrose for 10min and 30min, exposure to sucrose did not affect the hatching rate. The toxicity of PEG-8000 increased with concentration but did not have distinct difference between two exposure times. The toxicity of PVP increased with concentration and exposure time. The osmotic values of all solutions used in this study were measured. And the correlation between solution osmotic values and corresponding hatching rate of embryos were analyzed in each time group. The correlation between osmotic values of permeable cryoprotectants and corresponding hatching rate of embryos was significant (P<0.05) and the values of R2 of 10min, 30min and 1h group were 0.713, 0.685 and 0.725 respectively. The correlation between osmotic values of non-permeable cryoprotectants and corresponding hatching rate of embryos was not significant. Ice crystal damage was investigated by response of red sea bream embryos to rapid and slow cooling rates. The changes of heart-beating stage embryos were observed by a cryomicroscopy after immersing in five diffirent cryoprotectants (10%DMSO; 5%glycerol; 10%methanol; 20% 1, 2-propanediol; 10% ethylene glycol) for 30min. Three temperatures, oil globule turn to white, embryo flash, embryo turn to black, which represented oil globule ice formation (Toif); extra-embryo ice formation (Teif) and intraembryonic ice formation (Tiif), respectively were recorded. Cryomicroscopic observation revealed that Teif appeared after Tiif almost synchronously. Three values were reducing as the cooling rate increased. Different cryoprotectants show different effects on three temperatures. More significant decreasing of Tiif was shown by 20%1,2-propanediol. Two programmable freezing with slow rate and two vitrification protocols which used in the cryopreservation of embryos were simulated. Significant difference of the changing trend of temperatures (Toif, Teif, Tiif ) was not investigated between the groups of cooling rate with 3℃/min and the other two slow programmable cooling rate groups. Teif appeared after Tiif and ice formatted gradually in two simulated vitrification methods. Tiif were significantly decreased in two vitrification protocols with max cooling rate (-130℃/min). The morphologic change of post-thawed embryos displayed a relationship with the Tiif decreasing. A smaller decreasing contributed a good morphological of embryos. The use of methanol, 1,2-propanediol and vitrification in the cryopreservation of the red sea bream’s embryos was advised. The DNA damage was investigated in cryopreserved sperm of red sea bream. We used SCGE to study DNA stability of red sea bream spermatozoa by measuring alkali-induced DNA strand breaking, to find out under cryopreservation whether any hereditable materials were damaged or not. The SCGE examines outside harm to cell nuclear DNA, and widely used to various cell nucleus DNA，including mammalian sperms, now fish. Pretreat the sperm (collection, cleaning, freezing, and thaw). Electrophorese and unwind it in the alkaline electrophoresis solution, dye it with EB for 10min, observe it under fluorescence microscope, count the proportion of nuclei with tails in about 50 nuclei at random, then calculate the comet rate. The comet rate is defined as the percentage of the number of nuclei with DNA damaged that would appear in the comet tail, to the total nuclei numbers. Each sample is measured 2-4 times repeatedly. We evaluate the damage and divide them into 5 grades: no damage (G0), slightly damaged (G1), mildly damaged (G2), heavily damaged (G3), and totally damaged (G4). We found that most sperm with DNA damage are in the grades G1 and G2, and few were totally damaged nuclei. The measured comet rates in different cryoprotectant (DMSO) concentrations of 5％; 10％; 18％; 20％; 25％ and 30% were: 33.47％ ± 8.95％; 35.91％ ± 19.44％; 48.95％ ± 8.90％; 43.33％ ± 11.19％; 55.80％ ± 38.94％; and 81.99％ ± 19.05％ respectively. The comet rate of the fresh sperm was 31.43 ％ ± 2.68％. Compared with the DNA dam age states of red sea bream frozen sperm and the fresh sperm, the result indicates that the frozen sperm DNA in 30％ of DMSO had remarkable diference with the fresh sperm (P<0.05).
页数	69
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1539
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	徐西长. 真鲷(Pagrosomus major)胚胎冷冻保存损伤机理及冷冻精子遗传物质稳定性研究[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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