超低温冷冻对人精子糖萼的影响

发布时间：2019-03-30 12:29

【摘要】：精子的超低温冷冻保存技术是辅助生殖技术以及精子库的重要组成部分,此技术水平的提高以及复苏率的稳定性都直接影响着最终受孕率。然而精子经冷冻复苏后,其存活率、活力、DNA完整性、质膜蛋白组成以及线粒体基质密度等都发生了不同程度的损伤,并且其宫颈黏液穿透力、穿卵能力等生育力也显著降低。精子糖萼,又称糖被,是睾丸生成的精子在附睾成熟过程中,附睾上皮细胞产生的各种糖基化修饰的蛋白或脂质结合到精子膜表面而形成的,是雄性配子与外界环境相互作用的主要界面,在精子保存、精子穿透宫颈黏液以及精子与卵子的识别和结合过程中具有重要作用。精子糖萼损伤可能导致生育力降低甚至不育。然而由于精子糖萼结构复杂,以往的研究缺乏高通量、简便的分析手段,关于人精子经超低温冷冻后其表面糖萼的变化情况目前尚未报道。本文通过比较三种精子冷冻液,自配的以及商业化的Quinn和ORIGIO,对精子冷冻复苏率的影响,发现ORIGK)精子冷冻保护液的复苏效果最好;在此基础上,我们又进一步比较了人工降温法、程序降温法,以及一步熏蒸法三种冷冻方法对精子复苏率的影响,发现一步熏蒸法效果最佳。通过比较优化,我们选择了 ORIGIO精子冷冻保护液,以及一步熏蒸法进行后续研究超低温冷冻对精子糖萼的影响。本课题利用本实验室已建立的凝集素芯片检测人精子糖萼的技术,通过比较超低温冷冻前后人精子与凝集素结合谱,发现35种凝集素在精子冷冻复苏后具有显著性差异,其中与9种凝集素的结合显著增加,26种显著降低。我们选择了其中4种与精子结合增加的凝集素(ABA、AIA、UEAI和PSA)和2种与精子结合降低的凝集素(MAA和DSL),通过流式细胞仪验证,发现与精子结合增加的ABA、AIA和PSA以及与精子结合降低的MAA与芯片结果一致。这些凝集素未来可用于评价精子冷冻保护液或精子冷冻方法的优劣,为人类精子库或辅助生殖中心精子的冷冻条件的优化提供新的评价方法。另一方面,我们还通过比较耐冻组和不耐冻组精子与凝集素结合的差异,发现耐冻组精子样本与ABA的结合显著降低;通过大样本验证,与芯片结果完全一致,未来ABA可作为评价精子耐冻性能的潜在生物学标志物,为临床夫精冷冻及冷冻后精子质量评价提供新的评价指标。
[Abstract]:Cryopreservation of spermatozoa is an important part of assisted reproduction technology and sperm bank. The improvement of sperm cryopreservation level and the stability of recovery rate directly affect the final pregnancy rate. However, after cryopreservation, the survival rate, viability, DNA integrity, plasma membrane protein composition and mitochondrial matrix density were damaged in varying degrees, and the ability of cervical mucus penetration and the ability to penetrate eggs were also significantly decreased. Sperm glycosylated calyx, also known as sugar quilt, is formed by the binding of various glycosylated proteins or lipids produced by epididymal epithelial cells to the surface of sperm membranes during the maturation of testicular spermatozoa. It is the main interface between male gametes and external environment. It plays an important role in sperm preservation, sperm penetration through cervical mucus, and the recognition and binding of sperm and eggs. Damage to sperm sugar calyx may result in decreased fertility and even infertility. However, due to the complexity of the structure of sperm sugar calyx, the previous studies lack of high-throughput, simple analytical means, the changes of human sperm surface sugar calyx after cryopreservation have not been reported. By comparing the effects of three kinds of cryopreservation solution, self-made and commercial Quinn and ORIGIO, on the recovery rate of spermatozoa, it was found that the recovery effect of cryopreservation solution of ORIGK was the best. On this basis, we further compared the effects of artificial cooling method, programmed cooling method and one-step fumigation method on sperm recovery rate, and found that one-step fumigation method was the best one-step fumigation method. Through comparison and optimization, we chose ORIGIO cryopreservation solution and one-step fumigation to study the effect of cryopreservation on sperm sugar calyx. By comparing the binding spectrum of human sperm and agglutinin before and after cryopreservation, it was found that 35 lectins had significant difference after sperm cryopreservation, and the agglutinin microarray was used to detect the calyx of human spermatozoa by using the lectin microarray established in our laboratory. Among them, the binding to 9 lectins increased significantly, while 26 species decreased significantly. Four of these lectins (ABA,AIA,UEAI and PSA) with increased sperm binding and two lectins (MAA and DSL),) with decreased sperm binding were identified by flow cytometry, and ABA, with increased sperm binding was found. The results of AIA, PSA and MAA with decreased sperm binding were consistent with the results of microarray. These lectins can be used to evaluate the advantages and disadvantages of sperm cryopreservation solution or sperm freezing methods in the future, and provide a new evaluation method for the optimization of freezing conditions of sperm in human sperm bank or assisted reproduction center. On the other hand, we also compared the difference of sperm binding to lectin between freezing-resistant group and non-freezing-tolerant group, and found that the binding of sperm samples to ABA in freezing-resistant group was significantly lower than that in freezing-resistant group. The results of large samples confirmed that ABA could be used as a potential biomarker to evaluate the freezing resistance of spermatozoa and provide a new evaluation index for the quality evaluation of frozen and frozen spermatozoa.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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