Morshedi M, Oehninger S, Blackmore P, Bocca S, Coddington C, Hodgen G
Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Nor 23507, USA.
Int J Androl. 1995 Dec;18(6):279-86. doi: 10.1111/j.1365-2605.1995.tb00563.x.
The objective of the present studies was to assess the functional integrity of the sperm plasma membrane and metabolic and motility characteristics of the recovered motile fraction of human spermatozoa subjected to an automated freezing/quick-thawing method. Sperm membrane features examined included progesterone-induced changes in intracellular levels of calcium ([Ca2+]i), as measured by the fluorescent fura-2 indicator, and the tight binding of spermatozoa to homologous zonae pellucidae as assessed by the hemizona assay (HZA). Basal [Ca2+]i intracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels determined using chemiluminescence with luciferin-luciferase, and motility parameters determined using a computer-aided semen analyser (CASA) were studied concomitantly as an expression of metabolic/functional status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryopreserved-thawed samples, and fractions of each ejaculate (fresh and frozen-thawed) were subjected to parallel measurements of the same parameters at the same time frame. Basal and progesterone-induced increase in [Ca2+]i, and ATP levels (up to 24 h) were similar in fresh and frozen-thawed samples. HZA results showed a modest (26%) although significant (p = 0.008) decrease in binding in frozen-thawed samples. The ratios of ATP/ADP in fresh and frozen-thawed samples were also found to be similar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that the method was capable of preserving > 65% of motile spermatozoa in almost all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significantly impaired with regard to motility, mean linear velocity or linearity as compared to the fresh fractions in the first 4 h. Our results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the in-vitro fertilization (IVF) setting.
本研究的目的是评估采用自动冷冻/快速解冻方法处理后的人类精子活动部分的精子质膜功能完整性、代谢及运动特征。检测的精子膜特征包括：通过荧光fura-2指示剂测量孕酮诱导的细胞内钙水平（[Ca2+]i）变化，以及通过半透明带试验（HZA）评估精子与同源透明带的紧密结合。同时研究了使用荧光素-荧光素酶化学发光法测定的基础[Ca2+]i、细胞内三磷酸腺苷（ATP）和二磷酸腺苷（ADP）水平，以及使用计算机辅助精液分析仪（CASA）测定的运动参数，作为代谢/功能状态的一种表达。对来自生育男性（供体）的精液，在新鲜和冷冻解冻样本中对活动部分进行上浮分离后进行评估，并在同一时间框架内对每个精液样本（新鲜和冷冻解冻）的各部分进行相同参数的平行测量。新鲜和冷冻解冻样本中基础[Ca2+]i以及孕酮诱导的[Ca2+]i增加和ATP水平（长达24小时）相似。HZA结果显示，冷冻解冻样本中的结合能力虽有适度（26%）但显著（p = 0.008）下降。还发现新鲜和冷冻解冻样本中ATP/ADP的比率相似。虽然解冻后精子活力明显低于新鲜样本，但结果比较表明，该方法能够在几乎所有冷冻保存的样本中保存>65%的活动精子。此外，上浮法挽救了冷冻解冻样本中的活动部分，与新鲜部分相比，在前4小时内其活力、平均直线速度或直线性均未受到显著损害。我们的结果表明，这种自动冷冻-快速解冻方法会使精子与透明带的结合能力略有下降，并且在我们的实验条件下，新鲜和冷冻解冻样本中观察到的运动参数随时间的下降与ATP缺乏无关。这些体外结果与供体精子在体外受精（IVF）环境中的受精能力维持情况相符。
