Shalgi R, Seligman J, Kosower N S
Department of Embryology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Biol Reprod. 1989 May;40(5):1037-45. doi: 10.1095/biolreprod40.5.1037.
Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.
哺乳动物的精子在通过附睾时会经历成熟过程。成熟过程伴随着硫醇氧化为二硫键。二硫键可能参与精子染色质凝聚和尾部结构稳定。在本研究中，我们使用荧光硫醇标记剂单溴代双马来酰亚胺来确定大鼠精子头部和尾部在成熟过程中硫醇和二硫键的变化。精子取自睾丸、附睾（头、体、尾和输精管）以及射精精液。完整的精子用单溴代双马来酰亚胺标记，标记前或不进行二硫苏糖醇预处理。通过显微镜评估标记情况，并以标记的球蛋白为标准进行荧光分光光度法定量分析。样品也通过凝胶电泳进行分析。在精子成熟的整个过程中（26±0.5纳摩尔硫醇+二硫键/10⁶个精子），硫醇和二硫键的总量保持不变。然而，在附睾体和附睾尾之间，活性硫醇显著减少（从睾丸中占总量的90%以上和附睾体中的75%降至附睾尾中的约25%），在输精管和射出的精子中几乎没有进一步变化。用胰蛋白酶处理后通过蔗糖梯度分离精子头部和尾部。在附睾头精子的头部，硫醇占总SH + SS的84%，在尾部占74%，而在附睾尾精子中分别降至14%和45%。因此，活性硫醇的减少涉及头部和尾部，头部的硫醇氧化为二硫键的现象非常明显。电泳显示，在附睾成熟过程中，许多蛋白质组分中的硫醇都氧化为了二硫键。
