Gomez E, Buckingham D W, Brindle J, Lanzafame F, Irvine D S, Aitken R J
MRC Reproductive Biology Unit, Edinburgh, Scotland.
J Androl. 1996 May-Jun;17(3):276-87.
A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.
基于对NADH氧化还原酶活性的成像，已开发出一种用于量化人类精子中段残留细胞质的方法。该程序分别使用NADH和硝基蓝四唑作为电子供体和受体，结果是整个中段区域（包括残留细胞质）出现离散染色。然后使用图像分析技术生成中段的二值图像，据此可以对该细胞区域进行客观测量。在检测不到白细胞污染的精子群体中，发现这些数据与细胞质空间的生化标志物（如肌酸激酶（CK）和葡萄糖-6-磷酸脱氢酶（G-6-PDH））高度相关。还发现精子中段的形态计量分析反映了精液质量，因为它可以预测从Percoll梯度高密度区域回收的射精比例，并且与精液中精子的运动和形态呈负相关。过量残留细胞质保留的差异也与洗涤后精子制剂的功能能力差异有关，无论是在射精内还是射精间。因此，对高密度和低密度精子亚群进行射精内比较发现，低密度部分的精子功能存在相对破坏。这种破坏与中段中过量残留细胞质的存在、细胞质酶的高浓度以及活性氧（ROS）的产生增加有关。各个高密度Percoll制剂之间的功能差异也与中段面积以及精子产生ROS的相应能力呈负相关。这些发现表明，精子功能缺陷病因中涉及的因素之一是精子发生过程中生殖细胞细胞质的不完全挤出，其结果是精子经历了与氧化应激诱导相关的功能丧失。
