Stephan H, Polzar B, Rauch F, Zanotti S, Ulke C, Mannherz H G
Abteilung für Anatomie und Embryologie, Ruhr-Universität Bochum, Germany.
Histochem Cell Biol. 1996 Oct;106(4):383-93. doi: 10.1007/BF02473297.
The testis is a tissue of high proliferative activity. In this organ, sperm cells (spermatozoa) are produced from stem cells (spermatogonia) by two consecutive steps of cell multiplication and spermatid cytodifferentiation. Mitotic proliferation of spermatogonia generates primary spermatocytes which enter meiosis, leading to the generation of spermatids. The number of cells entering meiosis is held constant, since outnumbering spermatogonia or premeiotic spermatocytes are eliminated by apoptosis (programmed cell death). During apoptosis, the nuclear chromatin is internucleosomally degraded by the activity of a Ca2+, Mg2+-dependent endonuclease. Recent data indicate that deoxyribonuclease I (DNase I) is identical to the apoptotic endonuclease responsible for the internucleosomal DNA degradation. Previous results using primers specific for rat parotid DNase I in a polymerase chain reaction have demonstrated the presence of DNase I-specific gene transcripts in rat testis. We have therefore analysed the presence of DNase I in rat testis by immunohistochemistry and biochemical procedures. The presence of DNase I-like endonucleolytic activity was verified enzymatically. DNase I immunoreactivity was detected in the nuclei of a few spermatogonia and premeiotic spermatocytes, but within the acrosomic vesicle of all spermatids and spermatozoa. In situ hybridisation revealed the accumulation of DNase I-specific gene transcripts in a small number of spermatogonia and/or premeiotic spermatocytes, but in a large number of spermatids. The occurrence of apoptotic DNA fragmentation was investigated by in situ end-labelling (ISEL) of free 3'-OH DNA ends and gave positive nuclear staining of only very few spermatogonia. No positive ISEL staining was observed in maturing spermatids and/or spermatozoa. These data support the notion that, within the seminiferous epithelium, the number of primary spermatocytes entering meiosis is controlled by apoptosis. In addition, they demonstrated that mature sperm cells are equipped with an endonuclease that might be used for DNA degradation during their elimination at later stages of their life span. The expression and distribution of the tumour suppressor gene product, p53, was analysed by immunostaining. Strong p53 immunoreactivity was observed in the nuclei of a number of spermatogonia, of some premeiotic spermatocytes and probably in all spermatids. Thus, p53 expression appeared to parallel that of DNase I. In contrast, p53 immunoreactivity was absent in mature spermatozoa present in the lumen of the testicular tubules or the ductus epididymidis. It is therefore proposed that at later stages of spermatid maturation most probably before their release as mature spermatozoa-the p53 gene product was either degraded or retained in residual bodies, since p53 immunoreactivity was found to be concentrated within these organelles.
睾丸是具有高增殖活性的组织。在这个器官中，精子细胞（精子）由干细胞（精原细胞）通过细胞增殖和精子细胞分化的两个连续步骤产生。精原细胞的有丝分裂增殖产生初级精母细胞，初级精母细胞进入减数分裂，导致精子细胞的产生。进入减数分裂的细胞数量保持恒定，因为过量的精原细胞或减数分裂前的精母细胞通过凋亡（程序性细胞死亡）被清除。在凋亡过程中，核染色质通过钙、镁依赖性核酸内切酶的活性在核小体间被降解。最近的数据表明，脱氧核糖核酸酶I（DNase I）与负责核小体间DNA降解的凋亡核酸内切酶相同。先前在聚合酶链反应中使用针对大鼠腮腺DNase I的特异性引物的结果表明，大鼠睾丸中存在DNase I特异性基因转录本。因此，我们通过免疫组织化学和生化方法分析了大鼠睾丸中DNase I的存在情况。通过酶法验证了DNase I样核酸内切酶活性的存在。在少数精原细胞和减数分裂前的精母细胞的细胞核中检测到DNase I免疫反应性，但在所有精子细胞和精子的顶体泡内也有。原位杂交显示，DNase I特异性基因转录本在少数精原细胞和/或减数分裂前的精母细胞中积累，但在大量精子细胞中积累。通过对游离3'-OH DNA末端的原位末端标记（ISEL）研究凋亡DNA片段化的发生情况，结果显示只有极少数精原细胞的细胞核呈阳性染色。在成熟的精子细胞和/或精子中未观察到阳性ISEL染色。这些数据支持这样一种观点，即在生精上皮内，进入减数分裂的初级精母细胞数量受凋亡控制。此外，它们表明成熟的精子细胞配备有核酸内切酶，该酶可能在其寿命后期被清除时用于DNA降解。通过免疫染色分析了肿瘤抑制基因产物p53的表达和分布。在一些精原细胞、一些减数分裂前的精母细胞的细胞核中以及可能在所有精子细胞中观察到强烈的p53免疫反应性。因此，p53的表达似乎与DNase I的表达平行。相比之下，在睾丸小管或附睾管腔中的成熟精子中不存在p53免疫反应性。因此，有人提出，在精子细胞成熟的后期，很可能在它们作为成熟精子释放之前，p53基因产物要么被降解，要么保留在残余小体中，因为发现p53免疫反应性集中在这些细胞器内。
