Krishnamurthy H, Weinbauer G F, Aslam H, Yeung C H, Nieschlag E
Institute of Reproductive Medicine of the University, Münster, Germany.
J Androl. 1998 Nov-Dec;19(6):710-7.
Several studies have reported the occurrence and significance of programmed cell death (apoptosis) of testicular germ cells in mammals. In those studies, apoptotic germ cells were identified by morphological criteria or by in situ end labeling (TUNEL) and were enumerated from histological sections by semi-quantitative and time-consuming techniques. In the present study, we have established a flow cytometric technique for quantification of TUNEL-positive cells in the mouse testis. Groups of five adult mice each received 0, 650, or 1300 mg/kg (IP) of methoxyacetic acid (MAA), and testes were collected 24 hours later. MAA is known to induce germ cell apoptosis in rodent testes. MAA induced a significant (P < 0.01) dose-dependent decline in the percentage of pachytene spermatocytes (4C cells). DNA strand breaks generated by the activation of endogenous endonuclease in the apoptotic germ cells were detected by the in situ labeling of the 3'-OH termini with biotinylated dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL technique). Histologically, TUNEL-positive germ cells were observed in control testes, and the number of these cells was visibly increased following MAA exposure. As determined by flow cytometry, four cell populations contained TUNEL-positive cells: 1C cells (round spermatids), 2C cells (mainly spermatogonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes synthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analysis of the percentages of TUNEL-positive cells within each population yielded values of 1.57+/-0.23% for 1C cells, 1.65+/-0.27% for 2C cells, 6.26+/-1.03% for S-ph cells, and 3.24+/-0.39% for 4C cells. Hence, a substantial proportion of proliferating cells are undergoing apoptosis during normal spermatogenesis. The overall incidence of apoptotic cells among all testicular cells was around 2%. At 650 mg per kilogram of body weight, MAA induced a fourfold to eightfold increase (P < 0.001) in the percentage of TUNEL-positive cells, compared with saline-treated controls, and, overall, 17% of testicular cells were apoptotic. This effect of MAA was most pronounced for S-ph and 4C cells, with 25-30% of cells being affected in each of those populations. At 1300 mg per kilogram of body weight, MAA had no further effect. These quantitative data demonstrate that 1) in the normal testis, it is mainly proliferating cells that undergo apoptosis, and 2) MAA induces primary spermatocyte loss by germ cell apoptosis.
多项研究报道了哺乳动物睾丸生殖细胞程序性细胞死亡（凋亡）的发生及意义。在这些研究中，凋亡的生殖细胞通过形态学标准或原位末端标记法（TUNEL）得以鉴定，并通过半定量且耗时的技术从组织切片中进行计数。在本研究中，我们建立了一种流式细胞术技术，用于定量小鼠睾丸中TUNEL阳性细胞。每组五只成年小鼠分别腹腔注射0、650或1300 mg/kg的甲氧基乙酸（MAA），24小时后收集睾丸。已知MAA可诱导啮齿动物睾丸中的生殖细胞凋亡。MAA导致粗线期精母细胞（4C细胞）百分比出现显著（P<0.01）的剂量依赖性下降。在末端脱氧核苷酸转移酶存在的情况下，通过用生物素化的dUTP对凋亡生殖细胞中内源性核酸内切酶激活产生的3'-OH末端进行原位标记，来检测DNA链断裂（TUNEL技术）。组织学上，在对照睾丸中观察到TUNEL阳性生殖细胞，MAA处理后这些细胞的数量明显增加。通过流式细胞术测定，四个细胞群体含有TUNEL阳性细胞：1C细胞（圆形精子细胞）、2C细胞（主要是精原细胞）、S-ph细胞（正在合成DNA的精原细胞和前细线期精母细胞[S期]）以及4C细胞（初级精母细胞）。对每个群体中TUNEL阳性细胞百分比的分析得出，1C细胞的值为1.57±0.23%，2C细胞为1.65±0.27%，S-ph细胞为6.26±1.03%，4C细胞为3.24±0.39%。因此，在正常精子发生过程中，相当一部分增殖细胞正在经历凋亡。所有睾丸细胞中凋亡细胞的总体发生率约为2%。与生理盐水处理的对照组相比，650 mg/kg体重的MAA诱导TUNEL阳性细胞百分比增加了四倍至八倍（P<0.001），总体而言，17%的睾丸细胞发生凋亡。MAA对S-ph细胞和4C细胞的这种作用最为明显，这两个群体中各有25 - 30%的细胞受到影响。在1300 mg/kg体重时，MAA没有进一步的作用。这些定量数据表明：1）在正常睾丸中，主要是增殖细胞发生凋亡；2）MAA通过生殖细胞凋亡导致初级精母细胞丢失。
