Morphological and molecular cell biological study of apoptosis in mammalian morphogenesis

1. 采用硫酸尼罗蓝染色法，在妊娠11 ~ 15天连续观察小鼠胎儿发育肢体细胞凋亡情况。研究发现，在指间质形成过程中，程序性细胞死亡（PCD）始于指间质近端，随后向远端扩展。因此，发生在指间区的初始PCD可能决定了指分离的近端，也有助于掌（底）和指（趾）之间的划分。在数字分离过程中，发现第13天指间区PCD面积变大并向远端扩展，这可能是数字分离和确定指间区消失所必需的。此外，在手足板的桡侧（胫骨）和尺侧（腓骨）边缘观察到强烈的PCD，持续4-5天。这种边缘区域的PCD似乎可以防止多余数字的形成（更典型的是前和后轴polydac）和其他数字畸形。HSP105 （105k - da热休克蛋白）在妊娠第8天小鼠胚胎中很少检测到，但在妊娠第9 ~ 11天显著升高，到妊娠第14天逐渐下降。发现HSP105定位于凝集细胞和凝集体，表现出肢体指间区凋亡的典型特征。这些结果提示HSP105在小鼠胚胎发育过程中起重要作用。采用DNA缺口端标记、透射电镜（TEM）和电泳等方法观察了丝裂霉素C （MMC）对成年雄性小鼠生精细胞凋亡的影响。结果发现，在MMC处理小鼠的睾丸中，(1)DNA精化处理后的细胞凋亡是由MMC剂量依赖性诱导的；(2)凋亡细胞死亡在精原细胞中最常见，在精母细胞中较少发生；(3)MMC诱导的凋亡细胞死亡不局限于精精期小管。提示MMC的睾丸毒性可能与诱导生精细胞凋亡有关。少

1. Apoptotic cell death in the developing limb of mouse fetuses was examined sequentially on days 11-15 of gestation by means of Nile blue sulfate staining. It was found that during digit formation, programd cell death (PCD) begins at the proximal portion of the interdigital mesenchyme and subsequently expands distally. Therefore, the initial PCD that occurs in the interdigital zones may determine the proximal ends of digital separation and also contribute to the demarcation between the palm (sole) and digits (toes). During digit separation, the areas of PCD in the interdigital zones were found to become larger and expand distally on day 13, which may be necessary for the separtion of digits and for determining the the interdigital area to disappear.In addition, intense PCD was observed in the radila (tibial) and ulnar (fibular) margins of the hand and foot plates for 4-5 days. Such PCD at marginal areas seems to prevent the formation of supernumerary digits (pre-and poistaxial polydac … More tyly) and other digital malformations.2. HSP105 (105 k-Da heat shock protein) was detected scarcelyt in mouse embryos at gestational day (GD) 8, but its level increased markedly from GD9 to 11 and then decreased gradually by GD14. HSP105 was found to be localized in condensed cells and condensed bodies showing the typical characteristics of apoptosis at the interdigital region of the limb.. These findings suggest that HSP105 plays important roles during mouse embryo development.3. Mouse spermatogenic cell apoptosis was examined in mature male mice after administration of mitomycin C (MMC) using DNA nick end labeling, transmission electron microscopy (TEM) and electrophoresis. It was found tha in the testis of MMC-treated mice, (1) apoptotic cell treated with DNA feagmentation is induced by MMC dose-dependently, (2) apoptotic cell death was found to occur most commonly in spermatogonia and less frequently in spermatocytes, and (3) apoptotis cell death induced by MMC was not specific for the seminiferous stage of the tubules. It is suggested that spermatogenic cell apoptosis induced by MMC might be involved in its testicular toxicity. Less