Long C R, Dobrinsky J R, Garrett W M, Johnson L A
Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20705, USA.
Mol Reprod Dev. 1998 Sep;51(1):59-65. doi: 10.1002/(SICI)1098-2795(199809)51:1<59::AID-MRD7>3.0.CO;2-V.
In vitro-produced embryos exhibit decreased cell numbers, small inner cell masses and reduced pregnancy rates after transfer. Evaluation of intracellular components of in vitro-produced or -manipulated embryos will lead to improved methodology for embryo production. Whole mount techniques were developed to utilize terminal deoxynucleotidyl-transferase 3' nick end labeling (TUNEL) to detect broken DNA. Subsequent labeling of either tubulin or actin filaments provides further evidence of cytological damage. Porcine embryos produced in vitro or in vivo were evaluated throughout the cleavage and preimplantation stages of development. Early cleavage stages up to the 8-cell stage never contained TUNEL-labeled nuclei. However, TUNEL labeling of in vitro-produced morula revealed some blastomeres with broken DNA. Nearly all in vitro-produced blastocysts displayed some TUNEL positive cells, whereas in vivo-collected embryos at a similar stage displayed few, if any, TUNEL-labeled nuclei. The ratio of TUNEL-labeled DNA to total DNA area of in vitro-derived blastocysts was significantly greater than their in vivo counterparts (P < 0.05). Microtubule and microfilament labeling identified blastomeres of unequal size and shape that were losing cellular integrity. These data suggest that the combination of these labeling techniques may be useful in evaluating cellular damage in embryos produced under in vitro conditions.
体外生产的胚胎在移植后表现出细胞数量减少、内细胞团较小以及妊娠率降低。对体外生产或操作的胚胎的细胞内成分进行评估将有助于改进胚胎生产方法。开发了整装技术，利用末端脱氧核苷酸转移酶3' 缺口末端标记法（TUNEL）来检测断裂的DNA。随后对微管蛋白或肌动蛋白丝进行标记，可进一步证明细胞损伤。对体外或体内生产的猪胚胎在整个发育的卵裂和植入前阶段进行评估。直至8细胞期的早期卵裂阶段从未有过TUNEL标记的细胞核。然而，体外生产的桑椹胚的TUNEL标记显示一些卵裂球存在DNA断裂。几乎所有体外生产的囊胚都显示出一些TUNEL阳性细胞，而在类似阶段体内采集的胚胎则很少（如果有的话）有TUNEL标记的细胞核。体外来源的囊胚中TUNEL标记的DNA与总DNA面积的比率显著高于体内来源的囊胚（P < 0.05）。微管和微丝标记识别出大小和形状不等且正在丧失细胞完整性的卵裂球。这些数据表明，这些标记技术的组合可能有助于评估体外条件下生产的胚胎中的细胞损伤。
