Ivnitsky I, Torchinsky A, Gorivodsky M, Zemliak I, Orenstein H, Savion S, Shepshelovich J, Carp H, Fein A, Toder V
Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Israel.
Am J Reprod Immunol. 1998 Dec;40(6):431-40. doi: 10.1111/j.1600-0897.1998.tb00430.x.
The role of tumor necrosis factor (TNF)-alpha produced by embryonic cells in normal and abnormal development is poorly understood. To assess to what extent TNF-alpha may be involved in the process of induced dysmorphogenesis, the expression of TNF-alpha and TNF-alpha receptor (TNFRI) mRNA as well as TNF-alpha protein was evaluated in embryos responding to a cyclophosphamide (CP)-induced teratogenic insult. The effect of maternal immunostimulation increasing the embryo's tolerance to CP on TNF-alpha expression was also investigated.
ICR female mice were treated intraperitoneally with 40 mg/kg CP on day 12 of pregnancy. The immunostimulator, xenogeneic rat splenocytes, was injected intrauterine 21 days before mating. Embryos were collected on days 13, 14, or 15 of pregnancy. TNF-alpha mRNA, TNFRI mRNA, and TNF-alpha protein expression were evaluated by in situ hybridization and immunostaining techniques in control, teratogen-treated, and immuno-stimulated teratogen-treated embryos.
CP-treated embryos showed severe external brain and craniofacial anomalies already visible on day 14 of pregnancy. TNF-alpha mRNA transcripts were detected in cells of the brain and the head of 13-day embryos, which preceded the occurrence of CP-induced external craniofacial anomalies. On day 15 of pregnancy, when severe craniofacial anomalies increased, a significant increase in the intensity of TNF-alpha, TNFR1 mRNA transcripts, and TNF-alpha protein expression were observed in cells of the malformed regions of the head and the brain. In other nonmalformed organs of CP-treated embryos such as the liver (not macroscopically different from controls), neither TNF-alpha nor TNFR1 transcripts were detected. Immunostimulation substantially diminished the severity of CP-induced brain and craniofacial anomalies, decreased the resorption rate, and was associated with decreased intensity of TNF-alpha mRNA transcripts detected on day 15 of pregnancy in the head and the brain of CP-treated embryos.
TNF-alpha expressed in the embryo may be one of the molecules promoting the formation of CP-induced brain and craniofacial anomalies. The decrease of TNF-alpha expression in embryos of immunostimulated females may be one of the mechanisms responsible for the increased tolerance to the teratogenic insult.
胚胎细胞产生的肿瘤坏死因子（TNF）-α在正常和异常发育中的作用尚不清楚。为了评估TNF-α在诱导畸形发生过程中的参与程度，我们在对环磷酰胺（CP）诱导的致畸损伤做出反应的胚胎中，评估了TNF-α及其受体（TNFR1）mRNA以及TNF-α蛋白的表达。还研究了母体免疫刺激增强胚胎对CP耐受性对TNF-α表达的影响。
在妊娠第12天，对ICR雌性小鼠腹腔注射40mg/kg CP。在交配前21天，将免疫刺激剂异种大鼠脾细胞宫内注射。在妊娠第13、14或15天收集胚胎。通过原位杂交和免疫染色技术，评估对照、致畸剂处理以及免疫刺激致畸剂处理胚胎中的TNF-α mRNA、TNFR1 mRNA和TNF-α蛋白表达。
CP处理的胚胎在妊娠第14天就出现了严重的脑和颅面外部异常。在妊娠第13天胚胎的脑和头部细胞中检测到TNF-α mRNA转录本，这早于CP诱导的颅面外部异常的出现。在妊娠第15天，当严重的颅面异常增加时，在头部和脑的畸形区域细胞中观察到TNF-α、TNFR1 mRNA转录本强度以及TNF-α蛋白表达显著增加。在CP处理胚胎的其他未畸形器官如肝脏（肉眼观察与对照无差异）中，未检测到TNF-α和TNFR1转录本。免疫刺激显著减轻了CP诱导的脑和颅面异常的严重程度，降低了吸收率，并且与妊娠第15天在CP处理胚胎的头部和脑中检测到的TNF-α mRNA转录本强度降低有关。
胚胎中表达的TNF-α可能是促进CP诱导的脑和颅面异常形成的分子之一。免疫刺激雌性胚胎中TNF-α表达的降低可能是对致畸损伤耐受性增加的机制之一。
