TNF-alpha expression in embryos exposed to a teratogen.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Ivnitsky
I
I
Department of Embryology and Teratology, Sackler School of Medicine, Tel Aviv University, Israel.
Torchinsky
A
A
Gorivodsky
M
M
Zemliak
I
I
Orenstein
H
H
Savion
S
S
Shepshelovich
J
J
Carp
H
H
Fein
A
A
Toder
V
V
D000818:Animals; D001921:Brain / Q000002:abnormalities / Q000196:embryology; D019465:Craniofacial Abnormalities / Q000196:embryology; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000276:immunology; D005260:Female; D007114:Immunization; D017403:In Situ Hybridization; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013723:Teratogens / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D013723:Teratogens; D014409:Tumor Necrosis Factor-alpha; D003520:Cyclophosphamide