Amos Fein

TNF-α in pregnancy loss and embryo maldevelopment: A mediator of detrimental stimuli or a protector of the fetoplacental unit?

AbstractPurpose: Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, has been identified in the ovary, oviduct, uterus, and placenta, and is expressed in embryonic tissues. For many years TNF-α was mainly considered to be a cytokine involved in triggering immunological pregnancy loss and as a mediator of various embryopathic stresses. However, data collected during the last decade has characterized TNF-α not only as a powerful activator of apoptotic, but also antiapoptotic signaling cascades, as well as revealed its regulatory role in cell proliferation. This review summarizes and conceptualizes the studies addressing TNF-α-activated intracellular signaling and the possible functional role of TNF-α in embryonic development. Methods: Studies addressing the role of TNF-α in intercellular signaling, in vivo studies addressing the functional role TNF-α in spontaneous and induced pregnancy loss, and studies addressing the role of TNF-α in fetal malformations were reviewed. Comparative studies in TNF-α knockout and TNF-α positive mice were performed to evaluate embryonic death, structural anomalies in fetuses, the degree of apoptosis and cell proliferation, and the activity of molecules such as caspases 3 and 8, the NF-κB, (RelA), IκBα in some target embryonic organs shortly after exposure to embryopathic stresses. Results: It is proposed that the possible essential function of TNF-α may be to prevent the birth of offspring with structural anomalies. Conclusions: TNF-α will boost death signaling to kill the embryo if initial events (damages) triggered by detrimental stimuli may culminate in structural anomalies, and stimulate protective mechanisms if the repair of these damages may prevent maldevelopment.

Recycling to the Plasma Membrane is Delayed in EHD1 Knockout Mice

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N‐terminal domain that contains nucleotide‐binding motifs, a central coiled–coil domain involved in oligomerization and a C‐terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1–/– embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.

TNF‐α Protects Embryos Exposed to Developmental Toxicants

BACKGROUND:  Tumor necrosis factor α (ΤNF‐α) has been implicated in mediating post‐implantation embryo loss or the embryonic maldevelopment induced by development toxicants or maternal metabolic imbalances. In order to clarify the role of TNF‐α further, a comparative study was performed in TNF‐α knockout and TNF‐α positive mice, exposed to a reference teratogen, cyclophosphamide (CP).

Apoptosis in the Uterus of Mice with Pregnancy Loss

PROBLEM: The mechanisms mediating pregnancy loss induced by various agents are far from being understood. Thus, we investigated the possible involvement of one such mechanism, the apoptotic process, in pregnancy loss induced by lipopolysaccharide (LPS) or cyclophosphamide (CP) as well as the associated changes in the apoptosis‐regulating gene products p53 and bcl‐2.

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53−/− normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53+/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53−/− normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

TGFβ2 mRNA expression and pregnancy failure in mice

PROBLEM: We describe here a pattern of transforming growth factor (TGF) β2 mRNA expression at the fetomaternal interface in mice with high rate of resorptions as well as its expression following maternal immunopotentiation.

Immunostimulation increases the resistance of mouse embryos to the teratogenic effect of diabetes mellitus

Summary The present work was aimed to assess the possible effect of stimulation of the maternal immune system on the teratogenic potential of diabetes mellitus. ICR female mice were immunized with splenocytes of male rats 3 weeks before the beginning of mating and were injected with 240 mg/kg streptozocin (STZ) 10 days after immunization. Females with blood glucose levels over 27.8 mmol/l and HbA1 c levels over 6 standard deviations (SD) above the mean of intact animals were used for teratological studies. The rate of malformed fetuses, resorptions and fetal weights were evaluated for animals killed on day 19 of pregnancy using routine teratological methods. Also, phenotyping of spleen cells of these females was performed by fluorescein activated cell sorter analysis. Two main effects possibly due to immunostimulation of ICR females were observed: 1) immunostimulated females had significantly fewer litters with malformed fetuses than non-immunized females: only 4 litters out of 22 (18 %) compared to 10 out of 16 (63 %). Correspondingly, the incidence of malformed fetuses was also decreased: 2.1 compared to 8.9 %; 2) a significant increase in the pregnancy rate in immunized diabetic ICR mice: 69 % as compared to 44 % in non-immunized diabetic females. Also, immunostimulation resulted in a visible increase in spleen cellularity and a certain increase in the number of cells with mature T-cell and macrophage surface markers. These results strongly suggest that immunostimulation increases the tolerance of ICR females to the teratogenic effect of STZ-induced diabetes. [Diabetologia (1997) 40: 635–640]

NF-κB DNA-binding activity in embryos responding to a teratogen, cyclophosphamide

BackgroundThe Rel/NF-κB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-κB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFα-induced physiological apoptosis. This study assesses whether NF-κB may be involved in the embryos response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstraiting differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies).ResultsThe embryonic brain and liver were used as target organs. We observed that the Cyclophosphamide-induced excessive apoptosis in the brain, followed by the formation of severe craniofacial structural anomalies, was accompanied by suppression of NF-κB DNA-binding activity as well as by a significant and lasting increase in the activity of caspases 3 and 8. However, in the liver, in which cyclophosphamide induced transient apoptosis was not followed by dysmorphogenesis, no suppression of NF-κB DNA-binding activity was registered and the level of active caspases 3 and 8 was significantly lower than in the brain. It has also been observed that both the brain and liver became much more sensitive to the CP-induced teratogenic insult if the embryos were exposed to a combined treatment with the teratogen and sodium salicylate that suppressed NF-κB DNA-binding activity in these organs.ConclusionThe results of this study demonstrate that suppression of NF-κB DNA-binding activity in embryos responding to the teratogenic insult may be associated with their decreased resistance to this insult. They also suggest that teratogens may suppress NF-κB DNA-binding activity in the embryonic tissues in an organ type- and dose-dependent fashion.

TNF-α expression in embryos exposed to a teratogen

PROBLEM: The role of tumor necrosis factor (TNF)‐α produced by embryonic cells in normal and abnormal development is poorly understood. To assess to what extent TNF‐α may be involved in the process of induced dysmorphogenesis, the expression of TNF‐α and TNF‐α receptor (TNFRI) mRNA as well as TNF‐α protein was evaluated in embryos responding to a cyclophosphamide (CP)‐induced teratogenic insult. The effect of maternal immunostimulation increasing the embryos tolerance to CP on TNF‐α expression was also investigated.

The role of pro- and anti-apoptotic molecular interactions in embryonic maldevelopment

Toder V, Carp H, Fein A, Torchinsky A. The role of pro‐ and anti‐apoptotic molecular interactions in embryonic maldevelopment. AJRI 2002; 48:235–244