Increased TNF-alpha expression in cultured mouse embryos exposed to teratogenic concentrations of glucose.

Diabetes-induced early embryonic death is accompanied by an increased expression of tumour necrosis factor alpha (TNF-alpha) in the embryonic microenvironment. The aim of the present study was to evaluate whether diabetes-induced embryopathic stress may also alter the expression of TNF-alpha produced by the embryo itself. As a model, whole postimplantation embryos were cultured for 24 h in a medium with high concentrations of glucose, one of the main diabetes-associated teratogenic metabolites. An anomaly such as an open neural tube was used as an end-point characterizing the glucose-induced teratogenic effect and the number of somites was counted to evaluate growth retardation induced by glucose. The expression of TNF-alpha (by immunohistochemistry), apoptosis (by TdT-mediated dUTP nick-end labelling; TUNEL) and the activity of caspases 3 and 8 (by a fluorometric assay) were evaluated in normal and malformed embryos. Ninety-seven per cent of the embryos exposed to 1300 mg glucose dl(-1) exhibited an open neural tube. The percentage of malformed embryos was smaller in media containing 800 and 500 mg glucose dl(-1) (68 and 37%, respectively) but it still exceeded significantly the value registered in embryos developing in a normoglycaemic medium (12%). In addition, a significant decrease in the number of somites was observed in embryos developing in media containing 1300 and 800 mg glucose dl(-1). Malformed embryos exhibited a greater number of nuclei that were positive in the TUNEL assay as well as a higher amount of active caspase 8 compared with normal embryos (with closed neural folds). TNF-alpha expression was detected in the neuroepithelial layer of the neural tube of the malformed embryos, whereas the expression of this cytokine was weak, if detectable, in normal embryos. Together, these findings indicate that TNF-alpha produced by the embryo may be involved in regulating the response of embryos to diabetes-generated embryopathic stress.

Cytokine expression in the uterus of mice with pregnancy loss: effect of maternal immunopotentiation with GM-CSF.

It is believed that failure of the maternal immune system to actively support embryonic development, through production of the appropriate cytokine network, might be responsible for embryonic death. Thus, the aim of this study was to evaluate the possible involvement of cytokines such as tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta2 (TGF-beta2), which are crucial for normal embryonic development, in the early stages of mechanisms that mediate induced pregnancy loss. The early stages of the resorption process induced by lipopolysaccharide (LPS) were characterized by blood accumulation in the vicinity of the embryo, preceding any visible embryonic damage. At that time, immunohistochemical analysis revealed an increased expression of TNF-alpha in the primary and secondary decidua, which was reduced as the resorption process was completed. In contrast, TGF-beta2 expression was decreased in the primary and secondary decidua, as well as in the glandular epithelium, at all the times assessed. Maternal immunopotentiation with granulocyte macrophage-colony stimulating factor (GM-CSF), which controls maternal immune activities supporting normal embryonic development, decreased the resorption rate in LPS-treated mice while normalizing the expression of TNF-alpha and TGF-beta2 in the uterus of these animals throughout the ongoing resorption process. These results indicate a possible role for maternal immunopotentiation with GM-CSF in the mechanisms mediating the early stages of pregnancy loss, possibly via modulation of TNF-alpha and TGF-beta2 activity.

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. METHOD OF STUDY: Pregnancy loss was induced by LPS or CP on days 9 or 12 of pregnancy, respectively. LPS- or CP-associated apoptosis was assessed by the TdT mediated dUTP-biotin nick end labeling (TUNEL) method as well as by DNA fragmentation analysis, while p53 or bcl-2 expression was evaluated by immunohistochemistry. RESULTS: Lipopolysaccharide treatment initiated a resorption process that was accompanied by the appearance of apoptotic cells in the uterus, which increased in number by 24 hr after treatment. Induction of pregnancy loss with CP resulted in the appearance of some apoptotic cells in the uterus, reaching a peak at 72 hr after treatment. DNA fragmentation analysis revealed a DNA ladder at 24 hr after LPS as well as 72 hr after CP treatment. Immunohistochemical analysis demonstrated a continuous p53 expression in the uterus of LPS- or CP-treated mice, which was somewhat elevated at the peak of the apoptotic process. On the other hand, bcl-2 expression in LPS-treated mice could be reciprocally correlated with the apoptotic process, appearing only at its initiation or completion, while in CP-treated mice it was continuously expressed except for some elevation at the completion of the apoptotic process. CONCLUSIONS: Our results suggest a possible role for the apoptotic process in mechanisms mediating pregnancy loss and indicate an involvement of p53 and bcl-2 in its regulation.

Diabetes teratogenicity in mice is accompanied with distorted expression of TGF-beta2 in the uterus.

Early embryonic deaths as well as malformed newborns are among complications of the diabetic pregnancy. Cytokines and growth factors operating in the embryonic vicinity are found to be among factors that determine the sensitivity of embryos to external and internal detrimental stimuli, including diabetes. Transforming Growth Factor-beta2 (TGF-beta2) has been shown to be essential for embryonic development and survival. In the present work, we evaluated the pattern of TGF-beta2 expression in the uterus of streptozotocin-induced diabetic mice, demonstrating a decreased reproductive performance and elevated percentage of litters with severely malformed fetuses. Since stimulation of the maternal immune system was found to increase the resistance of mouse embryos to the teratogenic effect of diabetes, the effect of immunopotentiation on the expression of the cytokine was also investigated. TGF-beta2 expression was studied at the mRNA level by using the in situ hybridization technique and at the protein level by using the immunohistochemical analysis. A clear decrease in TGF-beta2 mRNA expression in the uterus of diabetic mice was observed at examined time points: days 1, 5, and 9 of pregnancy. Also, an evident reduction in TGF-beta2, the protein expression in the uterus of diabetic mice, was demonstrated at these time points. Maternal immunopotentiation that improved the reproductive performance of diabetic mice and reduced the number of the litters with malformed fetuses was also accompanied by a clear increase in the level of TGF-beta2 mRNA expression in the pregnant uteri. The above results clearly demonstrate that the embryotoxic effect of diabetes is accompanied by an alteration of TGF-beta2 expression. Immunopotentiation that was shown to improve the reproductive performance of the diabetic mice was accompanied by a partial normalization of TGF-beta2 expression in embryonic vicinity.

Expression of tumor necrosis factor-alpha in the pregnant uterus of diabetic mice: effect of maternal immunopotentiation.

PROBLEM: Tumor necrosis factor (TNF)-alpha mRNA and protein are expressed in the pregnant uterus of streptozotocin-induced diabetic mice at various stages of pregnancy. We intend to characterize their pattern and to evaluate whether the potentiation of the maternal immune system alters the pattern of the expression of the cytokine. METHOD OF STUDY: Diabetes was induced in ICR mice by streptozotocin injection. To modulate maternal immune responses, ICR mice were injected intrauterine with rat splenocytes 3 weeks before mating. The expression of TNF-alpha mRNA and protein was evaluated by in situ hybridization and immunohistochemistry techniques. RESULTS: The population of diabetic mice used in this study demonstrated a reduction in pregnancy rate and an increased number of litters with severely malformed fetuses. It has been observed that these disturbances are associated with a clear increase in TNF-alpha mRNA and protein expression in the uterus of these mice. Maternal immunopotentiation, while improving reproductive performance of these diabetic mice, was found to be accompanied by a reduced expression of TNF-alpha, both at the mRNA and protein level. CONCLUSIONS: The results of the present study suggest a possible involvement of TNF-alpha in mechanisms underlying diabetes-associated dismorphogenesis. Normalization of TNF-alpha expression by maternal immunopotentiation might represent a mechanism mediating its protective effect against diabetes-induced embryotoxic insult.

TGFbeta2 in embryos with inborn anomalies: effect of maternal immunopotentiation.

PROBLEM: TGFbetas are among the main immunoregulatory molecules contributing to successful embryonic development. Besides, our and other studies revealed that maternal immunopotentiation has a potential to increase the resistance of the embryo to the teratogenic insult. This work was designed to evaluate: (1) whether the formation of teratogen-induced anomalies is accompanied by an altered pattern of TGFbeta2 expression in embryonic cells and (2) whether maternal immunopotentiation modifies the pattern of TGFbeta2 expression in embryos responding to the teratogenic insult. METHOD OF STUDY: Experiments were performed in embryos of ICR mice exposed to 15 and 40 mg/kg of a reference teratogen, cyclophosphamide (CP) on day 12 of gestation. A group of mice was immunopotentiated with xenogeneic rat splenocytes 21 hr before the beginning of mating. Embryos were examined for the occurrence of gross structural anomalies 24 and 72 hr after CP treatment. Then, immunohistohemistry and in situ hybrydization assays were used to evaluate the expression of TGFbeta2 protein and mRNA in the brain, face, limbs and liver of these embryos. RESULTS: No external anomalies were observed in embryos examined 24 hr after CP treatment. Embryos examined 72 hr after CP treatment at 40 mg/kg exhibited agnathia, micrognathia, kinky tail, phocomelia, but no signs of dismorphogenesis were observed in the liver at the organ level. A significant increase in the expression of TGFbeta2 mRNA was observed in cells, residing in the brain, face and limbs but not in the liver of CP-exposed embryos tested 24 hr after CP injection in both doses. The level of TGFbeta2 protein in these embryos did not differ from that of controls. In embryos tested 72 hr after CP injection in the high dose both TGFbeta2 protein and mRNA expression were found to be elevated. Maternal immunopotentiation while enhancing the embryo's resistance to CP practically abolished an elevated expression of the TGFbeta2 mRNA detected in tested organ structures of embryos of non-immunopotentiated CP treated mice 24 hr after CP injection in both the low and the high doses. Also, a significant decrease in the level of TGFbeta2 mRNA expression was observed in embryos of immunopotentiated mice examined 72 hr after CP treatment. CONCLUSIONS: The results of this work show a possible involvement of TGFbeta2 in the formation of teratogen-induced structural anomalies and suggest that the stimulation of the maternal immune system may realize its protective effect by normalizing the level of TGFbeta2 expression in teratogen-targeted embryonic structures.

Ciprofloxacin affects pregnancy loss in CBA/JxDBA/2J mice possibly via elevation of interleukin-3 and granulocyte macrophage-colony stimulating factor production.

PROBLEM: The mechanisms mediating pregnancy loss are far from being understood, but it is believed that modulation of the maternal immune system, that is known to support pregnancy, might serve as a means for the treatment of habitual abortions. Thus, we examined the effect of the anti bacterial agent ciprofloxacin, which was shown to affect maternal immunoreactivity, on pregnancy loss in the resorption-prone CBA/JxDBA/2J mouse combination as well as associated changes in Interleukin (IL)-3 and Granulocyte macrophage-colony stimulating factor (GM-CSF) production. METHOD OF STUDY: CBA/J females mated to DBA/2J males were treated with ciprofloxacin for 5 consecutive days. On day 15 of pregnancy, the number of resorbed embryos was recorded and IL-3 mRNA expression as well as IL-3 and GM-CSF protein production by splenocytes were assayed by northern blotting and ELISA, respectively. RESULTS: Ciprofloxacin treatment resulted in a significant reduction in the resorption rate as compared to the effect of the control antibiotic ceftazidime or PBS only, while not affecting the number of implantation sites/mouse. Also, splenocytes from ciprofloxacin-treated CBA/J mice exhibited an increased level of IL-3 mRNA transcripts as well as an elevation in IL-3 and GM-CSF protein production. CONCLUSIONS: Our data demonstrate the ability of ciprofloxacin to reduce pregnancy loss in the CBA/JxDBA/2J mouse model, possibly via elevation of IL-3 and GM-CSF production.

Cellular events and the pattern of p53 protein expression following cyclophosphamide-initiated cell death in various organs of developing embryo.

This study was aimed at characterizing the temporal patterns of cell responses and p53 protein expression in the limbs, head, and liver of embryos responding to cyclophosphamide (CP)-induced teratogenic insult. ICR murine embryos were examined 24, 48, or 72 h after injection of 40 mg/kg CP on day 12 of pregnancy. The cellular events and temporal pattern of p53 protein expression were determined by FACS analysis and by TUNEL (apoptosis) in the head, limbs, and liver of the embryos. All tested organs showed apoptosis and a significantly decreased proportion of live cells after 24 h. Subsequent events were organ-dependent. In the liver, there were no dysmorphic events at any time and excessive cell death had been almost compensated for by 48 h. Compensation was preceded by G(1) arrest and accompanied by an increased level of p53 protein in surviving cells. Excessive cell death in the head and the limbs resulted in structural anomalies. In the head, there was an increased level of p53 protein and G(1) arrest after 24 h and the number of live cells at 48 h was equal to that seen in earlier samples, despite apoptosis. In the limbs, however, only isolated viable cells were seen by 48 h, but there was no increased level of p53 protein or G(1) arrest. Results of this study suggest that the differential sensitivity of tested organ systems to CP may be associated with differences in cellular events following CP-initiated cell death. They also suggest that the input of p53 in determining the response of these organ systems to CP-induced teratogenic insult may be different. Teratogenesis Carcinog. Mutagen. 19:353-367, 1999.

Colony-stimulating factor-1 (CSF-1) expression in the uteroplacental unit of mice with spontaneous and induced pregnancy loss.

CSF-1 plays an important role in female reproduction and normal embryo development. To understand further CSF-1 function in normal and, especially, in compromised pregnancy, we studied the pattern of its mRNA expression as well as expression of its receptor (c-fms) in the uteroplacental units of mice with induced (cyclophosphamide (CY)-treated) and spontaneous (CBA/J x DBA/2J mating combination) pregnancy loss. RNase protection analysis demonstrated the presence of two forms of CSF-1 mRNA in the uteroplacental unit corresponding to 1400- and 263-bp protective fragments. Densitometric analysis demonstrated that the level of 1400-bp mRNA form was decreased by 40% in the uteroplacental units of mice with CY-induced pregnancy loss compared with the control mice. About 20% decrease in 263-bp protective fragment was registered in resorbing versus non-resorbed placenta of CBA/J females mated to DBA/2J males. As judged by in situ hybridization assay, CSF-1 mRNA transcripts were localized in the uterine epithelium and stroma, while c-fms mRNA was found mainly in the trophoblast. The number of metrial gland cells as well as the number of uterine leucocytes expressing CSF-1 and c-fms mRNAs was substantially lower in the uteroplacental unit of mice with pregnancy loss than in control animals. Maternal immunostimulation, while significantly decreasing the resorption rate in mice with CY-induced pregnancy loss, also strengthened CSF-1 mRNA expression at the fetomaternal interface and resulted in reconstitution in the number of CSF-1+ uterine leucocytes and metrial gland cells. These data suggest a role for uterine CSF-1 in the physiology of normal and compromised pregnancy and demonstrate a possible involvement of CSF-1-associated signalling in mechanisms of placenta and endometrium repair following immunopotentiation.

TNF-alpha expression in embryos exposed to a teratogen.

PROBLEM: 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. METHOD OF STUDY: 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.

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

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 HbA1c 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.

Use of antibodies against the human antigen of erythroblasts for the detection of nucleated erythrocytes in the maternal circulation.

In the present study, we tried to detect nucleated erythrocytes in maternal blood by means of the HAE9 antibody that binds to the antigen of erythroblasts (Ag-Eb) on nucleated erythroid cells. Umbilical cord blood and placental cells, which are known to contain a high number of nucleated erythrocytes, indeed demonstrated a high percentage of Ag-Eb-positive cells. In comparison, HAE9 stained up to 4.5% of the peripheral blood cells collected from pregnant women, but not those of nonpregnant women or normal men. These results, demonstrating a simple and reliable method for the identification of nucleated erythrocytes in the maternal circulation using the anti-Ag-Eb antibody, might open new possibilities for the detection of nucleated erythrocytes in maternal blood.

Teratogen-induced apoptosis may be affected by immunopotentiation.

Intra-uterine immunization of mice with paternal allogeneic or xenogeneic (rat) splenocytes was found to increase embryo tolerance to cyclophosphamide (CP)-induced teratogenesis. As the CP-induced teratogenic effect was shown to be associated with apoptosis, the present study was designed to investigate whether the protective effect of immunopotentiation may be realized via an alteration of CP-induced apoptosis. Various doses of CP were injected intraperitoneally into ICR mice on day 12 of pregnancy. Intra-uterine immunization with xenogeneic rat splenocytes was carried out 3 weeks before mating. Implantation sites, resorptions, live and dead fetuses, as well as soft tissue anomalies and external malformations, were recorded to evaluate the CP-induced embryotoxic effect. In parallel, flow cytometric analysis and DNA fragmentation assay were used for evaluation of CP-induced apoptosis in limbs, tail and whole embryos. The treatment of mothers with a high dose of CP induced the death of almost all embryos and striking fetal growth retardation in survivors. This strong embryotoxic effect was accompanied by very prominent DNA degradation in cells collected from whole embryos. Immunostimulation caused a dramatic decrease of embryonal loss (by approximately 50%) and a significant (about 30%) increase in fetal weight. Such an increase in fetal survival and in fetal weight was found to be accompanied by a clear decrease in apoptosis level in embryo cell population as judged by DNA gel electrophoresis with subsequent quantitation of DNA fragmentation in negatives by an image analysis technique. After treatment with a low dose of CP, a decrease in the proportion of fetuses with limb and tail anomalies in immunized females was accompanied by a decrease in the proportion of apoptotic nuclei in cells taken from limbs and tails. The results of this study suggest that the teratogen-induced apoptosis may, at least partly, be dependent on fetomaternal immune interactions.

Anti-Mac-1 antibodies and early pregnancy loss in mice.

This study focused on the role of adhesion molecules in early pregnancy in mice. Injection of anti-Mac-1 antibodies during early pregnancy resulted in early pregnancy loss (only 30.7% of mice in the group injected with anti-Mac-1 antibody were pregnant compared with 87.5% of controls), while mice treated with anti-Mac-1 antibodies during late pregnancy did not show a significant abortive effect (68.8% mice in the treated group were pregnant compared with 92.9% of control mice). Anti-LFA-1 alpha, LFA-1 beta or mouse Ag-Eb antibodies, when injected during early pregnancy, caused a nonsignificant decrease in pregnancy rate ranging between 15% and 25% (P > 0.05), while anti-Thy-1.2 antibodies demonstrated a marginal effect only. Staining of uterine tissue sections, collected on days 4-6 of pregnancy, with anti-Mac-1 antibodies, demonstrated antibody bound to cells in the deep endometrium and in the myometrium but not in the uterine area close to the lumen or on the surface of the blastocyst. These results indicate a possible role for the Mac-1 antigen in early pregnancy.

Cyclophosphamide-induced teratogenesis in ICR mice: the role of apoptosis.

It is known that programmed cell death (apoptosis) is an important physiological determinant of embryonic development. In parallel, it may be one of the major events involved in induced teratogenesis. The present study was designated to evaluate to what extent is apoptosis involved in the formation of some final abnormalities induced by cyclophosphamide (CP) in ICR mice. The level of apoptosis in limbs, tail, liver, and whole embryo was assessed 24 h after administration of various doses of CP (day 12 of pregnancy) by flow cytometric analysis and by DNA fragmentation assay. In parallel, the rate of limb and tail malformations, resorptions, and growth retardation induced by various doses of CP was evaluated in animals sacrificed on day 19 of pregnancy using routine teratological methods. A striking correlation between the rate of CP-induced apoptosis in limb and tail cells and the severity of limb and tail anomalies was found after administration of CP ranging from 10 to 40 mg/kg. Thus, the percent of apoptotic cells collected from limbs and tails increased from 18 to 78%. In parallel, the severity of limb and tail anomalies increased from digit anomalies to amely and from crooked to short or absent tail. CP-induced embryolethality and fetal growth retardation also correlated with the level of apoptosis in cells collected from whole embryos but to a lesser extent. These results claim that CP-induced apoptosis is one of the inevitable events in the pathway leading to the formation of CP-induced abnormalities and also suggest that the extent of the involvement of apoptosis in the formation of different types of final abnormalities, may be different.