RESUMO
The involvement of NF-κB in the regulation of teratogen-induced apoptosis has not been established yet. Therefore, we tried to assess the involvement of the p65 subunit of NF-κB in the embryonic response to the anti-cancer drug Doxorubicin (DOX). Thus, exposure of p65 knockout (p65(-/-)) or wild type (WT) mouse embryonic fibroblasts (MEFs) to DOX resulted in a decrease in cell survival, culture density and cell proliferation, which was found to be more prominent in p65(-/-) MEFs. Those phenomena were accompanied by a DOX-induced increase in the proportion of apoptotic cells, which was demonstrated only in p65(-/-) cells and a G2/M arrest, which was found to be more prominent in WT cells. Furthermore, DOX-treated WT and p65(-/-) MEFs differed in their expression of various apoptosis-associated molecules, when the former demonstrated a decrease in the percentage of p65-positive and a more prominent decrease in the percentage of p53-positive cells, while a decreased percentage of IκBα-positive and a more prominent decrease in the percentage of bcl-2-positive cells was detected among the latter. The fact that the response of the cells to the teratogen was clearly p65-dependent implicates this molecule to be involved in the response of the embryonic cells to DOX.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Fator de Transcrição RelA/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fator de Transcrição RelA/genéticaRESUMO
Bax was shown previously to regulate apoptotic cell death in various experimental systems, however, its involvement in teratogen-induced apoptosis is not clear yet. Therefore, we explored the involvement of Bax in the response of mouse embryonic fibroblasts (MEFs) to the anti cancer drug methotrexate (MTX), using Bax wild type (WT) and knockout (Bax(-/-)) MEFs. Our results demonstrated a significant teratogen-induced dose- and time-dependant decrease in the survival and culture density of both cell lines, which were found to be somewhat more prominent in WT cells. Exposure to MTX resulted also in decreased cell proliferation of WT but not Bax(-/-) cells and accordingly, we observed an accumulation of cells in the S phase and an increased percentage of cells in the Sub-G(1) phase of the cell cycle and the appearance of condensed nuclei, which were found to be somewhat more prominent in WT MEFs. In parallel, WT MEFs demonstrated a MTX-induced increase in the percentage of Bax-positive cells and a significant decrease in the percentage of bcl-2-, p65- or IkappaBalpha-positive cells, which were not detected in Bax(-/-) MEFs. Altogether, the differential sensitivity of WT or Bax(-/-) MEFs to MTX suggests a possible involvement of this molecule in the response of embryonic cells to teratogens.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Metotrexato/toxicidade , Teratogênicos/toxicidade , Proteína X Associada a bcl-2/metabolismo , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Metotrexato/administração & dosagem , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Fator de Transcrição RelA/metabolismoRESUMO
NF-kappaB was shown previously to regulate apoptotic cell death processes in various experimental systems. However, its role in controlling teratogen-induced cell death has not been established yet. Therefore, the objective of the present study was to explore the involvement of the p65 subunit of NF-kappaB in the response of mouse embryonic fibroblasts (MEFs) to heat shock, using p65 knockout (p65-/-) cells. Indeed, we found p65-/- MEFs to be more susceptible to the exposure to heat shock, as compared with wild-type (WT) MEFs, as they demonstrated a more prominent decrease in cell survival and proliferation as well as the appearance of cells undergoing apoptotic cell death. These heat-shock-induced effects were preceded by a decrease in p65 expression in WT cells, which was accompanied by a decrease in IkappaBalpha expression in WT MEFs, while disappearing completely in p65-/- MEFs and accordingly, by an increase in p-IkappaBalpha expression in both cell lines, which was found to be more prominent in p65-/- MEFs. Interestingly, the heat shock-induced decrease in p65 expression was accompanied by an increase in HSP70 expression in both cell lines. However, it was again found to be more prominent in p65-/- MEFs. Taken together, our results suggest a protective role for the p65 subunit of NF-kappaB in mechanisms underlying the response of embryonic cells to heat shock.
Assuntos
Febre/fisiopatologia , Fibroblastos/fisiologia , Resposta ao Choque Térmico/fisiologia , NF-kappa B/fisiologia , Fator de Transcrição RelA/fisiologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas I-kappa B/genética , Proteínas I-kappa B/fisiologia , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Fator de Transcrição RelA/genéticaRESUMO
BACKGROUND: Leukemia inhibitory factor (LIF) is at present suggested to be essential for implantation in mammals. In parallel, the possibility that it may also be involved in the pathogenesis of stress-induced early embryonic death seems to emerge from studies, which addressed the embryotoxic potential of another cytokine, tumor necrosis factor-alpha (TNF-alpha). In this brief review, we discuss this possibility based on these studies as well as on those addressing TNF-alpha and LIF signaling. METHODS: Existing data were reviewed critically. RESULTS: Data summarized in this review suggest that: (1) TNF-alpha may act as a mediator of stress-induced early embryonic death, (2) TNF-alpha-mediated early embryonic death induced by some detrimental stimuli may be attributed to a dysfunction of mechanisms, which are critical for the ability of the uterus to become receptive to blastocysts, allowing implantation, (3) one such mechanism was shown to be associated with LIF signaling in uterine cells, and (4) TNF-alpha seems to have the potential to affect LIF signaling. CONCLUSION: Data presented in the this review suggest LIF as a good candidate for further studies addressing molecular mechanisms underlying stress-induced early embryonic death.
Assuntos
Morte Fetal/etiologia , Interleucina-6/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Blastocisto/imunologia , Feminino , Morte Fetal/imunologia , Fator Inibidor de Leucemia , Camundongos , Gravidez , Transdução de Sinais , Estresse Fisiológico/imunologia , Útero/imunologiaRESUMO
Insulin-dependent diabetes mellitus is a well-known teratogen, which might cause growth retardation, malformations and fetal death. We have previously shown, that potentiation of the maternal immune system (immunopotentiation) might protect the embryo from diabetes teratogenicity. Therefore, in the present study we further inquired whether diabetes teratogenicity might be associated with alterations in the level of immune effector cells in systemic and local lymphoid organs as well as in the uterus throughout pregnancy and whether the protection exerted by maternal immunopotentiation might be realized through its effect on those cells. Streptozotocin-induced diabetes in ICR mice was found to reduce pregnancy rate and fetal weight while increasing the resorption rate and the percentage of litters with malformed fetuses. These teratogenic effects were accompanied by a decreased percentage of cells expressing Mac-1, Thy-1.2, CD4 or CD8 in the spleen and inguinal as well as paraaortic lymph nodes, except for Mac-1 expression by splenocytes, which increased significantly in the beginning of pregnancy and decreased later. A different pattern was observed in the uterus, when the percentage of cells expressing these markers tended to increase in the beginning of pregnancy and decrease later. Intrauterine immunopotentiation with rat splenocytes was found to improve the reproductive performance of diabetic animals. This protective effect was accompanied by a general normalization of the level of the various cell surface markers, when in most cases their expression returned to that found in nonimmunopotentiated mice. Our results suggest that the protection exerted by maternal immunopotentiation on the embryo against diabetes teratogenicity might be mediated via its effect on the level of immune effector cells localized to uterus and lymphoid organs, which was found to be altered in diabetic mice.
Assuntos
Anormalidades Congênitas/patologia , Diabetes Mellitus Experimental/complicações , Tecido Linfoide/patologia , Macrófagos/patologia , Gravidez em Diabéticas/complicações , Subpopulações de Linfócitos T/patologia , Útero/patologia , Animais , Anormalidades Congênitas/etiologia , Anormalidades Congênitas/imunologia , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Feminino , Peso Fetal , Feto/imunologia , Feto/patologia , Citometria de Fluxo , Imunização , Linfonodos/imunologia , Linfonodos/patologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , Gravidez em Diabéticas/imunologia , Gravidez em Diabéticas/patologia , Ratos , Ratos Long-Evans , Baço/imunologia , Baço/patologia , Subpopulações de Linfócitos T/imunologia , Útero/imunologiaRESUMO
BACKGROUND: Cytokines operating in the embryo and embryonic microenvironment determine, to a significant extent, whether pregnancy is completed successfully or results in embryonic loss or maldevelopment. They act as activators of specific transcription factors, which control cell responses such as cell proliferation differentiation and apoptosis. One such transcription factor is the nuclear factor-kappaB (NF-kappaB), which is presently seen as a key molecule controlling the apoptosis process. In the light of evidence that a majority of embryopathic stresses, regardless of their nature, first disturb the apoptotic process, it is conceivable, that NF-kappaB may play an important role in regulating the resistance of embryos to embryopathic stresses. In this brief review, we discuss such a possibility based on data characterizing expression and function of NF-kappaB in the embryo and extraembryonic tissues during normal embryogenesis as well as after exposure to various embryopathic stresses. METHODS: Critical review of existing data. RESULTS: Data summarized in this review suggest that (a) practically all NF-kappaB/Rel family members are expressed in embryonic, trophoblast and uterine cells in a developmental stage- and cell type-specific manner; (b) NF-kappaB-mediated anti-apoptotic signaling in embryonic cells seems to be indispensable for proper development during the organogenesis stage, (c) NF-kappaB activity in stress-targeted embryonic and extraembryonic structures directly correlates with their ability to resist stress-induced process of embryo loss and maldevelopment. CONCLUSION: Data presented in this review suggest that NF-kappaB may act as a protector of embryos exposed to embryopathic stresses, possibly, because of the ability of NF-kappaB to prevent the induction of programmed cell death as well as to activate cell proliferation.
Assuntos
Apoptose/fisiologia , Embrião de Mamíferos/metabolismo , NF-kappa B/metabolismo , Estresse Fisiológico/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes rel/fisiologia , Humanos , Gravidez , Complicações na Gravidez/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/embriologiaRESUMO
AIMS/HYPOTHESIS: Activation of apoptosis in embryos is thought to be a key event in the pathogenesis of diabetes-induced embryopathies such as early embryonic death and inborn structural anomalies. TNF-alpha can activate apoptotic and anti-apoptotic signalling cascades, indicating its ability to contribute to and counteract diabetes-induced maldevelopment. To investigate how TNF-alpha regulates the response of embryos to diabetes-induced embryopathic stress, we used streptozotocin-induced diabetic TNF-alpha knockout mice. MATERIALS: To evaluate the reproductive performance, mated diabetic female mice were examined on days 4 and 8 of pregnancy for the presence of blastocysts or embryos in uterine horns. To evaluate the teratogenic effect, the female mice were killed on day 18 of pregnancy and fetuses were examined for gross external anomalies. In addition, apoptotic nuclei were localised by the TUNEL assay and DNA-binding activity of the transcription factor NF-kappaB was evaluated by electrophoretic mobility shift assay in 10- and 11-day-old embryos respectively. RESULTS: Severely diabetic TNF-alpha(+/+) female mice had a much greater decrease in pregnancy rate but a lower incidence of malformed fetuses in litters than severely diabetic TNF-alpha(-/-) female mice. Also, the intensity of excessive apoptosis was higher, but the amount of active NF-kappaB complexes was lower in malformed TNF-alpha(-/-) embryos than in TNF-alpha(+/+) embryos. CONCLUSIONS/INTERPRETATION: TNF-alpha contributes to death of peri-implantation embryos and possibly protects postimplantation embryos exposed to diabetes-induced teratogenic stimuli via activation of NF-kappaB-mediated anti-apoptotic signalling. It seems that TNF-alpha prevents the birth of malformed offspring in severely diabetic mice.
Assuntos
Anormalidades Congênitas/prevenção & controle , Diabetes Mellitus Experimental/patologia , Gravidez em Diabéticas/patologia , Teratogênicos , Fator de Necrose Tumoral alfa/fisiologia , Animais , Apoptose , Anormalidades Congênitas/patologia , Desenvolvimento Embrionário e Fetal , Feminino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Gravidez , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
Assuntos
Anormalidades Induzidas por Medicamentos/imunologia , Embrião de Mamíferos/imunologia , Glucose/toxicidade , Teratogênicos/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Caspases/metabolismo , Células Cultivadas , Meios de Cultura , Embrião de Mamíferos/química , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Fator de Necrose Tumoral alfa/análiseRESUMO
PURPOSE: Tumor necrosis factor alpha (TNF-alpha), a multifunctional cytokine, has been identified in the ovary, oviduct, uterus, and placenta, and is expressed in embryonic tissues. For many years TNF-alpha 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-alpha 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-alpha-activated intracellular signaling and the possible functional role of TNF-alpha in embryonic development. METHODS: Studies addressing the role of TNF-alpha in intercellular signaling, in vivo studies addressing the functional role TNF-alpha in spontaneous and induced pregnancy loss, and studies addressing the role of TNF-alpha in fetal malformations were reviewed. Comparative studies in TNF-alpha knockout and TNF-alpha 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-kappaB, (RelA), IkappaBalpha in some target embryonic organs shortly after exposure to embryopathic stresses. RESULTS: It is proposed that the possible essential function of TNF-alpha may be to prevent the birth of offspring with structural anomalies. CONCLUSIONS: TNF-alpha 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.
Assuntos
Aborto Espontâneo/imunologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Apoptose/imunologia , Feminino , Morte Fetal/imunologia , Humanos , Camundongos , Camundongos Knockout , Gravidez , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Aborto Espontâneo/imunologia , Adjuvantes Imunológicos/administração & dosagem , Citocinas/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Útero/imunologia , Animais , Feminino , Idade Gestacional , Imuno-Histoquímica/métodos , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C3H , Modelos Animais , Gravidez , Fator de Crescimento Transformador beta/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
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.
Assuntos
Aborto Espontâneo/patologia , Apoptose/fisiologia , Útero/fisiologia , Animais , Apoptose/efeitos dos fármacos , Ciclofosfamida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Feminino , Imunossupressores/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Útero/patologiaRESUMO
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.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Perda do Embrião/imunologia , Fator de Crescimento Transformador beta/metabolismo , Útero/metabolismo , Animais , Perda do Embrião/genética , Feminino , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Fatores de Tempo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta2RESUMO
PROBLEM: Pregnancy loss and the occurrence of inborn structural anomalies are often preceded by excessive apoptosis in targeted embryonic and extraembryonic tissues. Apoptogenic stimuli activate both death and survival, signaling cascades consisting of molecules acting as activators and effectors, or negative regulators of apoptosis. The interplay between these cascades determines whether the cell which is exposed to an apoptogenic stimulus dies or survives. This review summarizes the functioning of pro- and anti-apoptotic molecules in embryos responding to various teratogens. The effect of potentiation of the maternal immune system on these molecules is also discussed. METHODS OF STUDY: The data on the functioning of various pro- and anti-apoptotic molecules in embryos exposed to various developmental toxicants, and embryos developing in a diabetic environment are reviewed. Techniques such as the TUNEL method, DNA fragmentation assay, electromobility shift assay (EMSA), fluorometric assay, immunohistochemistry, Western blot, In situ hybridization, have been used in our studies to detect apoptosis, and evaluate the functioning of molecules such as TNFalpha, caspases, NF-kappaB and IkappaB, p53, and bcl-2 in different embryonic and extraembryonic tissues. RESULTS: Our and other data summarized in this review have demonstrated that the doses of developmental toxicants required to induce pregnancy loss and gross structural anomalies induce excessive apoptosis shortly after treatment. Depending on the intensity and type of targeted tissues, this apoptosis was accompanied by alterations in the activity of the molecules which act as activators and effectors (e.g. caspase 3, caspase 8, caspase 2, p53) or negative regulators (bcl-2, NF-kappaB) of apoptosis. Maternal immunopotentiation, which decreases the level of induced and spontaneous pregnancy loss and the incidence and severity of teratogen-induced structural anomalies has been shown to modulate the expression of these molecules both in embryonic tissues and at the feto-maternal interface. CONCLUSIONS: The data presented in this review suggest that molecules such as TNFalpha, caspase 3, caspase 8, NF-kappaB, p53 and bcl-2, which are involved in the regulation of apoptosis, may also be involved in determining the sensitivity of the embryo to developmental toxicants. Maternal immunopotentiation may modulate the functioning of these molecules.
Assuntos
Apoptose/fisiologia , Embrião de Mamíferos/anormalidades , Desenvolvimento Embrionário e Fetal/fisiologia , Animais , Apoptose/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Feminino , Camundongos , Gravidez/imunologia , Transdução de Sinais/efeitos dos fármacos , Teratogênicos/toxicidade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PROBLEM: Women with three or more unexplained miscarriages have a 60% chance of a subsequent live birth. Intravenous immunoglobulin (IVIG) has not been conclusively shown to improve this prognosis. This study assessed the effect of IVIG in patients expected to have a poor outcome if untreated, i.e. women with five or more abortions, who have aborted after paternal leukocyte immunization or who continue to abort despite expressing anti-paternal complement dependent antibody. METHODS: Seventy-six women received IVIG in a dose of 400 mg/kg body weight, in one day (total 30-45 g) in the follicular phase of a cycle in which pregnancy was planned. A booster dose was administered when pregnancy was diagnosed. Their results were compared to an untreated control group of 74 women. RESULTS: Thirty-five (49%) pregnancies in treated women have resulted in live births or passed their previous stages of abortion compared to 23 (31%) in control patients (P = 0.04). CONCLUSIONS: These figures indicate that IVIG may prevent further miscarriages in this poor prognosis population. These figures are especially significant considering the doubt concerning the efficacy of IVIG in patients with three miscarriages and therefore a relatively good prognosis.
Assuntos
Aborto Habitual/tratamento farmacológico , Imunoglobulinas Intravenosas/uso terapêutico , Aborto Habitual/prevenção & controle , Adulto , Feminino , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Masculino , Gravidez , Resultado da Gravidez , Taxa de Gravidez , PrognósticoRESUMO
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.
Assuntos
Diabetes Mellitus Experimental/imunologia , Expressão Gênica , Gravidez em Diabéticas/imunologia , Fator de Necrose Tumoral alfa/genética , Útero/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Mensageiro , Ratos , Ratos Long-Evans , Estreptozocina/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To assess the chromosomal aberrations in the abortus in recurrent miscarriage and the live birth rate after a euploid or aneuploid miscarriage. DESIGN: Retrospective analysis. SETTING: Tertiary referral unit in university hospital. PATIENT(S): One hundred sixty-seven patients with 3 to 16 miscarriages before 20 weeks. INTERVENTION(S): Material collected at curettage from 167 abortuses was analyzed by standard G-banding techniques. MAIN OUTCOME MEASURE(S): The incidence of aberrations and the outcome of the subsequent pregnancy were assessed according to the embryonic karyotype. RESULT(S): In this study 125 specimens were successfully karyotyped. Of these, 29% (36 of 125) had chromosome aberrations; 94% of the aberrations were aneuploidy, and 6% were structural. The most prevalent anomalies were chromosome 16, 18, and 21 trisomies, triploidy, and monosomy X. After an aneuploid miscarriage, there was a 68% subsequent live birth rate (13 of 19) compared to the 41% (16 of 39) rate after a euploid abortion. CONCLUSION(S): The low (29%) incidence of aberrations indicates that alternative mechanisms may be responsible for the majority of recurrent miscarriages. These figures provide a basis for assessing the efficacy of therapy for recurrent miscarriage. If further studies confirm that patients with karyotypically abnormal fetuses have a good prognosis, an informed decision can be made as to whether further investigations and treatment should be undertaken.
Assuntos
Aborto Habitual/genética , Aberrações Cromossômicas , Cariotipagem , Adulto , Aneuploidia , Feminino , Feto , Hospitais Universitários , Humanos , Recém-Nascido , Israel , Pessoa de Meia-Idade , Monossomia , Razão de Chances , Poliploidia , Gravidez , Resultado da Gravidez , Estudos Retrospectivos , Aberrações dos Cromossomos Sexuais , Translocação Genética , Trissomia , Cromossomo XRESUMO
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.
Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Transferência Adotiva , Fator de Crescimento Transformador beta/fisiologia , Anormalidades Induzidas por Medicamentos/metabolismo , Animais , Apoptose , Ciclofosfamida/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/análise , Ratos , Ratos Long-Evans , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/genética , Transplante HeterólogoRESUMO
Recurrent miscarriage may be due to an inherently abnormal embryo (e.g. chromosomal aberrations), or maternal factors (e.g. uterine anomalies or antiphospholipid antibodies). However, there may be another mechanism; fetuses may have anomalies induced by toxic maternal factors. Early ultrasound scanning has revealed structural anomalies in karyotypically normal embryos in pregnancies that have terminated in first-trimester missed abortion. The serum of recurrently miscarrying women is toxic to blastocysts, embryos and fetuses. Teratogens such as cyclophosphamide or toxins such as lipopolysaccharide cause fetal demise by excessive apoptosis. Excessive apoptosis may be mediated by tumor necrosis factor-alpha and other cytokines. Both immunomodulation and hormonal support (progesterone or human chorionic gonadotropin supplements) have been used to improve the live birth rate in recurrently aborting women. Each may modulate the balance between Th1 and Th2 cytokines. Th2 cytokines are thought to benefit the developing embryo by enhancing placental growth and function, and possibly by preventing inappropriate apoptosis. Although neither hormonal support nor immunopotentiation have proved to be beneficial, no trial has limited itself to pregnancies that are karyotypically normal. This review assesses fetal structural anomalies in humans and laboratory animals as causes of pregnancy loss, the role of cytokines in those anomalies and the role of immunomodulation and hormones in modifying these effects.
Assuntos
Aborto Habitual , Anormalidades Congênitas , Citocinas/fisiologia , Hormônios/fisiologia , Aborto Habitual/etiologia , Aborto Habitual/imunologia , Aborto Habitual/prevenção & controle , Gonadotropina Coriônica/administração & dosagem , Feminino , Humanos , Gravidez , Progesterona/administração & dosagem , Ultrassonografia Pré-NatalRESUMO
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.