RESUMO
In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MTT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro.
Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/virologia , Genes Mitocondriais , Infecções por Herpesviridae/patologia , Herpesvirus Bovino 5/fisiologia , Animais , Apoptose , Bovinos , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/virologia , Regulação da Expressão Gênica no Desenvolvimento , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/veterinária , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Oócitos/fisiologia , Oócitos/virologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.
Assuntos
Apoptose , Blastocisto/virologia , Ectogênese , Herpesvirus Bovino 5/metabolismo , Mitocôndrias/metabolismo , Peroxirredoxina III/metabolismo , Superóxido Dismutase/metabolismo , Animais , Blastocisto/metabolismo , Blastocisto/patologia , Bovinos , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/patologia , Fase de Clivagem do Zigoto/virologia , Feminino , Fertilização in vitro/efeitos adversos , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 5/isolamento & purificação , Técnicas de Maturação in Vitro de Oócitos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/virologia , Oócitos/fisiologia , Oócitos/virologia , Peroxirredoxina III/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.
Assuntos
Aborto Animal/virologia , Feto/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/virologia , Aborto Animal/embriologia , Animais , DNA Viral/análise , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/embriologia , Cavalos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e EspecificidadeRESUMO
Se evaluó la incidencia de alteraciones oculares en infantes nacidos de madres con titulación positiva contra el complejo TORCH y la infección por sífilis. Incluimos a 33 pacientes, captados en el periodo de septiembre de 1997 a junio de 1998. Se valoró la presencia de alteraciones oculares congénitas en infantes con antecedentes de infección intrauterina por el síndrome de TORCH. Se incluyeron 8 mujeres (24.2 por ciento) y 25 hombres (75.7 por ciento). Las lesione que más se encontró fue: catarata en 6 pacientes (18.1 por ciento), coloboma en 2 pacientes (6.0 por ciento), microftalmía, entropión, queratoconjuntivitis, masa retrobulbar, desprendimiento de retina, coriorretinitis y atrofia óptica en los restantes pacientes (3.0 por ciento). Las infecciones intrauterinas influyen en el dessarrollo del ojo. Estos datos sugieren que el diagnóstico temprano de tales infecciones por el complejo TORCH permite realizar el tratamiento adecuado