RESUMO
Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.
Assuntos
Bovinos/fisiologia , Fragmentação do DNA , Ectogênese , Fertilização in vitro/veterinária , Estresse Oxidativo , Espermatozoides/metabolismo , Matadouros , Animais , Apoptose , Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/citologia , Blastômeros/metabolismo , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Criopreservação/veterinária , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Cinética , Masculino , Malondialdeído/metabolismo , Análise do Sêmen/veterinária , Espermatozoides/citologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day, and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine embryonic stem cells and induced pluripotent stem cells.
Assuntos
Blastômeros/citologia , Blastômeros/metabolismo , Bovinos , Quimera/embriologia , Fase de Clivagem do Zigoto , Clonagem de Organismos/veterinária , Proteínas de Fluorescência Verde/genética , Animais , Animais Geneticamente Modificados , Bovinos/embriologia , Bovinos/genética , Bovinos/metabolismo , Fusão Celular/veterinária , Células Cultivadas , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/fisiologia , Clonagem de Organismos/métodos , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro/métodos , Proteínas de Fluorescência Verde/metabolismo , MasculinoRESUMO
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
Neural cadherin (N-cadherin) is a transmembrane glycoprotein involved in calcium-dependent cell-cell adhesion and signalling events. The previous evidence shows N-cadherin expression in the human gonads and gametes; however, N-cadherin subcellular localization in human spermatozoa and oocytes, and its involvement in fertilization remain to be characterized. In this study, expression of N-cadherin in human spermatozoa and testis was confirmed by RT-PCR and protein forms were identified using Western immunoblotting. N-cadherin localization in testicular and ejaculated spermatozoa, in cells that had undergone capacitation and acrosomal exocytosis, as well as in oocytes was assessed using immunocytochemistry. Participation of the adhesion protein in fertilization was evaluated using the HemiZona Assay (HZA) and the zona pellucida (ZP)-free hamster oocyte sperm penetration assay (SPA). Both the N-cadherin transcript and the mature protein form (135 kDa) were found in spermatozoa and testis. The protein was mainly immunolocalized in the acrosomal region of testicular, non-capacitated and capacitated spermatozoa, and was found in the equatorial segment after acrosomal exocytosis. N-cadherin was also detected in oocytes, in conjunction with beta-catenin, a member of the adhesion complex. Sperm incubation with anti N-cadherin antibodies did not affect their ability to interact with homologous ZP in the HZA; by contrast, presence of the antibodies in the SPA led to a significant (p < 0.01) reduction in the percentage of penetrated oocytes. In conjunction, results indicate that N-cadherin is a sperm protein of testicular origin localized in cellular regions involved in gamete interaction. N-cadherin would not participate in sperm-ZP interaction, but it would have a role in sperm-oolemma adhesion/fusion events.
Assuntos
Caderinas , Fertilização , Interações Espermatozoide-Óvulo , Espermatozoides/química , Espermatozoides/metabolismo , Acrossomo/metabolismo , Adulto , Animais , Anticorpos/análise , Anticorpos/metabolismo , Western Blotting , Caderinas/biossíntese , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Fase de Clivagem do Zigoto/metabolismo , Cricetinae , Feminino , Células Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oócitos/metabolismo , Proteínas/análise , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Capacitação Espermática , Testículo/metabolismo , Adulto Jovem , beta Catenina/análise , beta Catenina/metabolismoRESUMO
OBJECTIVE: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. DESIGN: Comparison between oocyte groups and between early embryo stages. SETTING: Laboratories of embryo manipulation and molecular biology from Departamento de Genética (FMRP) and Departamento de Ciências Básicas (FZEA)--University of São Paulo. SAMPLE(S): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. MAIN OUTCOME MEASURE(S): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). RESULT(S): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good- and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. CONCLUSION(S): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development.
Assuntos
Fase de Clivagem do Zigoto/metabolismo , Proteína 2 de Ligação a Metil-CpG/biossíntese , Oócitos/metabolismo , Proteínas de Ligação a Poli(A)/biossíntese , RNA Mensageiro/metabolismo , Animais , Bovinos , Biologia Computacional , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/metabolismo , Feminino , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas de Ligação a Poli(A)/genéticaRESUMO
In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (I) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and I (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22 h; and in ID at 26 h (45.0%) and 30 h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.
Assuntos
Adenina/análogos & derivados , Ionomicina/farmacologia , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Estrôncio/farmacologia , Adenina/administração & dosagem , Adenina/farmacologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Ciclo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Senescência Celular , Cromatina/metabolismo , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/efeitos dos fármacos , Fase de Clivagem do Zigoto/metabolismo , Feminino , Fertilização in vitro , Técnicas In Vitro , Ionomicina/administração & dosagem , Oócitos/citologia , Oócitos/metabolismo , Estrôncio/administração & dosagem , Tubulina (Proteína)/metabolismoRESUMO
The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.
Assuntos
Cromatina/metabolismo , Fertilização/fisiologia , Espermatozoides/metabolismo , Animais , Fase de Clivagem do Zigoto/metabolismo , Feminino , Variação Genética , Histonas/genética , Histonas/metabolismo , Masculino , Nucleossomos/metabolismo , Óvulo/metabolismo , Ouriços-do-MarRESUMO
Changes in protein synthesis during early Ambystoma mexicanum (axolotl) embryogenesis were monitored using two-dimensional (2-D) polyacrylamide gel electrophoresis. No change in synthesis patterns during progesterone-induced oocyte maturation was detected. In oocytes matured in vivo (unfertilized eggs), however, the synthesis of several oogenetic proteins ceased, only to be resumed later in development. At fertilization, one novel non-oogenetic protein was found. A cleavage-specific protein was also detected. Dramatic changes in protein synthesis patterns were detected at gastrulation in axolotl embryos. About 10% of the proteins synthesized at earlier stages ceased synthesis at gastrulation. Another 10% of the proteins synthesized during gastrulation were novel. A gastrulation-specific protein was also detected. After gastrulation additional novel non-oogenetic proteins were synthesized for most stages examined. A pronounced increase in the number of novel proteins synthesized was observed at the onset of neurulation and during neural fold fusion. Some of those proteins were specific to dorsal or axial structure tissue (AST) and some were specific to ventral or non-axial structure tissue (NAST). Actin and tubulin synthesis was also monitored during axolotl development. While the cytoplasmic gamma- and beta-actins were synthesized at all stages, muscle-specific alpha-actin synthesis began at the head-process stage (stage 23/25).