RESUMO
This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.
Assuntos
Criopreservação/veterinária , Oócitos/citologia , Vitrificação , Animais , Blastocisto/citologia , Bovinos , Criopreservação/métodos , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Meiose , Oócitos/metabolismo , Oócitos/ultraestruturaRESUMO
This study evaluates the post-hatching development of in vitro-produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post-hatching development (PHD) system. As a control group, in vivo-produced (IVV), Day-7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day-7 and Day-14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day-14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day-7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day-14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day-7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus.
Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Bovinos , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Perfilação da Expressão Gênica , Masculino , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Útero/fisiologiaRESUMO
Oocyte quality is one of the most important aspects of in vitro embryo development. Extensive epigenetic programming must occur during oocyte growth and maturation. A specific DNA methylation pattern of the imprinted genes must be established on differentially methylated regions (DMR). The insulin-like growth factor 2 (IGF2) gene is an important growth factor, and it is imprinted in several mammalian species. The aim of this study was to evaluate the methylation pattern on the DMR of the last exon of IGF2 in immature and mature bovine oocytes with different developmental competencies. Mature oocytes from large follicles were less methylated (28.93%) than immature oocytes from large follicles (77.38% P = 0.002), and there was also a tendency towards lower methylation in mature oocytes from large follicles (28.93%) compared with mature oocytes from small follicles (52.58% P = 0.07). Immature oocytes from small and large follicles showed 53.85% (7/13) and 91.66% (11/12) hypermethylated sequences, respectively, whereas mature oocytes from small and large follicles showed 61.11% (11/18) and 40% (4/10), respectively. The hypomethylation pattern in mature oocytes from large follicles may be related to the higher competence of these oocytes. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a molecular marker for oocyte competence in cattle and as a model for studies in other species.
Assuntos
Metilação de DNA , Fator de Crescimento Insulin-Like II/química , Fator de Crescimento Insulin-Like II/genética , Oócitos/metabolismo , Animais , Bovinos , Células do Cúmulo , Epigênese Genética , Feminino , Sequência Rica em GC , Impressão Genômica , Folículo Ovariano , Reação em Cadeia da PolimeraseRESUMO
Because bone-associated diseases are increasing, a variety of tissue engineering approaches with bone regeneration purposes have been proposed over the last years. Bone tissue provides a number of important physiological and structural functions in the human body, being essential for hematopoietic maintenance and for providing support and protection of vital organs. Therefore, efforts to develop the ideal scaffold which is able to guide the bone regeneration processes is a relevant target for tissue engineering researchers. Several techniques have been used for scaffolding approaches, such as diverse types of biomaterials. On the other hand, metallic biomaterials are widely used as support devices in dentistry and orthopedics, constituting an important complement for the scaffolds. Hence, the aim of this review is to provide an overview of the degradable biomaterials and metal biomaterials proposed for bone regeneration in the orthopedic and dentistry fields in the last years.
Assuntos
Materiais Biocompatíveis , Ortopedia , Regeneração Óssea , Odontologia , Humanos , Engenharia Tecidual , Alicerces TeciduaisRESUMO
During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production.
Assuntos
Blastocisto/fisiologia , Monoaminoxidase/genética , Inativação do Cromossomo X/genética , Alelos , Animais , Blastocisto/metabolismo , Bovinos , Células do Cúmulo , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Masculino , Monoaminoxidase/metabolismo , Fenótipo , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1-4 mL of Percoll, centrifuged for 20 min at 700 g; T2-800 microL of Percoll, centrifuged for 20 min at 700 g; and T3-800 microL of Percoll, centrifuged for 5 min at 5,000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P<0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P>0.05), but were affected by sire (P<0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.
Assuntos
Bovinos/embriologia , Fertilização in vitro , Hipergravidade , Povidona/farmacologia , Razão de Masculinidade , Dióxido de Silício/farmacologia , Animais , Centrifugação , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/fisiologia , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Hipergravidade/efeitos adversos , Masculino , Análise para Determinação do Sexo , Fatores de TempoRESUMO
Because bone-associated diseases are increasing, a variety of tissue engineering approaches with bone regeneration purposes have been proposed over the last years. Bone tissue provides a number of important physiological and structural functions in the human body, being essential for hematopoietic maintenance and for providing support and protection of vital organs. Therefore, efforts to develop the ideal scaffold which is able to guide the bone regeneration processes is a relevant target for tissue engineering researchers. Several techniques have been used for scaffolding approaches, such as diverse types of biomaterials. On the other hand, metallic biomaterials are widely used as support devices in dentistry and orthopedics, constituting an important complement for the scaffolds. Hence, the aim of this review is to provide an overview of the degradable biomaterials and metal biomaterials proposed for bone regeneration in the orthopedic and dentistry fields in the last years.
Assuntos
Humanos , Ortopedia , Materiais Biocompatíveis , Regeneração Óssea , Engenharia Tecidual , Odontologia , Alicerces TeciduaisRESUMO
Six geographically distinct isolates of Leishmania amazonensis causing diffuse cutaneous leishmaniasis (DCL) (from Bahia and Maranhão, in Brazil and Guarico, in Venezuela) were characterized by immunoblot analysis to see whether any geographical or strain-related differences existed in antigenic composition. Western blots of promastigote homogenates were reacted with polyclonal sera from patients infected with L. amazonensis with the various forms of clinical disease. The pattern of antigenic reactivity of these strains revealed the presence of shared antigenic components between geographically distinct L. amazonensis isolates causing DCL, when tested with the sera of the infected patients. In certain cases, however, some polyclonal sera also detected antigenic fractions unique to the strains examined. Variation was observed between the antigenic components of some isolates of L. amazonensis that were recognized by a single serum, and between the antigenic components of a single isolate of L. amazonensis that were recognized by the different patients' sera. However, no constant association was found between the antigenic components identified in these isolates and the geographical area of isolation. These results indicate that, although these parasites appear to be closely related antigenically, they also possess some strain-related antigenic differences.
Assuntos
Antígenos de Protozoários/análise , Leishmania mexicana/imunologia , Leishmaniose/imunologia , Animais , Western Blotting , HumanosRESUMO
The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.
Assuntos
Bovinos/embriologia , Fertilização in vitro/veterinária , Espermatozoides/citologia , Animais , Criopreservação/veterinária , Desenvolvimento Embrionário , Citometria de Fluxo/veterinária , Masculino , Análise do Sêmen/veterinária , Análise para Determinação do Sexo/veterináriaRESUMO
The purpose of this study was to evaluate the effect of cooling ovarian tissue on pig pre-antral follicles. Ovaries were maintained in saline solution (0.9%) at 4 or 20 degrees C for 6, 12 or 18 h. After storage, pre-antral follicles were morphologically evaluated. While primordial follicles were not affected by the storage, the percentage of morphologically normal growing follicles was significantly reduced in ovarian tissue stored at 20 degrees C for 12 or 18 h. To test the viability of stored follicles, growing follicles isolated from ovaries stored at 4 degrees C for 18 h and at 20 degrees C for 6 h were cultured for 3 days. Follicles stored in either condition presented the same growth pattern in vitro as fresh follicles. We conclude that storage of pig ovaries at 4 degrees C for up to 18 h or at 20 degrees C for up to 6 h does not affect the morphology of growing follicles or their ability to grow in vitro.
Assuntos
Preservação de Órgãos/veterinária , Folículo Ovariano/crescimento & desenvolvimento , Ovário/fisiologia , Suínos , Animais , Feminino , Preservação de Órgãos/métodos , Folículo Ovariano/ultraestrutura , Temperatura , Fatores de TempoRESUMO
A 12-year-old Simmental cow was presented with a moderately firm irregular whitish mass of approximately 5 cm in diameter, occupying the right orbit. Microscopically, a poorly differentiated neoplasm was observed. The immunohistochemical panel included cytokeratins, vimentin, epithelial membrane antigen, Factor VIII, CD34, Mart-1, Melan A, smooth muscle actin, desmin, chromogranin, neuron-specific enolase, S-100 protein, and MIB-1. The neoplasm was negative for all of them, with the exception of vimentin and S-100 protein. Transmission electron microscopy revealed abundant desmosomes. These findings support the diagnosis of orbital (retrobulbar) meningioma.
Assuntos
Doenças dos Bovinos/patologia , Meningioma/veterinária , Neoplasias Orbitárias/veterinária , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Feminino , Meningioma/diagnóstico , Meningioma/patologia , Neoplasias Orbitárias/diagnóstico , Neoplasias Orbitárias/patologiaRESUMO
The present study was designated to evaluate the antileishmanial activity of acid and basic fractions that were obtained after acid-basic extraction, from ethanolic 70% crude extract and pure compounds from the stem bark of Aspidosperma ramiflorum. The basic alkaloidal fraction presented a good activity against the extracellular form (promastigotes) of Leishmania (L.) amazonensis (LD(50) value<47 microg/ml). Based on these findings, the basic fraction was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording individual purified ramiflorines A and B. Both ramiflorines A and B showed significant activity against Leishmania (L.) amazonensis (LD(50) values of 16.3+/-1.6 microg/ml and 4.9+/-0.9 microg/ml, respectively). Our results are promising, showing that these compounds are biologically active against Gram-positive bacteria.
Assuntos
Antiprotozoários/farmacologia , Aspidosperma/química , Alcaloides Indólicos/farmacologia , Leishmania/efeitos dos fármacos , Animais , Antiprotozoários/química , Alcaloides Indólicos/química , Conformação Molecular , Fitoterapia , Casca de Planta/química , Caules de Planta/químicaRESUMO
A comparison of kDNA restriction-endonuclease fragment patterns from strains representing selected Endotrypanum zymodemes was done by schizodeme analysis. As the degree of heterogeneity within mini-circles varied among species or strains of Endotrypanum, the fingerprint obtained with each of the restriction enzymes was unique for each of these parasites. The data have revealed that this trypanosomatid genus is much more complex than it was originally thought to be.
Assuntos
DNA de Cinetoplasto/análise , Trypanosomatina/classificação , Animais , Heterogeneidade Genética , Variação Genética/genética , Isoenzimas/genética , Leishmania/classificação , Leishmania/enzimologia , Leishmania/genética , Polimorfismo Genético/genética , Trypanosomatina/enzimologia , Trypanosomatina/genéticaRESUMO
Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Epitopos/imunologia , Leishmania mexicana/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/isolamento & purificação , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Humanos , Especificidade da EspécieRESUMO
The opossum Didelphis marsupialis is known to be among the most important wild reservoirs of Trypanosoma cruzi and one in which the trypanosome may go through both the usual vertebrate intracellular cycle in its tissues and an extracellular cycle in the lumen of its scent glands. The species is highly resistant to heavy inocula and, depending on the parasite strain, experimental infections may be permanent or self limited. Aiming to understand the mechanisms involved in this parasite-host interaction we made a study of the acute phase of infection with different T. cruzi strains. Strains F, G-49 and G-327 produced durable infections with relatively high parasitemia and invasion of the scent glands, while equivalent inocula of the Y strain resulted in scanty parasitemia of short duration, no invasion of the SG, and no evidence of persistent parasitism. A smaller inoculum of G-49 produced only subpatent though persistent parasitemia and no invasion of the scent glands. The humoral immune response was less marked in the Y group; among the other groups IgM and IgG antibodies increased to high levels, higher in the G-49 group. The increase in IgG coincided with a drop of parasitemia to subpatent levels. Two opossums inoculated directly in the scent glands with culture forms of the Y strain had a short-lived subpatent parasitemia, but the parasites remained in the glands and serum Ig antibodies reached high levels. Immunoblot analysis showed that the sera of the inoculated opossums recognized few T. cruzi antigens (more in the F strain) in comparison with those of mice. However, with the only exception of those subcutaneously inoculated with the Y strain and including two naturally infected specimens, all the opossum's sera recognized a 90-kDa peptide in all T. cruzi strains. Our results confirm that opossums are able to selectively eliminate some strains of T. cruzi and indicate that the mechanism involved in this selection is probably not related to the humoral immune response. In infections by strains that are able to establish a permanent foothold in opossum tissues, there are indications that IgG antibodies participate in the control of the parasite population of the acute phase but are unable to prevent the chronic phase. It was once more demonstrated that the opossum infected scent glands function as diffusion chambers for parasite antigens but that, on the other hand, the parasites are here protected against the mechanisms developed by the host to control their population.