RESUMO
Human exposure to relatively low levels of methylmercury is worrying, especially in terms of its genotoxicity. It is currently unknown as to whether exposure to low levels of mercury (below established limits) is safe. Genotoxicity was already shown in lymphocytes, but studies with cells of the CNS (as the main target organ) are scarce. Moreover, disturbances in the cell cycle and cellular proliferation have previously been observed in neuronal cells, but no data are presently available for glial cells. Interestingly, cells of glial origin accumulate higher concentrations of methylmercury than those of neuronal origin. Thus, the aim of this work was to analyze the possible genotoxicity and alterations in the cell cycle and cell proliferation of a glioma cell line (C6) exposed to a low, non-lethal and non-apoptotic methylmercury concentration. Biochemical (mitochondrial activity) and morphological (integrity of the membrane) assessments confirmed the absence of cell death after exposure to 3 µM methylmercury for 24 hours. Even without promoting cell death, this treatment significantly increased genotoxicity markers (DNA fragmentation, micronuclei, nucleoplasmic bridges and nuclear buds). Changes in the cell cycle profile (increased mitotic index and cell populations in the S and G2/M phases) were observed, suggesting arrest of the cycle. This delay in the cycle was followed, 24 hours after methylmercury withdrawal, by a decrease number of viable cells, reduced cellular confluence and increased doubling time of the culture. Our work demonstrates that exposure to a low sublethal concentration of MeHg considered relatively safe according to current limits promotes genotoxicity and disturbances in the proliferation of cells of glial origin with sustained consequences after methylmercury withdrawal. This fact becomes especially important, since this cellular type accumulates more methylmercury than neurons and displays a vital role protecting the CNS, especially in chronic intoxication with this heavy metal.
Assuntos
Compostos de Metilmercúrio/toxicidade , Mutagênicos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Humanos , Compostos de Metilmercúrio/administração & dosagem , Testes de Mutagenicidade , Mutagênicos/administração & dosagem , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , RatosRESUMO
PURPOSE: Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. MATERIALS AND METHODS: In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). RESULTS: In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). CONCLUSIONS: Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.
Assuntos
Técnicas de Cocultura , Meios de Cultura , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Adulto , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células da Granulosa/citologia , Humanos , Camundongos , Proteínas de Transporte de Monossacarídeos/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , GravidezRESUMO
The efficiency of in vitro fertilization (IVF) depends on the viability of spermatozoa. For capuchin monkeys (Cebus apella), in vitro capacitation of spermatozoa is challenging because of their unique seminal coagulum. Motile spermatozoa can be obtained after liquefaction of the semen coagulum in coconut water-based solution. The objective of the present study was to establish an optimal in vitro maturation (IVM) protocol for capuchin monkeys and to observe the effect of follicle stimulating hormone (FSH) and luteinising hormone (LH) on IVF and parthenogenetic activation (PA) of oocytes collected from unstimulated females. We assessed spermatozoa quality after recovery from seminal coagulum using the solution ACP-118® as an extender. Oocytes were matured in vitro for 36 or 40 h and subjected to IVF or PA by applying ionomycin combined either with 6-dimethylaminopurine (6-DMAP) or roscovitine. In total, 87% of oocytes reached metaphase II (MII) after 40 IVM and 4-cell embryo production was obtained after IVF and parthenogenesis using ionomycin/6-DMAP. ACP-118® was used successfully to harvest viable spermatozoa from semen coagulum and in the preservation of spermatozoa, which were able to fertilize oocytes in vitro.
Assuntos
Embrião de Mamíferos/citologia , Fertilização in vitro , Metáfase/efeitos dos fármacos , Oócitos/citologia , Partenogênese/fisiologia , Espermatozoides/citologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Cebus , Células Cultivadas , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/farmacologia , Hormônios/farmacologia , Hormônio Luteinizante/farmacologia , Masculino , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Roscovitina , Espermatozoides/efeitos dos fármacosRESUMO
BACKGROUND: Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP. METHODS: The presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates. RESULTS: The [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates. CONCLUSIONS: The high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro.
Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Oócitos/metabolismo , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/metabolismo , Animais , Antioxidantes/farmacologia , Blastocisto/efeitos dos fármacos , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro/veterinária , Melatonina/farmacologia , Oócitos/efeitos dos fármacos , Quinona Redutases/metabolismoRESUMO
Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.