RESUMO
The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.
Assuntos
Bovinos/fisiologia , Células do Cúmulo/fisiologia , Temperatura Alta , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Animais , Feminino , Estresse FisiológicoRESUMO
The aim of this study was to verify whether the addition of catalase (20 IU/mL) at different steps of goat ovarian tissue vitrification affects ROS levels, follicular morphology and viability, stromal cell density, apoptosis and the expression of proteins related to DNA-damage signaling (γH2AX) and repair (53BP1). Goat ovarian tissues were analyzed fresh (control) or after vitrification: without catalase (VS-/WS-), with catalase in vitrification solutions (VS+/WS-), with catalase in washing solutions (VS-/WS+) or with catalase in both solutions (VS+/WS+). The vitrification without catalase had higher ROS levels than the control. The catalase, regardless the step of addition, maintained ROS levels similar to the control. There were no difference between treatments regarding follicular viability, stromal cell density and detection of γH2AX and 53BP1. There was no difference in follicular morphology and DNA fragmentation between groups vitrified. In conclusion, catalase addition to vitrification solutions prevents ROS formation in cryopreserved goat ovarian tissues.
Assuntos
Catalase/farmacologia , Criopreservação/veterinária , Folículo Ovariano/efeitos dos fármacos , Vitrificação , Animais , Apoptose/efeitos dos fármacos , Criopreservação/métodos , Feminino , Cabras , Histonas/metabolismo , Folículo Ovariano/citologia , Fosfoproteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The objective of this study was to determine the effect of different centrifugation forces in bovine sperm separation by discontinuous Percoll gradients for in vitro fertilization IVF. The semen samples from each bull were pooled or each bull were centrifuged separately and centrifuged in discontinuous Percoll gradients (30, 60 and 90%) at different forces: F1 (9000×g), F2 (6500×g), F3 (4500×g) and F4 (2200×g), according experiment. The sperm samples were evaluated to determine the concentration, motility, vigor, morphology, reactive oxygen species (ROS), integrity of the plasma membrane, lipid peroxidation, antioxidants and embryo development were also evaluated. No difference was observed in the concentration of sperm submitted to different centrifugation forces. The total percentage of motile sperm was increased after centrifugation at F3 and F4, and the ROS production at F1 was greater than the other forces. When the bulls semen were processed individually, no significant differences were observed for the sperm quality parameters between F1 and F4, including lipid peroxidation, antioxidants, cleavage rate and average time to the first cleavage. This work demonstrated for the first time that centrifugation at 2200×g enhanced the sperm penetration and fertilization rates without reducing sperm recovery compared to the typical centrifugation force (9000×g) currently used by the commercial bovine IVF industry.
Assuntos
Bovinos/fisiologia , Centrifugação com Gradiente de Concentração/veterinária , Fertilização in vitro/veterinária , Povidona , Dióxido de Silício , Espermatozoides/fisiologia , Animais , Bovinos/embriologia , Centrifugação com Gradiente de Concentração/métodos , Feminino , Masculino , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Análise do Sêmen/veterinária , Espermatozoides/citologiaRESUMO
Objetivou-se modificar o método de mini-Percoll para seleção espermática e redução da formação de ROS em espermatozoides bovinos. Dez palhetas de sêmen de um touro Bos taurus foram descongeladas, avaliadas, divididas em duas frações e depositadas sob gradientes de Percoll 90 e 45% (controle) e Percoll 90, 60 e 30% (tratado), sendo centrifugados por cinco minutos a 9000 x G. Após lavagem, foram efetuadas a avaliação morfofuncional e a quantificação de ROS. A proporção de espermatozoides com defeitos no grupo-controle (13,5%) foi igual (P > 0,05) ao do tratado (12,5%). A recuperação de 65,5 e 67 x 106 espermatozoides/mL, a motilidade progressiva de 54 e 63% e a integridade de membrana 23,7 e 22% não diferiram (P > 0,05) entre os grupos controle e tratado, respectivamente. Foi observada menor (P < 0,05) formação de ROS no grupo-tratado (3,03 ± 1,43 UF), comparado ao grupo-controle (7,24 ± 1,38 UF). A seleção espermática por gradientes de 90, 60 e 30% não afetou a recuperação e a qualidade dos espermatozoides, mas apresentou menor produção de ROS. Com base nesses resultados, pode-se concluir que o uso desses gradientes é eficiente na separação espermática, os quais podem ser utilizados em programas de PIV de embriões bovinos. (AU)
The objective was to modify the method for mini Percoll sperm selection and reducing the formation of ROS in bovine spermatozoa. Ten straws of semen from a bull Bos taurus were thawed, evaluated, divided into two fractions and deposited in Percoll gradients of 90 and 45% (Control) and Percoll 90, 60 and 30% (Treated), and centrifuged for 5' to 9000 xG. After washing was performed morphofunctional evaluation and quantification of ROS. Was similar (P > 0.05) the proportion of sperm with defects in the Control group (13.5%) and Treated (12.5%). The recovery of 65.5 and 67 x106 spermatozoa/mL, progressive motility of 54 and 63% and membrane integrity of 23.7 and 22% did not differ (P > 0.05) between the Control and Treated group, respectively. A lower formation of ROS (P < 0.05) was observed in the Treated group (3.03 ± 1.43 FU) compared to Control group (7.24 ± 1.38 FU). The sperm selection by Percoll gradients of 90, 60 and 30% did not affect the recovery and quality of sperm but showed a lower production of ROS. Based on these results we can conclude that the use these gradients is effective to separate sperm, can be used in programs of bovine IVP. (AU)
Assuntos
Animais , Masculino , Bovinos , Sêmen , Espécies Reativas de Oxigênio/farmacologia , Espermatozoides/química , Vírus da Imunodeficiência Felina , Fertilização in vitro/veterinária , Controle da População/métodos , OócitosRESUMO
Objetivou-se modificar o método de mini-Percoll para seleção espermática e redução da formação de ROS em espermatozoides bovinos. Dez palhetas de sêmen de um touro Bos taurus foram descongeladas, avaliadas, divididas em duas frações e depositadas sob gradientes de Percoll 90 e 45% (controle) e Percoll 90, 60 e 30% (tratado), sendo centrifugados por cinco minutos a 9000 x G. Após lavagem, foram efetuadas a avaliação morfofuncional e a quantificação de ROS. A proporção de espermatozoides com defeitos no grupo-controle (13,5%) foi igual (P > 0,05) ao do tratado (12,5%). A recuperação de 65,5 e 67 x 106 espermatozoides/mL, a motilidade progressiva de 54 e 63% e a integridade de membrana 23,7 e 22% não diferiram (P > 0,05) entre os grupos controle e tratado, respectivamente. Foi observada menor (P < 0,05) formação de ROS no grupo-tratado (3,03 ± 1,43 UF), comparado ao grupo-controle (7,24 ± 1,38 UF). A seleção espermática por gradientes de 90, 60 e 30% não afetou a recuperação e a qualidade dos espermatozoides, mas apresentou menor produção de ROS. Com base nesses resultados, pode-se concluir que o uso desses gradientes é eficiente na separação espermática, os quais podem ser utilizados em programas de PIV de embriões bovinos.
The objective was to modify the method for mini Percoll sperm selection and reducing the formation of ROS in bovine spermatozoa. Ten straws of semen from a bull Bos taurus were thawed, evaluated, divided into two fractions and deposited in Percoll gradients of 90 and 45% (Control) and Percoll 90, 60 and 30% (Treated), and centrifuged for 5' to 9000 xG. After washing was performed morphofunctional evaluation and quantification of ROS. Was similar (P > 0.05) the proportion of sperm with defects in the Control group (13.5%) and Treated (12.5%). The recovery of 65.5 and 67 x106 spermatozoa/mL, progressive motility of 54 and 63% and membrane integrity of 23.7 and 22% did not differ (P > 0.05) between the Control and Treated group, respectively. A lower formation of ROS (P < 0.05) was observed in the Treated group (3.03 ± 1.43 FU) compared to Control group (7.24 ± 1.38 FU). The sperm selection by Percoll gradients of 90, 60 and 30% did not affect the recovery and quality of sperm but showed a lower production of ROS. Based on these results we can conclude that the use these gradients is effective to separate sperm, can be used in programs of bovine IVP.
Assuntos
Masculino , Animais , Bovinos , Espermatozoides/química , Espécies Reativas de Oxigênio/farmacologia , Fertilização in vitro/veterinária , Sêmen , Vírus da Imunodeficiência Felina , Controle da População/métodos , OócitosRESUMO
Research strategies have been developed to characterize parameters in peripheral tissues that might easily be measured in humans as surrogate markers of damage, dysfunction or interactions involving neural targets of toxicants. The similarities between platelet and neuron may even be clinically important, as a number of biochemical markers show parallel changes in the central nervous system (CNS) and platelets. The purpose of our research was to investigate the effect of Hg(2+), Pb(2+) and Cd(2+) on the [(3)H]-glutamate binding and [(3)H]-glutamate uptake in human platelets. The involvement of oxidative stress in the modulation of glutamatergic system induced by heavy metals was also investigated. The present study clearly demonstrates that Hg(2+), Cd(2+), and Pb(2+) inhibited [(3)H]-glutamate uptake in human platelets. Hg(2+) inhibited [(3)H]-glutamate binding, while Cd(2+) and Pb(2+) stimulated [(3)H]-glutamate binding in human platelets. Hg(2+), Cd(2+) and Pb(2+) increased lipid peroxidation levels and reactive oxygen species (ROS) measurement in platelets. The present limited results could suggest that glutamatergic system may be used as a potential biomarker for neurotoxic action of heavy metals in humans.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ácido Glutâmico/sangue , Metais Pesados/toxicidade , Cádmio/toxicidade , Humanos , Técnicas In Vitro , Chumbo/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Mercúrio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Acute effects of mercury on mouse blood, kidneys, and liver were evaluated. Mice received a single dose of mercuric chloride (HgCl2, 4.6 mg/kg, subcutaneously) for three consecutive days. We investigated the possible beneficial effects of antioxidant therapy (N-acetylcysteine (NAC) and diphenyl diselenide (PhSe)2) compared with the sodium salt of 2,3-dimercapto-1-propanesulfonic acid (DMPS), an effective chelating agent in HgCl2 exposure in mice. We also verified whether metallothionein (MT) induction might be involved in a possible mechanism of protection against HgCl2 poisoning and whether different treatments would modify MT levels and other toxicological parameters. The results demonstrated that HgCl2 exposure significantly inhibited delta-aminolevulinate dehydratase (delta-ALA-D) activity in liver and only DMPS treatment prevented the inhibitory effect. Mercuric chloride caused an increase in renal non-protein thiol groups (NPSH) and none of the treatments modified renal NPSH levels. Urea concentration was increased after HgCl2 exposure. NAC plus (PhSe)2 was partially effective in protecting against the effects of mercury. DMPS and (PhSe)2 were effective in restoring the increment in urea concentration caused by mercury. Thiobarbituric acid-reactive substances (TBARS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities and ascorbic acid levels were not modified after mercury exposure. Mercuric chloride poisoning caused an increase in hepatic and renal MT levels and antioxidant treatments did not modify this parameter. Our data indicated a lack of therapeutic effect of the antioxidants tested.
Assuntos
Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Cloreto de Mercúrio/toxicidade , Intoxicação por Mercúrio/metabolismo , Metalotioneína/biossíntese , Animais , Derivados de Benzeno/uso terapêutico , Peso Corporal/efeitos dos fármacos , Quelantes/uso terapêutico , Quimioterapia Combinada , Injeções Subcutâneas , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Intoxicação por Mercúrio/etiologia , Intoxicação por Mercúrio/prevenção & controle , Camundongos , Compostos Organosselênicos/uso terapêutico , Sintase do Porfobilinogênio/antagonistas & inibidores , Sintase do Porfobilinogênio/metabolismo , Compostos de Sulfidrila/metabolismo , Unitiol/uso terapêutico , Ureia/sangueRESUMO
We investigated the effects of dimercaprol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercapto-1-propanesulphonic acid (DMPS) on human blood delta-aminolevulinate dehydratase (delta-ALA-D) activity, the most reliable indicator of lead intoxication in humans, in the presence of lead in vitro. Furthermore, we studied the effects of the chelating agents, administered subcutaneously, on delta-ALA-D activity in blood and tissues of mice submitted to sub-acute lead exposure (50 mg/kg for 15 consecutive days, subcutaneously). In vitro results demonstrated that human blood delta-ALA-D activity was significantly inhibited (62%) by lead acetate. Lead acetate (1-1000 microM) pre-incubated with human blood increased the inhibitory potency of this compound on delta-ALA-D when compared to the assay without pre-incubation (89%). Chelating agents caused a marked potentiation of delta-ALA-D inhibition induced by lead, in vitro. One of the most notable observations in the present study was the correspondence between in vitro and ex vivo effects. In fact, BAL and DMPS increase the inhibitory effect of lead on delta-ALA-D activity from mice blood. The complexes formed (lead and chelators) were more inhibitory than lead alone in kidney and liver enzyme activity, ex vivo.
Assuntos
Quelantes/farmacologia , Dimercaprol/farmacologia , Inibidores Enzimáticos/farmacologia , Chumbo/farmacologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Succímero/farmacologia , Unitiol/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismoAssuntos
Dimercaprol/administração & dosagem , Succímero/administração & dosagem , Unitiol/administração & dosagem , Ácido Aminolevulínico/antagonistas & inibidores , Ácido Aminolevulínico/química , Animais , Química Encefálica/efeitos dos fármacos , Dimercaprol/farmacocinética , Dimercaprol/toxicidade , Epilepsia Tônico-Clônica/induzido quimicamente , Injeções Subcutâneas , Rim/química , Rim/efeitos dos fármacos , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Camundongos , Succímero/farmacocinética , Succímero/toxicidade , Compostos de Sulfidrila/química , Fatores de Tempo , Testes de Toxicidade Aguda/métodos , Unitiol/farmacocinética , Unitiol/toxicidade , Zinco/químicaRESUMO
The effects of dithiol chelating agents meso-2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane-1-sulfonic acid (DMPS), and 2,3-dimercaptopropanol (BAL) on delta-aminolevulinate dehydratase (delta-ALA-D) from human erythrocytes were evaluated. Furthermore, possible protective effects of zinc chloride (ZnCl(2)), dithiothreitol (DTT), and cysteine were studied. delta-ALA-D activity from human erythrocytes was inhibited by dithiol chelating agents in a concentration-dependent manner. Cysteine, at all concentrations tested, did not protect the inhibitory effect of 1 and 4 mM DMPS and DMSA, but protected 1 mM BAL inhibition. Dithiotreitol was able to protect the inhibition caused by 1 mM BAL (28%), DMPS (56%), and DMSA (40%) in a concentration-dependent manner. Zinc chloride protected and restored 1 mM BAL inhibitory effect on delta-ALA-D. Zinc chloride at 500 microM and 1 mM, respectively, protected inhibitory effects of DMPS and DMSA (1 and 4 mM), but did not reverse its effects. The preincubation of dithiol chelating agents with enzyme demonstrated that DMSA was the most potent delta-ALA-D inhibitor of human erythrocytes. These data are in agreement with delta-ALA-D activity from purified enzyme. ZnCl(2) (1 microM) added, in the reaction mixture, increased enzyme activity and DTT (100 microM) totally restored the enzyme activity for all chelating agents tested.