RESUMO
Heparin is the most commonly used in vitro capacitation inducer in the bovine. However, hyaluronic acid (HA) has been recently used for capacitation induction as well as for other reproductive biotechnologies, such as sperm selection and in vitro fertilization (IVF). Our aim was to induce sperm capacitation with heparin or HA in order to study mAC and TK intracellular signals and their relation with cleavage and blastocyst rates after IVF as well as with the oxidative status of the potential bovine embryos. 2,5-dideoxyadenosine and genistein were used as mAC and TK inhibitors, respectively. Sperm capacitation was analyzed using CTC technique, sperm plasma membrane and acrosome integrity were determined using trypan blue stain and differential interference contrast, and mitochondrial activity was evaluated using fluorochrome JC-1. Cleavage rate was analyzed 48h and blastocyst production 7-8 days after IVF, while cytosolic oxidative activity was determined using RedoxSensor Red CC-1 fluorochrome 7h after IVF. When mAC and TK inhibitors were added to sperm samples, only capacitation decreased significantly both in HA and heparin treated samples (P < 0.05), but plasma membrane and acrosome integrity percentages were not affected in any of these groups (P > 0.05). Sperm mitochondrial membrane potential only decreased in heparin treated samples in the presence of both inhibitors (P < 0.05). Oocytes activated with HA sperm treated samples with the addition of 2,5-dideoxyadenosine and genistein presented a lower cytosolic oxidative status than those activated with sperm treated with HA alone (P < 0.05). On the other hand, oocytes activated with heparin treated sperm samples presented a lower cytosolic oxidative status only in the presence of 2,5-dideoxyadenosine (P < 0.05). Therefore, mAC and TK present a differential participation in heparin and HA sperm induced capacitation and mitochondrial function as well as in IVF.
Assuntos
Adenilil Ciclases/metabolismo , Fertilização in vitro/veterinária , Ácido Hialurônico/farmacologia , Proteínas Tirosina Quinases/metabolismo , Capacitação Espermática/efeitos dos fármacos , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Criopreservação/veterinária , Didesoxiadenosina/administração & dosagem , Didesoxiadenosina/farmacologia , Quimioterapia Combinada , Genisteína/administração & dosagem , Genisteína/farmacologia , Heparina/administração & dosagem , Heparina/farmacologia , MasculinoRESUMO
Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo-osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.
Assuntos
Criopreservação/veterinária , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Suínos , alfa-Tocoferol/farmacologia , Acrossomo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Clortetraciclina , Crioprotetores/farmacologia , Peroxidação de Lipídeos , Masculino , Imagem Óptica , Consumo de OxigênioRESUMO
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Isocitrato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Fosfofrutoquinases/metabolismo , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/enzimologia , Animais , Bicarbonatos/farmacologia , Feminino , Líquido Folicular/fisiologia , Isocitrato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/antagonistas & inibidores , Masculino , Fosfofrutoquinases/antagonistas & inibidores , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Sus scrofa , Tartronatos/farmacologiaRESUMO
The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO2 and tri-gas. A group of porcine oocytes maturated in vitro were injected with vitrified-warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO2 or tri-gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified-warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO2 or under tri-gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO2 and tri-gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 106 spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.
Assuntos
Injeções de Esperma Intracitoplásmicas/veterinária , Sus scrofa/fisiologia , Vitrificação , Animais , Criopreservação/métodos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro/veterinária , Gases , Masculino , Oócitos , Espermatozoides , Zigoto/crescimento & desenvolvimentoRESUMO
The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H2O2 or O2â(-) production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O2â(-) and H2O2 scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O2â(-) and H2O2 during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Suínos , Animais , Meios de Cultura , Oxigênio/farmacologiaRESUMO
Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2-oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10(-5) and (2.54 ± 0.32) 10(-5) , and for MDH, the U were (4.72 ± 0.42) 10(-5) and (4.38 ± 0.25) 10(-5) for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10(-3) and (0.94 ± 0.12) 10(-3) , and for MDH (9.08 ± 0.93) 10(-3) and (1.89 ± 0.10) 10(-3) for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2-oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Malato Desidrogenase/metabolismo , Oócitos/enzimologia , Fosfofrutoquinases/metabolismo , Suínos/fisiologia , Animais , Sobrevivência Celular , Células do Cúmulo , Regulação Enzimológica da Expressão Gênica/fisiologia , Ácidos Cetoglutáricos/farmacologia , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/genética , Meiose/fisiologia , Fosfofrutoquinases/antagonistas & inibidores , Fosfofrutoquinases/genética , Tartronatos/farmacologiaRESUMO
The purpose of this work was to assess commercially available Cryotech Vitrification Kit, in terms of survival, in vitro development and pregnancy rate for bovine embryos. Cumulus-oocyte complexes (COCs) were recovered from ovaries obtained from slaughtered cows and then matured in vitro for 22 h. COCs were fertilized by sex-sorted sperm in IVF-mSOF and cultured in IVC-mSOF for 7 days to the blastocyst stage. Blastocysts were vitrified with the Cryotech Vitrification Kit(®) and then either warmed to check viability or transferred to synchronized heifers. We observed 100% survival of the in vitro produced blastocysts and obtained the same pregnancy rate (46.8%) as that obtained using fresh in vitro produced blastocysts. We thus conclude that the Cryotech vitrification method is a valid alternative to other vitrification or slow-cooling methods in the bovine species and that it is ready for livestock production.
Assuntos
Blastocisto/citologia , Bovinos/embriologia , Criopreservação/veterinária , Vitrificação , Animais , Sobrevivência Celular , Criopreservação/métodos , Transferência Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , GravidezRESUMO
The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen-thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7-24âh from activation) by 2',7'-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19âh (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9âh (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24âh, presenting peaks around 7, 19, and 24âh (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17âh (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.
Assuntos
Bovinos/fisiologia , Ectogênese , Oócitos/fisiologia , Partenogênese , Espécies Reativas de Oxigênio/metabolismo , Interações Espermatozoide-Óvulo , Zigoto/metabolismo , Matadouros , Animais , Animais Endogâmicos , Divisão do Núcleo Celular , Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Cinética , Masculino , Oócitos/citologia , Oxirredução , Preservação do Sêmen/veterinária , Zigoto/citologiaRESUMO
The anatomy and histology of the male genital tract of the lesser anteater were studied. Fine details of spermatozoa regarding their genesis and morphology were also studied in six adult specimens. The testes lie in the pelvic cavity. The deferent duct emerges from the epididymis and opens into the ejaculatory duct, which drains into the membranous urethra. Accessory glands (prostate, seminal vesicle and bulbourethral gland) are histologically similar to those described in other mammals. The short penis presents an urethral orifice, while the corpus spongiosum becomes thinner at the end indicating the absence of a histologically defined glans. The seminiferous epithelium shows: (1) Sertoli cells with deep nuclear indentations, (2) spermatogonia with crusty-like chromatin, (3) spermatocytes at different stages of maturation and (4) three morphologically distinct stages of spermatid differentiation according to nuclear shape, acrosome development and chromatin condensation. Sperm heads appear oval. The length of the spermatozoa averages 67.33 ± 1.60 µm. Two specimens with inactive spermatogenesis were azoospermic. Their testes and epididymis presented sizes smaller than those with active spermatogenesis. These studies together with others in anteaters may contribute to successful breeding in conservation programmes.
Assuntos
Genitália Masculina/anatomia & histologia , Genitália Masculina/fisiologia , Espermatogênese/fisiologia , Xenarthra/fisiologia , Animais , Masculino , Estações do AnoRESUMO
Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.
Assuntos
Glicólise/fisiologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Via de Pentose Fosfato/fisiologia , Suínos , 6-Aminonicotinamida/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Células do Cúmulo , Feminino , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Ácido Láctico/biossíntese , NADP/farmacologia , Oócitos/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Fluoreto de Sódio/farmacologiaRESUMO
The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A(1) (with a dense cumulus), A(2) (with a translucent cumulus), B(1) (with the corona radiata), B(2) (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A(1) COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.
Assuntos
Células do Cúmulo/fisiologia , Gonadotropinas/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Suínos , Amônia/metabolismo , Animais , Contagem de Células , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Fertilização in vitro/veterinária , Hormônio Foliculoestimulante/farmacologia , Glucose/metabolismo , Glicólise , Ácido Láctico/metabolismo , Lipídeos/análise , Lipólise , Hormônio Luteinizante/farmacologia , Metaboloma , Oócitos/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/análiseRESUMO
Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.
Assuntos
Bovinos , Filtração/métodos , Espermatozoides/citologia , Espermatozoides/fisiologia , Animais , Contagem de Células , Separação Celular/métodos , Criopreservação/veterinária , Feminino , Vidro , Masculino , Sêmen , Análise do Sêmen , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Interações Espermatozoide-Óvulo/fisiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
The morphological and histological features of the unusual reproductive tract of the female lesser anteater, Tamandua tetradactyla (Myrmecophagidae, Xenarthra), are described for the first time. The present study aimed to establish the main similarities and differences between this species and other xenarthrans. The populations of this species are declining rapidly for a number of reasons and our study is relevant to diverse programs related to its conservation. Studies were carried out on five female genital tracts of adult specimens. Ovaries were ovoid, presenting a medulla completely surrounded by the cortex, differently from that described in other xenarthans. Like in Dasypus but different from all other armadillos studied, single oocyte follicles were observed and a simple the uterus. The uterovaginal canal connects the uterus with the urogenital sinus. The simple columnar epithelium of the uterovaginal canal ends abruptly at a septum which resembles a hymen, where the transitional epithelium of the urogenital sinus appears. This ancestral feature is shared with that of other armadillos, except Tolypeutes matacus, which has a true vagina. Characteristics of the reproductive tract and sperm morphology of other Xenarthra are comparatively discussed. These observations suggest that important reproductive features are shared between the family Myrmecophagidae and the genus Dasypus, a basal group in the phylogeny of Xenarthra.
Assuntos
Genitália Feminina/anatomia & histologia , Sistema Urogenital/anatomia & histologia , Xenarthra/anatomia & histologia , Animais , Feminino , Ovário/anatomia & histologia , Oviductos/anatomia & histologia , Útero/anatomia & histologiaRESUMO
The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.
Assuntos
Cafeína/farmacologia , Criopreservação , Heparina/farmacologia , Preservação do Sêmen , Capacitação Espermática/efeitos dos fármacos , Adenosina/farmacologia , Animais , Bicarbonatos/farmacologia , Bovinos , Clortetraciclina/química , Clortetraciclina/farmacocinética , Criopreservação/métodos , Criopreservação/veterinária , Feminino , Fertilização in vitro , Fluorescência , Líquido Intracelular/efeitos dos fármacos , Masculino , Sêmen/efeitos dos fármacos , Sêmen/metabolismo , Sêmen/fisiologia , Análise do Sêmen , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Transdução de Sinais/efeitos dos fármacos , Capacitação Espermática/fisiologia , Superóxidos/farmacologiaRESUMO
Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7'-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.
Assuntos
Embrião de Mamíferos/metabolismo , Espécies Reativas de Oxigênio , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Dióxido de Carbono , Bovinos , Meios de Cultura/metabolismo , Embrião de Mamíferos/citologia , Feminino , Fertilização in vitro , Fluoresceínas/farmacologia , Técnicas In Vitro , Oócitos/metabolismo , Ovário/metabolismo , Oxigênio/metabolismo , Sêmen/metabolismo , Espectrometria de Fluorescência , Espectrofotometria , Temperatura , Fatores de TempoRESUMO
Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39°C in 5 CO2: 95 humidified air. In vitro fertilization was carried out in IVF-mSOF with frozenthawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90 N2: 5 CO2: 5 O2, ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7' -dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P<0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time
Assuntos
Bovinos , Animais , Desenvolvimento Embrionário e Fetal , Espécies Reativas de Oxigênio , Radicais Livres , Estresse OxidativoRESUMO
Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39ºC in 5 CO2: 95 humidified air. In vitro fertilization was carried out in IVF-mSOF with frozenthawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90 N2: 5 CO2: 5 O2, ROS was determined in denuded oocytes and embryos at su
Assuntos
Bovinos , Animais , Espécies Reativas de Oxigênio , Estresse Oxidativo , Radicais Livres , Desenvolvimento Embrionário e FetalRESUMO
In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Bovinos/fisiologia , Fertilização in vitro/veterinária , Oócitos/fisiologia , alfa-Tocoferol/farmacologia , Animais , Bovinos/embriologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão/veterinária , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5
CO2: 95
humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90
N2: 5
CO2: 5
O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2,7-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.
RESUMO
Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.