RESUMO
Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS.
RESUMO
This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus-oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.
Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro/veterinária , Oócitos/efeitos dos fármacos , Partenogênese , Vitrificação , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Bovinos , Criopreservação/métodos , Feminino , Fertilização in vitro/métodos , Masculino , Oócitos/citologia , Oogênese/efeitos dos fármacos , Oogênese/fisiologiaRESUMO
Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17ß-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control -ve). The addition of 5.0-mM L-arg increased (P < 0.05) the percentage of oocytes at the germinal vesicle breakdown stage after 7 hours of cultivation compared with control -ve. All concentrations of L-arg (2.5, 5.0, and 10.0 mM) increased the percentage of oocytes that reached the metaphase I (MI) at 15 hours (P < 0.05) but do not affect the progression from MI to metaphase II (P > 0.05) at 22 hours. All concentrations of L-arg tested increased (P < 0.05) the percentage of cumulus cells with plasma membrane integrity at 22 hours of cultivation. L-arginine did not change (P > 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control -ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control -ve and treatment with 5-mM L-arg progressively decreased (P < 0.05) after the first hour of cultivation; however, COCs treated with 5.0-mM L-arg had higher concentrations of cGMP at 1 hour of cultivation (P < 0.05). The cAMP concentration of COCs supplemented or not with 5.0-mM L-arg progressively increased until 3 hours of cultivation and at, 6 hours, decreased (P < 0.05). The results show, in using this system, that (1) the mechanisms that give the oocyte the ability to restart the meiosis until MI after adding 5.0-mM L-arg do not involve changes in the concentration of nitrate/nitrite, P4, and E2 in the culture medium and (2) L-arg acts on a pathway that involves changing the cGMP concentration but does not involve changing cAMP concentration. More studies are needed to assess whether the observed effects of L-arg during IVM using this system are via NO or not and what the role is in increasing the viability of cumulus cells in the resumption and progression of meiosis until MI.
Assuntos
Arginina/farmacologia , Bovinos , Células do Cúmulo/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Animais , Células do Cúmulo/fisiologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologiaRESUMO
In this work, a promising approach to increase the advantageous properties of melatonin through its encapsulation into lipid-core nanocapsules (LNC) was examined. Oocytes were treated during in vitro maturation with non-encapsulated melatonin (Mel), melatonin-loaded lipid-core nanocapsules (Mel-LNC), and unloaded LNC. Cytotoxicity, meiotic maturation rate, development to the blastocyst stage, reactive oxygen species (ROS) and glutathione levels, mean cell number and apoptotic cell/blastocyst, and mRNA quantification were evaluated. Both Mel and Mel-LNC enhanced in vitro embryo production, however, Mel-LNC proved to be more effective at decreasing ROS levels and the apoptotic cell number/blastocyst, increasing the cleavage and blastocyst rates, up-regulating the GPX1 and SOD2 genes, and down-regulating the CASP3 and BAX genes. Mel-LNC could penetrate into oocytes and remain inside the cells until they reach the blastocyst stage. In conclusion, when melatonin was encapsulated in LNC and applied during in vitro oocyte maturation, some quality aspects of the blastocysts were improved.
Assuntos
Antioxidantes/administração & dosagem , Melatonina/administração & dosagem , Nanocápsulas/administração & dosagem , Oócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Bovinos , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Glutationa/metabolismo , Glutationa Peroxidase/genética , Oócitos/fisiologia , Superóxido Dismutase/genética , Proteína X Associada a bcl-2/genética , Glutationa Peroxidase GPX1RESUMO
This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 µM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 µM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 µM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.
Assuntos
Insulina/farmacologia , Oócitos/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Selênio/farmacologia , Transferrina/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Bovinos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Meiose/efeitos dos fármacos , Oócitos/citologia , Oócitos/fisiologia , Quinolonas/farmacologia , Fatores de TempoRESUMO
A qualidade do oócito é um fator limitante na fertilidade das fêmeas e reflete seu intrínseco potencial ao desenvolvimento embrionário subsequente. As alterações moleculares e bioquímicas no processo de maturação dos oócitos são necessárias para permitir a fecundação destes. Sob influência das gonadotrofinas, uma cascata de eventos é desencadeada, alterando a expressão gênica e a estrutura dos folículos. A maturação ocorre pelo intercâmbio entre o oócito e as células do cumulus que irão fornecer fatores para o desenvolvimento do oócito e criar o microambiente necessário para garantir o sucesso na maturação. A ação do FSH sobre a retomada da meiose ocorre, possivelmente, por ativação da proteína quinase C (PKC). A via de sinalização desta proteína parece estar envolvida na ativação da quinase ativada por mitógeno (MAPK) em oócitos e células do cumulus, na maturação induzida por FSH e LH, além de regular a síntese do Fator de Crescimento Epidermal (EGF). Deste modo, o objetivo do presente trabalho foi avaliar a ação da PKC na maturação de oócitos bovinos e se esta ativação envolve o EGF. Para tal foram realizados dois experimentos. Em ambos, a progressão do ciclo celular foi avaliada utilizando a sonda fluorescente Hoechst 33342. A expansão das células do cumulus foi avaliada utilizando-se o software Image Pro Plus 5.1 para análise das imagens dos oócitos geradas em microscópio Olympus IX81. O maior diâmetro de cada complexo cumulus oócito foi adotado como parâmetro de mensuração da expansão. A dosagem de progesterona do meio de cultivo foi realizada pela técnica de RIA. A ativação da PKC e da MAPK foi avaliada pela técnica de Western blot. Os dados foram avaliados pelo software SigmaPlot versão 12.2 e submetidos ao teste de normalidade (Shapiro-Wilk). Quando necessário, os dados foram transformados. Para comparação entre dois tratamentos, utilizou-se o teste t-student. Para mais de dois tratamentos foi realizada análise de variância e teste de comparação de médias (TUKEY), considerando-se 0,05 para rejeitar a hipótese de nulidade. No experimento 1 foi avaliado se a ativação da PKC foi estimulada por gonadotrofinas. Os oócitos foram maturados in vitro tratados com gonadotrofinas, na presença ou ausência do inibidor de PKC. A presença do inibidor de PKC diminuiu as taxas de quebra de vesícula germinativa e a expansão das células do cumulus, sem alterar a esteroidogênese. Estes resultados demonstram que a PKC participa da via de sinalização da retomada da meiose. No experimento 2 foi avaliado se o EGF está envolvido na via regulada pela PKC. Os oócitos foram maturados in vitro, na presença ou ausência de LH e FSH, do inibidor de PKC e do EGF. O EGF foi capaz de reverter os efeitos do inibidor de PKC, aumentando as taxas de quebra de vesícula germinativa e a expansão de células do cumulus. Não foi possível detectar, nas condições deste experimento, a ativação das proteínas PKC e MAPK através do Western Blot. Este trabalho permite concluir que a via de sinalização da maturação de oócitos bovinos envolve a PKC e sugere a participação do EGF nesta via
Oocyte quality is a limiting factor in female fertility and reflects its potential to the subsequent embryonic development. Molecular and biochemical alterations during the oocyte maturation process are needed to allow fecundation. Under gonadotropin influence, cascade of events occurs changing gene expression and follicle structure. Maturation depends on the interaction between oocyte and cumulus cells interaction, which provides factors for oocyte development and create the ideal microenvironment for the success of the maturation process. The FSH stimulation of meiosis resumption probably occurs through PKC activation. The signaling pathway of PKC might be involved by the mitogen activated protein kinase (MAPK) in oocytes and cumulus cells during FSH-LH induced maturation. Furthermore, MAPK regulates the epidermal growth factor (EGF) synthesis. The aim of the present study was to evaluate PKC function during bovine oocyte maturation and if its activity involves EGF. Two experiments were performed. In both experiments, the cell cycle progression was analyzed by Hoechst 33342 fluorescent dye. The cumulus cells expansion was performed using software Image Pro Plus 5.1 by the analysis of oocyte images taken in Olympus IX81 microscope. The highest diameter of each cumulus oocyte complex was recorded as the expansion value. The RIA and Western Blot techniques were used to measure progesterone concentration in the culture media and the PKC and MAPK activity, respectively. Data was analyzed by SigmaPlot software, version 12.2. The Shapiro-Wilk test was used to assess for normality and, when needed, the data was transformed. Student t tests were carried out to compare two treatments. Differences between more than two means were assessed by analysis of variance followed by Tukey test, considering P-value lower than 0.05 as statistically significant. Experiment 1 studied whether PKC function was stimulated by gonadotropins. FSH and LH were used for oocyte maturation in vitro, with or without PKC inhibitor. The presence of PKC inhibitor decreased germinal vesicle breakdown and the cumulus cells expansion, but did not alter the steroidogenesis. These results show that PKC participates in the signaling pathway of meiosis resumption. The Experiment 2 evaluated whether EGF influences PKC signaling pathway. The oocytes were matured in vitro, in the presence or absence of LH and FSH, PKC inhibitor and EGF. Epidermal Growth Factor was able to reverse PKC inhibitor effects, increasing germinal vesicle breakdown rates and cumulus cells expansion. The Western Blot technique was not able to detect PKC and MAPK activity, considering the conditions of this study. In conclusion, PKC is involved in the signaling pathway of bovine oocytes maturation and its pathway is mediated by EGF
Assuntos
Animais , Bovinos , Fertilidade , Hormônio Liberador de Gonadotropina , Proteína Quinase C/análise , Proteína Quinase C/efeitos adversos , Fator de Crescimento Epidérmico , Gonadotropinas , Meiose , Oócitos/crescimento & desenvolvimentoRESUMO
Non-invasive oocyte quality assessment remains a major challenge of routine bovine in vitro embryo production (IVP). There is a major need for techniques allowing early selection of developmentally competent oocytes on the basis of a simple, quick, economic and feasible protocol. The availability of such a technique would clearly in crease IVP efficiency since only competent oocytes would be used, maximising blastocyst yield by ameliorating culture conditions. This mini-review summarizes briefly the currently available techniques that allow high throughput non-invasive oocyte quality assessment and indicates their possibilities and limitations.(AU)
Assuntos
Animais , Oócitos/citologia , Embrião de Mamíferos/embriologia , Blastocisto/citologia , Bovinos/classificaçãoRESUMO
Non-invasive oocyte quality assessment remains a major challenge of routine bovine in vitro embryo production (IVP). There is a major need for techniques allowing early selection of developmentally competent oocytes on the basis of a simple, quick, economic and feasible protocol. The availability of such a technique would clearly in crease IVP efficiency since only competent oocytes would be used, maximising blastocyst yield by ameliorating culture conditions. This mini-review summarizes briefly the currently available techniques that allow high throughput non-invasive oocyte quality assessment and indicates their possibilities and limitations.