Corrigendum to “Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro

High oxygen levels during in vitro culture (IVC) can induce oxidative stress through accumulation of reactive oxygen species (ROS), negatively affecting embryo development. This study evaluated the effect of different O2 tensions during IVC on bovine blastocyst development and transcriptional status, considering transcription factors that play an essential role during early embryo development. For this purpose, embryos were produced in vitro by conventional protocols and cultured in two different oxygen tensions, physiological (5%) and atmospheric (20%). Expanded blastocysts were subjected to transcript quantitation analysis by RT-qPCR with Biomark™ HD System (Fluidigm, US), using 67 TaqMan assays specific for Bos taurus. Differences were observed in genes related to oxidation-reduction processes, DNA-dependent transcription factors, and factors related to important functional pathways for embryo development. Blastocyst rate was higher in the 5% O2 group and the number of cells was assessed, with the 5% O2 group having a higher number of cells. ROS concentration was evaluated, with a higher ROS presence in the 20% O2 group. Taken together, these results allow us to conclude that IVC of embryos at atmospheric O2 tension affects the expression of important transcription factors involved in multiple cell biology pathways that can affect embryo development, quality, and viability

O papel da espécies reativas de oxigênio e do antioxidante astaxantina em oócitos bovinos submetidos ao estresse térmico in vitro

Condições ambientais adversas, tais como temperatura e umidade elevada, limitam a exploração do máximo potencial genético animal. Em bovinos, o estresse térmico materno compromete a fertilidade de vacas leiteiras, resultando em prejuízos econômicos para indústria de leite. Sabe-se que o oócito e o embrião no estágio pré-implantacional inicial são os principais alvos dos efeitos deletérios causados pelo estresse térmico materno, entretanto, os mecanismos celulares desencadeados pela temperatura elevada e o papel do estresse oxidativo neste contexto são pouco conhecidos. Estudos recentes indicaram que a exposição de embriões bovinos ao choque térmico aumentou a concentração de espécies reativas de oxigênio (EROs) intracelular embrionária e o uso de antioxidantes, tais como a astaxantina, reverteram estes efeitos. Com isto, o presente estudo visou avaliar os efeitos do choque térmico e do antioxidante astaxantina durante a maturação in vitro (MIV) de oocitos bovinos na (1) concentração das EROs, (2) competência oocitária, (3) atividade de enzimas antioxidantes e (4) peroxidação lipídica em complexos cumulus-oócito (CCOs) bovinos. O experimento 1 avaliou os efeitos de diferentes concentrações de astaxantina (0, 50, 100, 200 ou 500 µM) na competência oocitária à 38,5°C (etapa 1) e os efeitos de diferentes concentrações de astaxantina (0, 12,5, 25, 50, 100 ou 50 000 nM) na competência de oocitos bovinos submetidos ao choque térmico (etapa 2). As doses de 100, 200 e 500 µM de astaxantina reduziram (P< 0,05) a proporção de embriões clivados que atingiu o estágio de blastocisto. A exposição de oocitos bovinos ao choque térmico reduziu (P< 0,05) as taxas de clivagem e blastocistos. No entanto, a adição de astaxantina em oocitos submetidos ao modelo de choque térmico resgatou a clivagem embrionária (P<0,05); doses de 12,5, 25, 50, 100 e 50 000 nM de astaxantina) e o desenvolvimento a blastocisto (P< 0,05; doses de 12,5 e 25 nM de astaxantina) quando comparadas ao grupo controle-veículo 41°C. O experimento 2 visou padronizar a técnica de ressonância paramagnética eletrônica (RPE) em CCOs bovinos. Para tanto, foram realizados vários estudos preliminares a fim de estabelecer o protocolo de RPE em CCOs. No entanto, não foi possível detectar as EROs em CCOs na maioria dos procedimentos conduzidos. Quando CCOs submetidos aos tratamentos Controle (38,5°C por 14 horas) e Choque Térmico (41°C por 14 horas) foram incubados com 100 µM do pró-oxidante menadiona e 50 mM de PBN em meio MIV e PBS 10 mM 1:1 as EROs foram detectadas apenas no grupo choque térmico. Os experimentos 3 e 4 avaliaram os efeitos da astaxantina (0, 12,5 e 25 nM) e da temperatura (38,5 ou 41°C) durante as primeiras 14 horas da MIV na atividades das enzimas antioxidantes (superóxido dismutase - SOD, glutationa peroxidase - GPX e catalase - CAT) e os níveis de peroxidação lipídica no meio MIV. O choque térmico não afetou a atividade das enzimas GPX, SOD e CAT em oócitos ou células do cumulus e a taxa de peroxidação lipídica no meio MIV. Em oocitos submetidos ao choque térmico as doses de 12,5 (P=0,09) e 25 nM (P<0,05) astaxantina estimularam a atividade de GPX quando comparado aos oócitos do grupo controle veiculo 41°C. Em contraste, a dose de 25 nM de astaxantina reduziu (P<0,05) a atividade de GPX em células do cumulus independente de temperatura. A atividade da enzima SOD foi estimulada (P<0,05) em oocitos do grupo controle maturados com 25 nM de astaxantina em relação a 0 nM de astaxantina. A dose de 12,5 nM de astaxantina estimulou (P<0,05) a atividade de SOD em oocitos do grupo choque térmico em relação a mesma dose no grupo controle. O perfil de atividade da SOD em células do cumulus variou com a dose. Enquanto a dose de 12,5 nM de astaxantina inibiu a SOD,a dose de 25 nM estimulou a atividade de SOD no grupo choque térmico. Houve tendência (P=0,09) da dose de 12,5 nM de astaxantina em aumentar a atividade de CAT em oócitos apenas durante o choque térmico. Foi também observada uma tendência da dose de 12,5 nM de astaxantina em aumentar (P= 0,09) a formação do MDA/TBA no grupo estressado termicamente, quando comparado a mesma dose durante a temperatura controle. Em conclusão, o choque térmico afetou a competência oocitária e a astaxantina reverteu esse efeito resgatando a competência oocitária. É possível que este efeito da astaxantina tenha sido mediado pelo aumento na atividade de enzimas GPX, SOD e CAT em oócitos submetidos ao choque térmico. As vias pelas quais este antioxidante exerce efeito termoprotetor ainda não foram completamente compreendidas.Adverse environmental conditions such as temperature and high humidity, limiting the exploitation of animal genetic maximum potential. In cattle, maternal heat stress compromises the fertility of dairy cows, resulting in economic losses to the dairy industry. It is known that the oocyte and embryo in early preimplantation stage are the main targets of the deleterious effects caused by maternal heat stress, however, the cellular mechanisms triggered by elevated temperature and the role of oxidative stress in this context are poorly understood. Recent studies have indicated that exposure of bovine embryos to heat shock increased the concentration of reactive oxygen species (ROS) and the use of embryonic intracellular antioxidants, such as astaxanthin, reversed these effects. With this, the present study aimed to evaluate the effects of heat shock and antioxidant astaxanthin during in vitro maturation (IVM) of bovine oocytes in (1) concentration of ROS, (2) oocyte competence, (3) the activity of antioxidant enzymes and (4) lipid peroxidation in cumulus - oocyte complexes (COCs) cattle. The first experiment evaluated the effects of different concentrations of astaxanthin (0, 50, 100, 200 or 500 mM) in oocyte competence to 38.5°C (step 1) and the effects of different concentrations of astaxanthin (0, 12.5, 25, 50, 100 or 50 000 nM) in the competence of bovine oocytes subjected to heat shock (step 2) . Doses of 100, 200 and 500 mM of astaxanthin decreased (P < 0.05) than the proportion of cleaved has reached the blastocyst stage embryos. Exposure of bovine oocytes to heat shock reduced (P < 0.05) rates of cleavage and blastocyst. However, the addition of astaxanthin in oocytes subjected to heat shock model rescued the embryonic cleavage (P < 0.05; doses of 12.5, 25, 50, 100, and 50 000 nM astaxanthin) and development to blastocyst ( P < 0.05; doses of 12.5 and 25 nM of astaxanthin) compared to the vehicle control group 41°C. Experiment 2 aimed to standardize the technique of electron paramagnetic resonance (EPR ) in bovine oocytes. To this end, several preliminary studies were conducted to establish the protocol RPE in COCs. However, it was not possible to detect ROS in oocytes conducted in most procedures. When subjected to the control oocytes (38.5°C for 14 hours) and hot water (41°C for 14 hours) treatments were incubated with 100 mM of pro-oxidant menadione and 50 mM PBN in IVM medium and 10 mM PBS 1:1 ROS were detected only in group thermal shock. The experiments 3 and 4 assessed the effects of astaxanthin (0, 12.5 and 25 nM) and temperature (38.5 or 41°C) during the first 14 hours of the IVM in the activities of antioxidant enzymes (superoxide dismutase – SOD, glutathione peroxidase - GPX and catalase - CAT) and lipid peroxidation levels in IVM medium. Heat shock did not affect the activity of GPX, SOD and CAT enzymes in oocytes and cumulus cells and the rate of lipid peroxidation in the IVM medium. In oocytes subjected to thermal shock doses of 12.5 (P = 0.09 ) and 25 nM (P <0.05) astaxanthin stimulated GPX activity when compared to the vehicle control group oocytes 41°C. In contrast, a dose of 25 nM astaxanthin decreased (P <0.05) GPx activity in cumulus cells independent of temperature. The activity of SOD was stimulated (P < 0.05) in the control group oocytes matured with 25 nM astaxanthin compared to 0 nM astaxanthin. The dose of 12.5 nM astaxanthin stimulated (P <0.05) the SOD activity in the group of oocytes thermal shock compared to the same dose in the control group. The profile of SOD activity in cumulus cells varied with dose. While the dose of 12.5 nM astaxanthin inhibited the SOD dose of 25 nM stimulated the activity of SOD in thermal shock group. There was a trend (P = 0.09) in a dose of 12.5 nM astaxanthin increase in CAT activity in oocytes only during thermal shock. We also observed a trend of dose of 12.5 nM astaxanthin to increase (P = 0.09) the formation of MDA/TBA in thermally stressed animals compared with the same dose for the temperature control . In conclusion , the thermal shock affect oocyte competence and astaxanthin reversed this effect rescuing the oocyte competence. It is possible that this effect of astaxanthin was mediated by an increase in the activity of GPX, SOD and CAT enzyme in oocytes subjected to thermal shock. The pathways by which this antioxidant exerts termoprotetor effect have not been fully understood.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

MOESM4 of Genome-wide screening of DNA methylation in bovine blastocysts with different kinetics of development

Additional file 4. Hypermethylations in SB

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos.

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8&nbsp;h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells

Role of insulin-like growth factor 1 on cross-bred Bos indicus cattle germinal vesicle oocytes exposed to heat shock

Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100 ng mL(-1)) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5 degrees C (control) or 41 degrees C (heat shockHS) for 14 h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41 degrees C increased (P < 0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25 ng mL(-1), IGF1 tended to minimise these negative effect of HS (P = 0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P < 0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P < 0.05) by 12.5 ng mL(-1) IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.FAPESPCNPqSao Paulo State Univ, UNESP, Inst Biosci, Dept Pharmacol, Rubiao Jr S-N, BR-18618970 Botucatu, SP, Univ Fed Sao Paulo, Dept Biol Sci, 275 Prof Artur Riedel St, BR-09972270 Diadema, SP, BrazilUniv Sao Paulo, Dept Anim Reprod, 87 Prof Dr Orlando Marques de Paiva Av, BR-05508010 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biol Sci, 275 Prof Artur Riedel St, BR-09972270 Diadema, SP, BrazilFAPESP: 2007/53323-0FAPESP: 2010/04728-0CNPq: 478558/2008-2Web of Scienc

Thermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock

The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 degrees C for 22 hours) and heat shock (41 degrees C for 14 hours followed by 38.5 degrees C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 degrees C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration. (C) 2016 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Sao Paulo, Dept Biol Sci, Diadema, SP, BrazilState Univ Sao Paulo, Inst Biosci, Botucatu, SP, BrazilUniv Sao Paulo, Dept Anim Reprod, Sao Paulo, BrazilDepartment of Biological Sciences, Universidade Federal de São Paulo (UNIFESP), Diadema, SP, BrazilFAPESP: 2007/53323-0CNPq: 478558/2008-2CNPq: 501205/2009-8Web of Scienc

Tricarboxylic Acid Cycle Metabolites as Mediators of DNA Methylation Reprogramming in Bovine Preimplantation Embryos

In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells