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

Influência da adição de fator de crescimento fibroblástico 10 (FGF10), durante a maturação oocitária, na produção in vitro de embriões bovinos

The Brazilian livestock stands out for having the world largest commercial herd of cattle and leads meat exportation and production of bovine embryos. The in vitro production (IVP) of embryos is considered an effective option to overcome problems such as infertility in cows with high economic value and also for genetic improvement of cattle. The in vitro oocyte maturation is an essential step to the success of IVP, but is still considered poor when compared to in vivo maturation. Recent studies have suggeested an important role of Fibroblast Growth Factor 10 (FGF10) on the in vitro maturation of oocytes, which favored the expression of genes related to oocyte maturation and cumulus cell expansion. Aware that maturity stage influences the final production of blastocysts, we aimed study to verify if the addition of FGF10 into the maturation medium is able to affect positively the IVP of bovine embryos. Hence, FGF10 was added to maturation in five different concentrations: 0.5 ng/mL (group 0.5), 2.5 ng/mL (group 2.5), 5 ng/mL (group 5), 10 ng/mL (group 10) and 50 ng/mL (group 50). Additionally, two other maturation groups were used, group BSA (Bovine Serum Albumin, 4 mg/mL) and group FCS (Fetal Calf Serum, 10%). The rates of cleavage, morula and blastocyst were analyzed by Analysis of Variance (ANOVA), differences of P<0.05 were considered significant. Cleavage rates did not differ between the seven groups. On the other hand, morula rate on FCS group was higher than groups BSA, 0.5, 10 and 50 (P<0.05), but did not differ among groups treated with intermediate doses of FGF10 (2.5 and 5). FCS group presented higher blastocyst rate compared to all other groups that were well below the FCS group (P<0.0001). Therefore, the use of FGF10 during oocyte maturation did not affect positively embryo development on the IVP of bovine embryosA pecuária brasileira destaca-se mundialmente por possuir o maior rebanho comercial de gado e liderar a exportação de carne e a produção de embriões bovinos. A técnica de produção in vitro (PIV) de embriões é considerada boa alternativa para superar problemas como infertilidade de vacas com alto valor econômico e para melhoramento genético de rebanhos. A maturação oocitária in vitro é etapa determinante para o sucesso da PIV, porém insatisfatória quando comparada à maturação in vivo. Estudos recentes demonstraram a participação do Fator de Crescimento Fibroblástico 10 (FGF10) durante a maturação in vitro de oócitos bovinos, onde favoreceu a expressão de genes relacionados a maturação oocitária e expansão das células do cumulus. Cientes de que a fase de maturação influencia a produção final de blastocistos, objetivamos com o presente estudo analisar se a adição do FGF10 no meio de maturação é capaz de interferir positivamente na PIV de embriões bovinos. Para tal, o FGF10 foi adicionado à maturação em 5 diferentes concentrações: 0,5 ng/mL (grupo 0,5), 2,5 ng/mL (grupo 2,5), 5 ng/mL (grupo 5), 10 ng/mL (grupo 10) e 50 ng/mL (grupo 50). Adicionalmente, dois outros grupos de maturação foram utilizados, grupo BSA (Albunina Sérica Bovina, 4 mg/mL) e o grupo SFB (Soro Fetal Bovino, 10%). As taxas de clivagem, mórula e blastocisto foram analisadas por Análise de Variância (ANOVA), sendo consideradas significantes as diferenças com P<0,05. A taxa de clivagem não diferiu entre os sete grupos experimentais. Por outro lado, a taxa de mórula do grupo SFB foi superior quando comparado aos grupos BSA, 0,5, 10 e 50 (P<0,05), porém não diferiu dos grupos tratados com dose intermediária de FGF10 (2,5 e 5). Efeito superior do grupo SFB também foi observado na taxa de blastocisto, visto que a taxa de produção de blastocisto dos demais seis grupos foram muito aquém ..

Perfil gênico no oviduto bovino de fêmeas Nelore e Aberdeen angus

The oviduct has an important role in mammal reproduction, promoting a favorable microenvironment for oocyte maturation, sperm storage and capacitation, fertilization, transport of gametes and early embryo development. Anatomically and functionally, the oviduct is divided in three regions: infundibulum, ampulla and isthmus. The oocyte and the sperm enter in opposite sides of the oviduct, respectively infundibulum and isthmus, and are transported to the fertilization site, the ampulla. Reproductive success is directly related to appropriate timing of gamete transport to the fertilization site, as well as a precise time of embryo transport to the uterus, to obtain the capacity of implantation. The coordination and regulation of oviductal functions are complex and under endocrine, paracrine and autocrine effects, which temporally and spatially alter the transcription and translation of several factors. Therefore, this study aimed to evaluate the effect of reproductive biotechnologies, specifically ovarian superstimulation, as well as genetic and physiological reproductive characteristics in the transcriptional profile of several factors in the bovine oviduct. To do so, we evaluated the effects of inducing multiple ovulation in Nelore cows (data presented in the first manuscript), and the effects of the influence of genetic selection of animals with high follicle count in Nellore and Aberdeen Angus heifers, in the initial period post-ovulation (data presented in the second manuscript), in gene expression related to gametes transport and fertilization. The results demonstrated that ovarian superstimulation modulates the expression of some genes related to oviductal contractility in Nelore cows and ovulation is the main factor responsible for transcriptional control in bovine oviduct, with less or no impact of breed and ovarian follicle countO oviduto possui papel essencial na reprodução de mamíferos, promovendo um microambiente favorável para a maturação oocitária, estocagem e capacitação do espermatozoide, fertilização, transporte dos gametas e desenvolvimento inicial do embrião. Anatomicamente e funcionalmente, o oviduto é dividido em três regiões principais: infundíbulo, ampola e istmo. O oócito e o espermatozoide entram nos lados opostos do oviduto, respectivamente no infundíbulo e istmo, e são transportados até a ampola, local onde ocorre a fertilização. O sucesso reprodutivo está diretamente ligado a temporização apropriada do transporte dos gametas ao local da fertilização, bem como, a precisão no tempo de transporte do embrião até o útero, para a aquisição da capacidade de implantação. A coordenação e regulação das funções do oviduto são complexas e estão sob efeitos endócrinos, parácrinos e autócrinos, os quais alteram temporalmente e espacialmente a transcrição e tradução de diversos fatores. Diante disso, o presente trabalho visou avaliar o efeito de biotecnologias reprodutivas, especificamente da superestimulação ovariana, bem como de características genéticas e fisiológicas reprodutivas no perfil transcricional de diversos fatores no oviduto bovino. Para tanto, foram avaliados os efeitos da indução de múltiplas ovulações em vacas da raça Nelore (dados apresentados no primeiro manuscrito), bem como os efeitos da influência da seleção genética de animais com alta contagem folicular em novilhas da raça Nelore e Aberdeen Angus, no período inicial pós ovulação (dados apresentados no segundo manuscrito), na expressão de genes relacionados ao transporte de gametas e fertilização. Os resultados demonstram que a superestimulação ovariana modula a expressão de alguns genes relacionados à contratilidade do oviduto em vacas da raça Nelore e que a ovulação é principal fator responsável por controlar ...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Can the antral follicular count modulate the gene expression of bovine oviducts in Aberdeen Angus and Nelore heifers?

The number of visible ovarian antral follicles (antral follicle count-AFC) is repeatable in bovine individuals, but highly variable between animals, and with differences between Bos taurus and Bos indicus breeds. Several studies have tried to determine the correlation between AFC and increased fertility in cattle. While the impacts of AFC on embryo production, hormonal levels, and pregnancy rates have been described, the molecular effects of AFC on bovine oviducts have not yet been investigated. Here, the aim was to investigate the impact of breeds, such as Aberdeen Angus and Nelore heifer with high or low AFC, on abundance of transcripts and protein related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction in the oviducts. In summary, the ovulation side was the major factor that affected transcript abundance on bovine oviducts. However, a discreet effect among AFC and cattle breeds was also observed. Based on this, we concluded and reinforced here that differential microenvironments between ipsilateral and contralateral oviducts have a major effect on modulating the transcripts related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction. However, we cannot exclude that there is minimal effect of AFC or breed on regulation of some genes (such as AGTR1, ACE1, FUCA1, and VEGFA) in bovine oviducts

Modulation of long-chain Acyl-CoA synthetase on the development, lipid deposit and cryosurvival of in vitro produced bovine embryos.

In this study, we evaluated the modulation effect of long-chain Acyl-CoA synthetase during early embryo development. Bovine embryos were cultured in four groups: positive modulation (ACS+) with GW3965 hydrochloride, negative modulation (ACS-) with Triacsin C, association of both modulators (ACS±), and control. Embryo development rates were not altered (P>0.05) by treatments. Embryonic cytoplasmic lipid content increased in ACS+ but reduced in ACS- compared to the control (P 0.05) between groups; however, an increased apoptotic cells percentage was found in ACS- compared to control. Twenty-four hours after warming, ACS+ and control grade I embryos presented the best hatching rates, whereas the ACS+ group equaled the hatching rates between their embryos of grades I, II and III 48 hours after warming. The relative abundance of transcripts for genes associated with lipid metabolism (ACSL3, ACSL6, ACAT1, SCD, and AUH), heatshock (HSP90AA1 and HSF1), oxidative stress (GPX4), and angiogenesis (VEGF), among other important genes for embryo development were affected by at least one of the treatments. The treatments were effective in modulating the level of transcripts for ACSL3 and the cytoplasmic lipid content. The ACS- was not effective in increasing embryonic cryosurvival, whereas ACS+ restored survival rates after vitrification of embryos with low quality, making them equivalent to embryos of excellent quality

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