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In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.

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The oviduct provides a suitable microenvironment from the gametes' final maturation until initial embryo development. Dynamic functional changes are observed in the oviduct cells, mainly controlled by steroid hormones and well-orchestrated during the estrous cycle. However, based on the roles played by the oviduct, additional layers of complexity might be present in its regulatory process. There is a cellular process that includes metabolic adaptation that can guide molecular modifications. This process is known as metaboloepigenetics. Therefore, we aimed to better understand how this crosstalk occurs in oviductal epithelial cells (OEC). Due to limited in situ access to the oviduct, we used the primary in vitro cell culture as a culture model and glucose as a metabolic disturbed factor. For that, cells derived from the oviductal epithelial layer were collected from cows at either follicular or luteal stages (n = 4 animals per group). They were cultured on a monolayer culture system under normoglycemic (2.7 mM glucose) or hyperglycemic conditions (27 mM glucose). On day five of culture, attached cells were submitted to analysis of mitochondrial metabolism (mitochondrial membrane potential - MMP) and epigenetics markers (5- methylcytosine - 5 mC and histone 3 lysine 9 acetylation - H3K9ac). Moreover, the culture media were submitted to the metabolites analysis profile by Raman spectrometry. Data were analyzed considering the effect of glucose level (normoglycemic vs. hyperglycemic), stages when OEC were harvested (follicular vs. luteal), and their interaction (glucose level * cycle stage) by two-way ANOVA. As a result, the high glucose level decreased the H3K9ac and MMP levels but did not affect the 5 mC. Regardless of the metabolic profile of the culture media, the glucose level was the only factor that changed the Raman shifts abundance. Although this present study evaluated oviductal epithelial cells after being submitted to an in vitro monolayer culture system, which is known to lead to cell dedifferentiation, yet, these results provide evidence of a relationship between epigenetic reprogramming and energy metabolism under these cell culture conditions. In conclusion, the levels of metabolites in culture media may be crucial for cellular function and differentiation, meaning that it should be considered in studies culturing oviductal cells.

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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 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.

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Lipids play a crucial role in various biological functions, including membrane composition, energy storage, cell signalling, and metabolic and epigenetic processes. Abnormal lipid accumulation and metabolism during in vitro maturation (IVM) of oocytes have been linked to the use of fetal bovine serum (FBS), even though it provides several beneficial molecules, contributing to the oocyte competence. Delipidating agents have been used to mitigate these deleterious effects, but they can have adverse effects on embryonic development. In this study, we explored how lipids present in fetal bovine serum (FBS) can impact the composition of oocytes and their resulting blastocysts in vitro. For that, we used organic solvents to separate the polar and nonpolar (lipid enriched) phase of FBS. Oocytes were in vitro matured in the presence of 10% whole FBS (control), 10% FBS plus 10% nonpolar lipids (lipid enriched - OL) or 10% polar lipids only (partially delipidated - ODL). After 24 h, part of the matured oocytes was collected and those remaining in each group underwent in vitro fertilization (IVF) and culture (IVC) under the same conditions and expanded blastocysts were collected at day 7 (control, BL and BDL). Oocytes and embryos were analysed by Multiple Reaction Monitoring mass spectrometry (MRM-MS) to determine their lipid composition. Interestingly, principal component analysis (PCA) revealed a clear distinction in the lipid profile of oocytes and blastocysts from both treatments compared to the control group. Control oocytes and blastocysts had higher triacylglycerol and cholesterol ester enrichment while the OL, ODL, BL and BDL groups had higher amounts of free fatty acids (FFAs). The structural and signalling phospholipids also differed among groups. Our findings suggest that the lipid-enriched fraction of FBS can be manipulated for IVM to ensure proper maturation, resulting in oocytes and blastocysts with less accumulated intracellular lipids and an improved metabolic status.

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Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.

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In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.

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Supplementing embryonic culture medium with fetal bovine serum (FBS) renders this medium undefined. Glucose and growth factors present in FBS may affect the results of cell differentiation studies. This study tested the hypothesis that FBS supplementation during in vitro culture (IVC) alters cell differentiation in early bovine embryo development. Bovine embryos were produced in vitro and randomly distributed into three experimental groups at 90 h post insemination (90 hpi): the KSOM-FBS group, which consisted of a 5% (v/v) FBS supplementation; the KSOM33 group, with the renewal of 33% of medium volume; and the KSOM-Zero group, without FBS supplementation nor renewal of the culture medium. The results showed that the blastocyst rate (blastocyst/oocytes) at 210 hpi in the KSOM-FBS group was higher than in the KSOM-Zero group but not different from the KSOM33 group. There were no significant changes in metabolism-related aspects, such as fluorescence intensities of CellROX Green and MitoTracker Red or reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+). Immunofluorescence analysis of CDX2 revealed that the lack of FBS or medium supplementation reduced the number of trophectoderm (TE) cells and total cells. Immunofluorescence analysis revealed a reduction of SOX17-positive cell numbers after FBS supplementation compared with the KSOM33 group. Therefore, we concluded that FBS absence reduced blastocyst rates; however, no reduction occurred when there was a 33% volume renewal of the medium at 90 hpi. We also concluded that FBS supplementation altered TE and primitive endoderm cell allocation during early bovine embryo development.

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Metaboloepigenetic regulation has been reported in stem cells, germ cells, and tumor cells. Embryonic metaboloepigenetics, however, have just begun to be described. Here we analyzed RNAseq data to characterize the metaboloepigenetic profiles of human, mouse, and bovine pre-implantation embryos. In embryos, metaboloepigenetic reprogramming was species-specific, varied with the developmental stage and was disrupted with in vitro culture. Metabolic pathways and gene expressions were strongly correlated with early embryo DNA methylation and were changed with in vitro culture. Although the idea that the in vitro environment may influence development is not new, there has been little progress on improving pregnancy rates after decades using in vitro fertilization. Hence, the present data will contribute to understanding how the in vitro manipulation affects the metaboloepigenetic status of early embryos, which can be used to establish culture strategies aimed at improving the in vitro environment and, consequently, pregnancy rates and offspring health.

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Metabolism and epigenetics, which reciprocally regulate each other in different cell types, are fundamental aspects of cellular adaptation to the environment. Evidence in cancer and stem cells has shown that the metabolic status modifies the epigenome while epigenetic mechanisms regulate the expression of genes involved in metabolic processes, thereby altering the metabolome. This crosstalk occurs as many metabolites serve as substrates or cofactors of chromatin-modifying enzymes. If we consider the intense metabolic dynamic and the epigenetic remodelling of the embryo, the comprehension of these regulatory networks will be important not only for understanding early embryonic development, but also to determine in vitro culture conditions that support embryo development and may insert positive regulatory marks that may persist until adult life. In this review, we focus on how metabolism may affect epigenetic reprogramming of the early stages of development, in particular acetylation and methylation of histone and DNA. We also present other metabolic modifications in bovine embryos, such as lactylation, highlighting the promising epigenetic and metabolic targets to improve conditions for in vitro embryo development.

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The kinetics of the first cleavages is a predictor of blastocyst development and implantation. For bovine embryos, this attribute was previously related to distinct metabolic, molecular and epigenetic profiles, including DNA and histone modifications. In the present work, we described the dynamics of trimethylation of lysine 27 on histone H3 (H3K27me3) in fast and slow developing embryos and verified if this epigenetic mark was also influenced by the speed of the first cleavages. In vitro-produced bovine embryos were classified as fast (4 or more cells) or slow (2 cells) at 40 hr post fertilization (hpf) and either collected or cultured until 96 hpf or 186 hpf. Immunofluorescence analysis was performed in these three time points and showed that although both groups presented the same levels of H3K27me3 at 40 hpf, slow embryos presented a pronounced increase in this mark at 186 hpf when compared to fast embryos, resulting in blastocysts with remarkable differences in H3K27me3 levels. In conclusion, the increased levels of this repressive histone post-translation modification (PTM) might be an attempt of slow embryos to promote gene expression control and chromatin integrity, since it was already reported that these embryos present reduced levels of other epigenetic repressive marks as DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3).

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Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mechanisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 × 106 cells/mL) were incubated for 1 h at 38.5 °C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p ˂ 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to influence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to increase SMGT protocols, without altering the sperm viability.

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Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

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The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

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Understanding preimplantation embryonic development is crucial for the improvement of assisted reproductive technologies and animal production. To achieve this goal, it is important to consider that gametes and embryos are highly susceptible to environmental changes. Beyond the metabolic adaptation, the dynamic status imposed during follicular growth and early embryogenesis may create marks that will guide the molecular regulation during prenatal development, and consequently impact the offspring phenotype. In this context, metaboloepigenetics has gained attention, as it investigates the crosstalk between metabolism and molecular control, i.e., how substrates generated by metabolic pathways may also act as players of epigenetic modifications. In this review, we present the main metabolic and epigenetic events of pre-implantation development, and how these systems connect to open possibilities for targeted manipulation of reproductive technologies and animal production systems.

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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.

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Previous studies have discussed the importance of an optimal range of metabolic activity during preimplantation development. To avoid factors than can trigger an undesirable trajectory, it is important to learn how nutrients and metabolites interact to help launching the correct developmental program of the embryo, and how much the in vitro culture system can impair this process. Here, using the bovine model, we describe a factorial experimental design used to investigate the biochemical and molecular signature of embryos in response to different combinations of morphological features-i.e. speed of development-and external stimuli during in vitro culture-i.e. different oxygen tensions and glucose supplementation. Our analyses demonstrate that the embryos present heterogeneous metabolic responses depending on early morphological phenotypes and the composition of their surroundings. However, despite the contribution of each single stimulus for the embryo phenotype, oxygen tension is determinant for such differences. The lower oxygen environment boosts the metabolism of embryos with faster kinetics, in particular those cultured in lower glucose concentrations.

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The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.

Assuntos

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Understanding preimplantation embryonic development is crucial for the improvement of assisted reproductive technologies and animal production. To achieve this goal, it is important to consider that gametes and embryos are highly susceptible to environmental changes. Beyond the metabolic adaptation, the dynamic status imposed during follicular growth and early embryogenesis may create marks that will guide the molecular regulation during prenatal development, and consequently impact the offspring phenotype. In this context, metaboloepigenetics has gained attention, as it investigates the crosstalk between metabolism and molecular control, i.e., how substrates generated by metabolic pathways may also act as players of epigenetic modifications. In this review, we present the main metabolic and epigenetic events of pre-implantation development, and how these systems connect to open possibilities for targeted manipulation of reproductive technologies and animal production systems.(AU)

Assuntos

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Understanding preimplantation embryonic development is crucial for the improvement of assisted reproductive technologies and animal production. To achieve this goal, it is important to consider that gametes and embryos are highly susceptible to environmental changes. Beyond the metabolic adaptation, the dynamic status imposed during follicular growth and early embryogenesis may create marks that will guide the molecular regulation during prenatal development, and consequently impact the offspring phenotype. In this context, metaboloepigenetics has gained attention, as it investigates the crosstalk between metabolism and molecular control, i.e., how substrates generated by metabolic pathways may also act as players of epigenetic modifications. In this review, we present the main metabolic and epigenetic events of pre-implantation development, and how these systems connect to open possibilities for targeted manipulation of reproductive technologies and animal production systems.

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Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8-16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

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