Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Unraveling the Consequences of Oxygen Imbalance on Early Embryo Development: Exploring Mitigation Strategies.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.

Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Insulin-like growth factor-1 (IGF-1) selectively modulates the metabolic and lipid profile of bovine embryos according to their kinetics of development.

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.

Species-specific molecular differentiation of embryonic inner cell mass and trophectoderm: A systematic review.

A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal model, 22.2 % (8/36) pigs, 16.7 % (6/36) cattle, 5.5 % (2/36) humans, and 2.8 % (1/36) goats as well as 2.8 % (1/36) equine. Our results demonstrated that each of the first cell lineages of embryos requires a certain pattern of expression to establish the cellular determination of fate. Moreover, these patterns are shared by many species, particularly for those molecules that have already been identified in the literature as biomarkers. In conclusion, the present study integrated carefully chosen studies regarding embryonic development and first cellular decisions in mammalian species and summarized the information about the differential characterization of the first cell lineages and their possible relationship with specific gene expression.

Lipid characterization of in vitro-produced bovine embryos with distinct kinetics of development.

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.

Follicular environment as a predictive tool for embryo development and kinetics in cattle.

Follicular fluid composition and the transcription pattern of granulosa cells were analysed to better comprehend associations between embryo development and morphokinetics. Bovine follicles were punctured and their respective follicular fluid and granulosa cells were collected. Cumulus-oocyte complexes derived from these follicles were matured and fertilised invitro. Embryo morphology and kinetics were evaluated at 40h after insemination, when embryos were classified as fast (FCL, four or more cells), slow (SCL, 2-3 cells) or non-cleaved (NCL). Their development was followed until the blastocyst stage. Glucose, pyruvate, cholesterol and oestradiol were quantified in the follicular fluid and the transcription pattern of 96 target genes was evaluated in granulosa cells by large-scale quantitative reverse transcription polymerase chain reaction. Follicular fluid from the blastocyst group had increased levels of glucose, total cholesterol and pyruvate compared to the non-blastocyst group, whereas higher levels of oestradiol were observed in the follicular fluid of embryos and blastocysts with fast cleavage. The transcriptional pattern revealed altered metabolic pathways between groups, such as lipid metabolism, cellular stress and cell signalling. In conclusion, both follicular fluid and granulosa cells are associated with the possibility of identifying follicles that may generate embryos with high potential to properly develop to the blastocyst stage.

Influence of follicle size on bovine oocyte lipid composition, follicular metabolic and stress markers, embryo development and blastocyst lipid content.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

Corrigendum to "Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro Produced Embryos".

[This corrects the article DOI: 10.1155/2017/1502489.].

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

BACKGROUND: The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed. RESULTS: Genome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation. CONCLUSION: The kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.

Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro Produced Embryos.

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.

Effective individual culture system for in vitro production of bovine embryos / Sistema eficaz de cultivo individual de embriões bovinos produzidos in vitro

A new and effective protocol to culture bovine embryos without coculture and with individualized culture media has been established, which would allow the study of a single embryo's metabolism. For this purpose, bovine embryos were produced in vitro by standard protocols in three different types of media: KSOM, SOFaa, and KSOM followed by SOFaa at day 2. Presumptive zygotes were divided into six groups: control, cultured in groups (C-KSOM, C-SOFaa, and C-KS), and individual well system (W-KSOM, W-SOFaa, and W-KS). Cleavage and blastocyst rates were assessed on days 2 and 7 respectively. Relative quantification of transcripts related to important metabolic processes (GLUT1, GLUT3, GSK3, SOD1, HSPD1, G6PD) were assessed in C-KS and W-KS blastocysts. Results show that cleavage was significantly higher only in W-KSOM when compared to C-KSOM, while blastocyst rates differ only between C-SOF and W-SOF. All the other comparisons did not present statistical difference. Moreover, gene expression analysis revealed that blastocysts cultured in groups and in the individual well system present similar transcription patterns. Thus, the obtained conclusion was that the individual well system performed could be used as an effective alternative protocol for individual culture of bovine embryos, since the rates are similar to routine group culture.(AU)

Estabeleceu-se um protocolo novo e eficaz de cultivo individual de embriões bovinos sem o uso de cocultivo e sem compartilhamento de meio visando à análise do metabolismo individual do embrião. Para isso, embriões foram produzidos in vitro por protocolos convencionais em três diferentes tipos de meio: KSOM, SOFaa e KSOM seguido por SOFaa no dia 2. Os zigotos presumíveis foram divididos em seis grupos: controles (cultivo em grupo ­ C-KSOM, C-SOFaa e C-KS) e sistema de poços individuais (W-KSOM, W-SOFaa e W-KS). As taxas de clivagem foram avaliadas nos dias 2 e 7, respectivamente. Além disso, a quantificação relativa de transcritos relacionados a importantes processos metabólicos (GLUT1, GLUT3, GSK3, SOD1, HSPD1 e G6PD) foi avaliada nos blastocistos dos grupos C-KS e W-KS. Os resultados mostram que as taxas de clivagem foram maiores apenas no grupo W-KSOM quando comparado ao grupo C-KSOM, enquanto a taxa de blastocistos diferiu apenas entre os grupos C e W-SOF. Além disso, a análise da expressão gênica mostrou que blastocistos cultivados em grupo ou em sistema de poços individuais são semelhantes quanto à expressão gênica. Assim, a conclusão obtida foi que o sistema individual proposto pode ser utilizado como um protocolo alternativo eficiente para o cultivo individual de embriões de bovino, uma vez que suas características permanecem semelhantes àquelas do sistema convencional de produção de embriões.(AU)

Raman-based noninvasive metabolic profile evaluation of in vitro bovine embryos.

The timing of the first embryonic cell divisions may predict the ability of an embryo to establish pregnancy. Similarly, metabolic profiles may be markers of embryonic viability. However, in bovine, data about the metabolomics profile of these embryos are still not available. In the present work, we describe Raman-based metabolomic profiles of culture media of bovine embryos with different developmental kinetics (fast x slow) throughout the in vitro culture. The principal component analysis enabled us to classify embryos with different developmental kinetics since they presented specific spectroscopic profiles for each evaluated time point. We noticed that bands at 1076 cm(−1) (lipids), 1300 cm(−1) (Amide III), and 2719 cm(−1) (DNA nitrogen bases) gave the most relevant spectral features, enabling the separation between fast and slow groups. Bands at 1001 cm(−1) (phenylalanine) and 2892 cm(−1) (methylene group of the polymethylene chain) presented specific patterns related to embryonic stage and can be considered as biomarkers of embryonic development by Raman spectroscopy. The culture media analysis by Raman spectroscopy proved to be a simple and sensitive technique that can be applied with high efficiency to characterize the profiles of in vitro produced bovine embryos with different development kinetics and different stages of development.

Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc.

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups.

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2-methylacrylic acid 2-[(2-(dimethylamino)ethyl)methylamino]ethyl ester44} (POEGMA70-b-P(OEGMA10-co-DAMA44). Block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (R(H) ≈ 65-85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment.

Photobiological effect of low-level laser irradiation in bovine embryo production system.

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism.