Background The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. Results We show that SOX17 starts to be expressed in 16–32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. Conclusions This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 μM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion. Electronic supplementary material The online version of this article (10.1186/s12861-019-0193-9) contains supplementary material, which is available to authorized users.
The efficiency of ethanol fermentation, as affected by grain source (maize and decorticated red sorghum), total sugar concentration (13 or 20° Plato) and type of microorganism (Saccharomyces cerevisiae or Zymomonas mobilis) was studied. Maize mashes yielded 0.32 l ethanol kg(-1) ground grain whereas mashes prepared with decorticated red sorghum produced 0.28 l ethanol kg(-1). Both microorganisms yielded similar amounts of ethanol. However, high-gravity mashes (20° Plato) yielded lower amounts of ethanol compared to counterparts adjusted to 13° Plato (0.28 vs. 0.22 l ethanol kg(-1) ground grains). In decorticated sorghum mashes adjusted to 20° P, Z. mobilis produced 40 ml kg(-1) more ethanol compared to S. cerevisiae. In addition, Z. mobilis had a lower dependency on nitrogenous compounds.
Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification.
ObjectivesThe use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated.ResultsProtocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals’ iPS cells.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3627-8) contains supplementary material, which is available to authorized users.
BACKGROUND Bermuda grass is an irrigated forage crop adapted to arid and subtropical zones. This grass can be effectively bioconverted into second‐generation fuel ethanol. The aim was to optimize different acid hydrolyses conditions to maximize the release of C5 and C6 sugars from the forage with minimal production of microbial inhibitors. RESULTS The optimum conditions for H2SO4 were 12.5% solid to liquid, an acid concentration of 1.25%, and 75 min hydrolysis. The efficiencies after dilute acid and enzymatic treatments were 92.5, 73.5 and 89.9% for glucose, xylose and arabinose, respectively and the amounts of inhibitors were 8.2 kg m‐3 of acetic acid, 0.5 kg m‐3 of HMF, and 1.7 kg m‐3 furfural. For the HCl pretreatment, the optimum conditions were 12.5% solid to liquid fraction, 1.25% acid concentration and 37.5 min hydrolysis. These conditions released 56.2% glucose, 86.2% xylose and 48.3% of arabinose and generated 9 kg m‐3 of acetic acid, 0.5 kg m‐3 of HMF, and 2.6 kg m‐3 of furfural. CONCLUSION Dual chemical and enzyme conversions are promising for production of second‐generation ethanol from Bermuda grass. © 2013 Society of Chemical Industry
The pre-conceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of Assisted Reproductive Technologies (ART; ~25%), additives and adjuvants, such as glucocorticoids, are utilized to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study we present a comprehensive single-cell transcriptome, methylome and small RNA atlas in the day 7 human embryo. We demonstrate that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the TE lineage and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sub-lineages, supported by the presence of extravillous trophoblast markers in the polar sub-lineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation (DNA methylation and microRNAs (miRNAs)) likely underlie the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.
Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions and a complex coordination of physical cues, molecular mechanisms and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is comprised of more specified cellular lineages, that will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal regulation of lineage markers and responses to cell signaling perturbations. In this review, recent findings pertaining to the processes of preimplantation development, with an emphasis on specification of the first cellular lineages, in the mouse and human are compared side-by-side. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight and critique the latest advancement in Reproductive research, the development of the 3D stem cell-based models known as ‘blastoids’. This knowledge has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.
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