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 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.

Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells.

Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

Similarities between bovine and human germline development revealed by single-cell RNA sequencing.

The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes and exist transiently during development until they differentiate into oogonia in females, or spermatogonia in males. Little is known about the molecular characteristics of primordial germ cells in cattle. By performing single-cell RNA-sequencing, quantitative real-time PCR, and immunofluorescence analyses of fetal gonads between 40 and 90 days of fetal age, we evaluated the molecular signatures of bovine germ cells at the initial stages of gonadal development. Our results indicate that at 50 days of fetal age, bovine primordial germ cells were in the early stages of development, expressing genes of early primordial germ cells, including transcriptional regulators of human germline specification (e.g. SOX17, TFAP2C, and PRDM1). Bovine and human primordial germ cells also share expression of KIT, EPCAM, ITGA6, and PDPN genes coding for membrane-bound proteins, and an asynchronous pattern of differentiation. Additionally, the expression of members of Notch, Nodal/Activin, and BMP signaling cascades in the bovine fetal ovary, suggests that these pathways are involved in the interaction between germ cells and their niche. Results of this study provide insights into the mechanisms involved in the development of bovine primordial germ cells and put in evidence similarities between the bovine and human germline.

Efficient One-Step Knockout by Electroporation of Ribonucleoproteins Into Zona-Intact Bovine Embryos.

Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.

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.

Livestock Gene Editing by One-step Embryo Manipulation.

The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs.

Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Epigenetics in preimplantation mammalian development.

Fertilization is a very dynamic period of comprehensive chromatin remodeling, from which two specialized cells result in a totipotent zygote. The formation of a totipotent cell requires extensive epigenetic remodeling that, although independent of modifications in the DNA sequence, still entails a profound cell-fate change, supported by transcriptional profile modifications. As a result of finely tuned interactions between numerous mechanisms, the goal of fertilization is to form a full healthy new individual. To avoid the persistence of alterations in epigenetic marks, the epigenetic information contained in each gamete is reset during early embryogenesis. Covalent modification of DNA by methylation, as well as posttranslational modifications of histone proteins and noncoding RNAs, appears to be the main epigenetic mechanisms that control gene expression. These allow different cells in an organism to express different transcription profiles, despite each cell containing the same DNA sequence. In the context of replacement of spermatic protamine with histones from the oocyte, active cell division, and specification of different lineages, active and passive mechanisms of epigenetic remodeling have been revealed as critical for editing the epigenetic profile of the early embryo. Importantly, redundant factors and mechanisms are likely in place, and only a few have been reported as critical for fertilization or embryo survival by the use of knockout models. The aim of this review is to highlight the main mechanisms of epigenetic remodeling that ensue after fertilization in mammals.

Mechanisms of epigenetic remodelling during preimplantation development.

Epigenetics involves mechanisms independent of modifications in the DNA sequence that result in changes in gene expression and are maintained through cell divisions. Because all cells in the organism contain the same genetic blueprint, epigenetics allows for cells to assume different phenotypes and maintain them upon cell replication. As such, during the life cycle, there are moments in which the epigenetic information needs to be reset for the initiation of a new organism. In mammals, the resetting of epigenetic marks occurs at two different moments, which both happen to be during gestation, and include primordial germ cells (PGCs) and early preimplantation embryos. Because epigenetic information is reversible and sensitive to environmental changes, it is probably no coincidence that both these extensive periods of epigenetic remodelling happen in the female reproductive tract, under a finely controlled maternal environment. It is becoming evident that perturbations during the extensive epigenetic remodelling in PGCs and embryos can lead to permanent and inheritable changes to the epigenome that can result in long-term changes to the offspring derived from them, as indicated by the Developmental Origins of Health and Disease (DOHaD) hypothesis and recent demonstration of inter- and trans-generational epigenetic alterations. In this context, an understanding of the mechanisms of epigenetic remodelling during early embryo development is important to assess the potential for gametic epigenetic mutations to contribute to the offspring and for new epimutations to be established during embryo manipulations that could affect a large number of cells in the offspring. It is of particular interest to understand whether and how epigenetic information can be passed on from the gametes to the embryo or offspring, and whether abnormalities in this process could lead to transgenerationally inheritable phenotypes. The aim of this review is to highlight recent progress made in understanding the nature and mechanisms of epigenetic remodelling that ensue after fertilisation.

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.

Human-induced pluripotent stem cells produced under xeno-free conditions.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. While cell differentiation protocols have been successfully developed, and animal models of human disease have proved that these cells have the potential to treat human diseases and conditions produced as a consequence of aging, degeneration, injury, and birth defects, logistical issues still remain unsolved and hamper the possibility of testing these cells in human clinical trials. Among them is the widely spread use of animal products for the generation and culture of iPSCs. We report here a xeno-free iPSC generation system that addresses all the steps of iPSCs production including the isolation and culture of adult skin fibroblasts, and iPSCs generation, expansion, and maintenance. iPSCs generated with a polycistronic lentiviral vector under xeno-free conditions displayed markers of pluripotency and gave rise to embryoid bodies (EBs) displaying indicators of the 3 primary germ layers. Xeno-free iPSCs injected into nude mice produced classic teratomas, and teratoma explants cultured under conditions favoring fibroblastic cells gave rise to cells morphologically indistinguishable from input cells. Protocols here described will facilitate the implementation of new cellular therapies for preclinical and clinical studies, potentially reducing the regulatory burden without compromising the differentiation potential of the cells.

Full developmental potential of mammalian preimplantation embryos is maintained after imaging using a spinning-disk confocal microscope.

Fluorescent live imaging of cells and embryos at subcellular resolution poses significant challenges for biologists due to morbidity and mortality ensuing from phototoxicity. Here we report the use of a spinning-disk confocal microscope to image mouse and bovine preimplantation embryos without impairing their developmental potential. We also present data indicating that this imaging technique does not affect the functionality of subcellular components as assessed by reactive oxygen species (ROS) production, caspase activity, and DNA integrity. Spinning-disk confocal microscopy was also useful in determining cell number and allocation in transgenic bovine blastocysts. We conclude that this imaging method is suitable for monitoring preimplantation embryos.

Estradiol benzoate given 0 or 24 h after the end of a progestagen treatment in postpartum suckled beef cows.

The objective of Experiment 1 was to compare the effects of estradiol benzoate (EB) given 0 or 24h after the end of a progestagen treatment on ovulation and CL formation in anestrous cows. Twenty cows were treated with an intravaginal sponge containing 250 mg of medroxiprogesterone acetate (MPA). At sponge insertion, each cow received 3 mg EB and 10 mg MPA im. At device removal, cows received 0.7 mg EB either at that time (EB0) or 24h later (EB24). Ultrasound examinations and blood sampling to determine plasma progesterone concentrations were performed to detect ovulation and CL formation. Ovulation occurred in 77.8 and 81.8% cows in the EB0 and EB24 groups, respectively. Diameter of the ovulatory follicle (EB0 = 10.9 +/- 0.5mm; EB24 = 12.1 +/- 0.8 mm; P = 0.26) and the interval from sponge removal to ovulation (median = 3 days; P = 0.64) did not differ between treatments. Among the cows that ovulated (n = 16), short-lived CL were present in 2/7 and 2/9 cows in the EB0 and EB24 groups, respectively. Plasma progesterone concentrations and CL area did not differ between treatments (P > 0.05). In Experiment 2, cows were treated with the same protocol as in Experiment 1, but at sponge withdrawal all cows received 250 microg cloprostenol and timed artificial insemination (TAI) was performed 48 h after sponge removal. In Replicate 1 (n = 204 multiparous cows), pregnancy rates were 45.0 and 47.5% for EB0 and EB24, respectively (P > 0.05). In Replicate 2 (n = 69 primiparous cows) pregnancy rate did not differ between EB0 and EB24 (51.4% versus 52.9%). In conclusion, EB given 0 or 24h after the end of a progestagen treatment had the same effect on ovulation rate, time to ovulation, diameter of the ovulatory follicle, incidence of short-lived CL, luteal tissue area, and plasma progesterone concentrations of normal lifespan CL, and pregnancy rate after TAI in suckled beef cows.