Bovine Preimplantation Embryos Research Articles

Transcript profiling of bovine embryos implicates specific transcription factors in the maternal-to-embryo transition.

Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Hyaluronic acid and epidermal growth factor improved the bovine embryo quality by regulating the DNA methylation and expression patterns of the focal adhesion pathway.

Focal adhesion pathway is one of the key molecular pathways affected by suboptimal culture conditions during embryonic development. The epidermal growth factor (EGF) and hyaluronic acid (HA) are believed to be involved in the focal adhesion pathway function by regulating the adherence of the molecules to the extracellular matrix. However, regulatory and molecular mechanisms through which the EGF and HA could influence the embryo development is not clear. Therefore, this study aimed to investigate the effect of continued or stage specific supplementation of EGF and/or HA on the developmental competence and quality of bovine preimplantation embryos and the subsequent consequences on the expression and DNA methylation patterns of genes involved in the focal adhesion pathway. The results revealed that, the supplementation of EGF or HA from zygote to the blastocysts stage reduced the level of reactive oxygen species and increased hatching rate after thawing. On the other hand, HA decreased the apoptotic nuclei and increased blastocyst compared to EGF supplemented group. Gene expression and DNA methylation analysis in the resulting blastocysts indicated that, combined supplementation of EGF and HA increased the expression of genes involved in focal adhesion pathway while supplementation of EGF, HA or a combination of EGF and HA during the entire preimplantation period changed the DNA methylation patterns of genes involved in focal adhesion pathway. On the other hand, blastocysts developed in culture media supplemented with EGF + HA until the 16-cell stage exhibited higher expression level of genes involved in focal adhesion pathway compared to those supplemented after the 16-cell stage. Conversely, the DNA methylation level of candidate genes was increased in the blastocysts obtained from embryos cultured in media supplemented with EGF + HA after 16-cell stage. In conclusion, supplementation of bovine embryos with EGF and/or HA during the entire preimplantation period or in a stage specific manner altered the DNA methylation and expression patterns of candidate genes involved in the focal adhesion pathway which was in turn associated with the observed embryonic developmental competence and quality.

Aflatoxin compromises development of the preimplantation bovine embryo through mechanisms independent of reactive oxygen production

Aflatoxin is a potent carcinogen often found in animal feedstuffs. Although it reportedly impairs development of the preimplantation pig embryo, it is not known whether it adversely affects development of the preimplantation bovine embryo. We conducted 3 experiments to investigate this possibility and determine whether deleterious effects of aflatoxin were caused by increased production of reactive oxygen species (ROS). Experiments were conducted with embryos produced in vitro and cultured after fertilization with various concentrations of aflatoxin. For experiment 1, embryos were treated with 0 (control), 40, 400, or 4,000 µg/L of aflatoxin B1 (AFB1). Treatment at all concentrations of AFB1 tended to reduce cleavage rate, with the 2 highest concentrations having significant effects. As compared with the control, 40 µg/L AFB1 reduced the percentage of oocytes becoming blastocysts and the percentage of cleaved embryos becoming blastocysts (19.7 vs. 8.1% and 30.3 vs. 14.3%, respectively). Complete inhibition of blastocyst formation occurred at concentrations of 400 and 4,000 µg/L of AFB1. Experiments 2 and 3 involved a 2 × 2 factorial design with effects of AFB1 (0 and 40 µg/L), the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble analog of vitamin E; 0 and 5 µM), and their interaction on production of ROS in putative zygotes (experiment 2) and development to the blastocyst stage (experiment 3). Production of ROS was increased by AFB1, and this effect was reversed by Trolox. However, Trolox did not prevent the reduction in development to the blastocyst stage caused by AFB1. Thus, the anti-developmental effects of AFB1 are not caused solely by increased ROS production. Rather, other underlying mechanisms exist for the adverse effects of aflatoxin on embryonic development. Overall, results indicate the potential for feeding aflatoxin-contaminated feed to cause embryonic loss in cattle.

Prostaglandin F2\u03b1 (PGF2\u03b1) production possibility and its receptors expression in the early- and late-cleaved preimplantation bovine embryos

BackgroundProstaglandin F2α (PGF2α) is an important component for the physiology of female reproductive processes. In the literature, the data pertaining to the synthesis and action of PGF2α in early embryonic bovine development are limited. In our study, we used the bovine in vitro culture model based on the time of first cleavage to determine the mRNA expression and immunolocalization of PGF2α synthase and its receptor in bovine embryos from the 2-cell stage to the hatched blastocyst stage. We also evaluated PGF2α production at 2, 5 and 7 days of in vitro culture.ResultsWe found a significantly higher proportion of blastocysts obtained from the early-cleaved embryos than from the late-cleaved embryos (37.7% vs. 26.1% respectively, P < 0.05). The PGFS mRNA expression was significantly higher in the late-cleaved group than in the early-cleaved group at the 2-, 4- and 16-cell stages (P < 0.05). For PTGFR, we observed that within the late-cleaved group, the mRNA abundance was significantly higher in embryos at the 2- and 16-cell stages than in embryos at the 4- and 8-cell stages (P < 0.05). We observed that PTGFR mRNA expression was significantly higher in the 2- and 16-cell embryos in the late-cleaved group than that in the early-cleaved group embryos (P < 0.05). Among the blastocysts, the PGFS and PTGFR expression levels showed a trend towards higher mRNA expression in the late-cleaved group than in the early-cleaved group. Analysis of PGF2α production showed that within the early-cleaved group, the content of PGF2α in the in vitro culture medium was significantly higher on day 7 than it was on day 2 (P < 0.05).ConclusionsThe mRNA expression levels of PGF2α synthase and its receptor depend on the developmental stage and the embryo quality. Analyses of PGFS and PTGFR expression in bovine blastocysts and of PGF2α embryo production suggest that prostaglandin F2α can act in an autocrine and paracrine manner in bovine in vitro-produced preimplantation embryos. Moreover, the tendency of PTGFR and PGFS mRNA expression to be upregulated in embryos with low developmental potential can indicate a compensation mechanism related to high PGFS and PTGFR mRNA expression levels in low-quality embryos.

The pre-hatching bovine embryo transforms the uterine luminal metabolite composition in vivo

In cattle, conceptus development after elongation relies on well-characterized, paracrine interactions with the hosting maternal reproductive tract. However, it was unrecognized previously that the pre-hatching, pre-implantation bovine embryo also engages in biochemical signalling with the maternal uterus. Our recent work showed that the embryo modified the endometrial transcriptome in vivo. Here, we hypothesized that the embryo modulates the biochemical composition of the uterine luminal fluid (ULF) in the most cranial portion of the uterine horn ipsilateral to the corpus luteum. Endometrial samples and ULF were collected post-mortem from sham-inseminated cows and from cows inseminated and detected pregnant 7 days after oestrus. We used quantitative mass spectrometry to demonstrate that the pre-hatching embryo changes ULF composition in vivo. Embryo-induced modulation included an increase in concentrations of lipoxygenase-derived metabolites [12(S)-HETE, 15(S)-HETE] and a decrease in the concentrations of amino acids (glycine), biogenic amines (sarcosine), acylcarnitines and phospholipids. The changed composition of the ULF could be due to secretion or depletion of specific molecules, executed by either the embryo or the endometrium, but initiated by signals coming from the embryo. This study provides the basis for further understanding embryo-initiated modulation of the uterine milieu. Early embryonic signalling may be necessary to guarantee optimal development and successful establishment of pregnancy in cattle.

Finding of bands of higher molecular weight than expected in three proteins in bovine preimplantation embryos

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

Acute in vitro exposure to environmentally relevant atrazine levels perturbs bovine preimplantation embryo metabolism and cell number

Atrazine is a widely used herbicide known to negatively alter endocrine systems and perturb metabolism. Preimplantation exposure to pesticides may adversely affect long-term health, however few studies examine the effect of environmental levels and whether specific periods of development are particularly sensitive. In this study, the effect of acute, preimplantation atrazine exposure (days 3.5–7.5 post-fertilization) at levels detected and deemed safe in drinking water (0.02 and 20 μg/L respectively) on in vitro bovine embryo development, quality, metabolism, and gene expression was investigated. Atrazine exposure had no effect on development or quality, but significantly reduced blastocyst total cell numbers, attributable to a decrease in trophectoderm cells. Notably, atrazine (20 μg/L) markedly increased carbohydrate metabolism. Therefore, short-term exposure to environmentally relevant atrazine concentrations perturbs bovine preimplantation embryo metabolism and cell number, highlighting a potential mechanism by which atrazine can mediate embryo viability and health.

Expression of enzymes involved in the synthesis of prostaglandin E2 in early- and late-cleaved bovine embryos at different stages of preimplantation development

Prostaglandin (PG) E2 plays a role in numerous aspects of mammalian reproduction, such as oviductal transport of gametes, hatching from the zona pellucida in blastocysts and early embryonic development. Despite the evident role of PGE2 in the regulation of female reproductive processes, in the literature, there is very little information concerning the expression of PGE2 synthesizing enzymes and the exact amount of PGE2 produced by bovine embryos in vitro. In the present study, we aimed to determine the mRNA levels and immunolocalization of the enzymes responsible for PGE2 synthesis (PTGS2, mPGES1, mPGES2 and cPGES) in embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, early blastocyst, blastocyst, expanded blastocyst and hatched blastocyst stages, using a well-defined bovine model of oocyte developmental competence based on the time of first cleavage. PTGS2, mPGES2 and cPGES transcripts and proteins were detected in all stages of embryos, whereas the mPGES1 transcript and protein were not detected in embryos from the 2- to 16-cell stage. The results showed different transcription profiles of the enzymes involved in PGE2 synthesis in early- and late-cleaved embryos during the early stages of their in vitro preimplantation development. We also found that all the analysed stages of bovine preimplantation embryos released PGE2, with the highest concentration on Day 7 of culture in both the early- and late-cleaved groups. The present study is the first to demonstrate PGE2 synthesis and production by bovine early- and late-cleaved embryos at different stages of preimplantation development. Bovine embryos can produce PGE2, which may exert paracrine regulation during development. The transcription levels of PGE2 synthases were affected by the embryonic stage of development and quality. Our results indicate that the different transcription profiles of PTGS2, mPGES1, mPGES2 and cPGES, as well as PGE2 concentration, in early-versus late-cleaved embryos are dependent on the quality of the oocytes from which the embryos were obtained, which could reveal the association of PGE2 production during bovine preimplantation development with more advanced stages of embryo development.

Gene expression analysis of single preimplantation bovine embryos and the consequence for developmental potential

Preimplantation embryo development typically involves sequential morphological events connecting embryonic cleavage, morula compaction and blastocyst formation, and occurs in parallel with transcriptional regulation, specifically, the maternal to embryonic transition. The underlying homeostatic and metabolic mechanisms governing embryo development are influenced by both genetic and epigenetic factors that respond to environmental stimuli and may impact development during later gestational and fetal growth. There is a renewed interest in the identification and characterization of developmentally important genes during embryonic and fetal development. Perturbations in gene expression, resulting from environmental conditions, can have serious consequences on further embryonic development, homeostasis and disease pathogenesis. The bovine embryo is, however, capable of tolerating and adapting to a wide range of conditions, although little is known of the molecular fingerprint required for oocyte maturation, fertilization and development to term. The genomic revolution united with promising new technologies offer greater opportunity to elucidate the mechanisms behind this well-orchestrated biological process. This paper reviews the current literature on gene expression in the bovine embryo with reference to environmental interference and the development of new technologies to observe this biological process. Defining the difference in molecular signalling between in vivo and in vitro systems will undoubtedly improve the safety and efficiency of assisted reproductive technologies. The future challenge is to devise culture conditions that mimic the changing environment required by developing embryos to allow the correct temporal and spatial expression of a cohort of developmental genes in a manner similar to that seen in

Improves the In Vitro Developmental Competence and Reprogramming Efficiency of Cloned Bovine Embryos by Additional Complimentary Cytoplasm.

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.

60 Quercetin protects bovine pre-implantation embryos against oxidative stress via activation of Nrf2 signaling pathway

High atmospheric oxygen level during in vitro embryo culture system is reported to induce oxidative stress, leading to an increased intracellular reactive oxygen species (ROS) accumulation and lower the quality and development of the embryos. The Nrf2 is a redox sensitive transcription factor, which regulates the antioxidant machinery against oxidative stress in bovine pre-implantation embryos (Amin et al. 2014Mol. Reprod. Dev. 81, 497-513). Quercetin is a plant-derived flavonoid found in fruits and vegetables with antioxidant, anti-inflammatory, and anti-apoptotic properties. The effect of these antioxidants on the development and quality of bovine pre-implantation embryos with respect to the activation of the Nrf2 signalling pathways is not yet discovered. Here, we aimed to investigate the effect of quercetin supplementation on the activation of the Nrf2 signalling pathway and the subsequent impact on the quality of bovine embryos. For this, presumptive bovine zygotes were cultured in SOFaa embryo culture media supplemented with or without 10 µM quercetin (482 and 485 zygotes, respectively) and incubated under high oxygen level (20%) conditions. Day 7 and Day 8 blastocysts were analysed for the accumulation intracellular ROS and mitochondrial activity using H2DCFDA and MitoTracker® Red, respectively. Thereafter, blastocysts from both groups were subjected to total RNA isolation using Norgen total RNA purification plus kit (BioTek, Winooski, VT, USA). The relative abundance of Nrf2 and its downstream antioxidant genes (NQO1, PRDX1, SOD1, and CAT) was quantified using quantitative PCR. Moreover, the protein level of Nrf2 was detected using immunofluorescence staining. Data from 4 independent biological replicates were statistically analysed using a 2-tailed Student’s t-test. A significant difference in the mean value among treatments was determined at P=0.05. Results showed that quercetin supplementation in embryo culture medium significantly increased the blastocyst total cell number both at Day 7 (125±7.98 v. 100±3.74; P=0.007) and Day 8 (128±4.49 v. 95±3.15; P&amp;lt;0.0001). However, no difference was observed in the quercetin-treated group compared with control with respect to the cleavage rate (85.89±1.11v. 83.86±1.27; P=0.2) as well as the blastocyst rate at Day 7 (24.57±4.94 v. 27.23±1.68; P=0.64) and Day 8 (41.17±3.70 v. 38.87±0.85; P=0.58). Interestingly, quercetin-supplemented blastocysts showed a significant reduction in intracellular ROS level and increment in the mitochondrial activity compared with untreated counterparts. Supplementation of quercetin activated the Nrf2 transcriptional factor both at the mRNA and protein level, which subsequently activated the transcription of the aforementioned downstream antioxidant genes. In conclusion, supplementation of quercetin to embryo culture media protects embryos against oxidative stress by activating the Nrf2-mediated oxidative stress response mechanism, which leads to reduced accumulation of ROS and elevated mitochondrial activity.

41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos

Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

67 Optimizing a protocol for isolating extracellular vesicles from medium conditioned by bovine embryos in vitro

In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P&amp;lt;0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).

2 Role of histone H3 lysine 9 trimethylation during bovine pre-implantation embryonic development

Histones play an important role in DNA’s compaction and organisation into the cellular nucleus. Depending on which histone modification occurs, chromatin can take a conformation of heterochromatin or euchromatin, which are associated with gene repression or expression, respectively. Histone H3 lysine 9 (H3K9) trimethylation (H3K9me3) is associated with gene silencing. At least 3 methyltransferases are able to change the methylation status of H3K9: SUV39H1, SUV39H2, and SETDB1. In several mammalian species, modulation of H3K9 methylation status has been demonstrated to be necessary to achieve a successful pre-implantation embryonic development after IVF or somatic cell NT. The aim of this work was to study the role of H3K9me3 in IVF pre-implantation bovine embryos. For this purpose, immunostaining of H3K9me3 at different pre-implantation stages of development was performed. Further, the relative abundances of the methyltransferases SUV39H1 and SUV39H2 were measured by real-time PCR using luciferase transcript as an exogenous gene for normalization. Finally, to evaluate H3K9me3 involvement during pre-implantation embryonic development, we generated SUV39H1 or SUV39H2 knockout embryos by the CRISPR/Cas9 system. We designed guide RNA targeting SUV39H1 or SUV39H2 and co-injected the presumptive zygote’s cytoplasm 18h post-fertilization with Cas9 protein. At Day 8 post-fertilization, the number of blastocysts was assessed and embryos were immunostained to evaluate H3K9me3. Results were analysed using Student’s t-test or ANOVA with the post-hoc Tukey test depending on data set (P ≤ 0.05) and reported as means±standard errors of the mean. Oocytes at germinal vesicle stage and metaphase II as well as embryos at different stages of pre-implantation development (2, 4, and 8 cells, morula, and blastocyst; n=6) were immunoreactive for H3K9me3. Expression of SUV39H1 and SUV39H2 mRNA decreased significantly as embryonic development progressed, reaching undetectable levels at stages where genome activation had already occurred (morula and blastocyst; P&amp;lt;0.0001, n=3). When zygotes were co-injected with the guide RNA targeting SUV39H1/Cas9, embryonic production showed a significant increase compared with the control [42.26%±5.03 (28/65) v. 23.86%±3.99 (21/88), respectively; P=0.034, n=4], and H3K9me3 immunostaining was reduced in treated embryos. Editing efficiency was estimated at 66%. In contrast, no statistical differences were found in embryonic production or H3K9me3 immunostaining in embryos co-injected with the guide RNA targeting SUV39H2/Cas9 (P=0.57, n=3). In conclusion, we were able to characterise H3K9me3 and determine transcript levels of methyltransferases SUV39H1 and SUV39H2 in oocytes and different stages of pre-implantation embryonic development. We also demonstrated that SUV39H1 deletion led to an increased embryonic production, suggesting that H3K9me3 removal would allow a greater relaxation of the heterochromatin and consequently a successful activation of embryonic genes. This highlights the essential role of H3K9me3 during bovine pre-implantation embryonic development.

178 Cellular and extracellular expression of stress response transcription factors in male and female bovine pre-implantation embryos under oxidative stress conditions

Pre-implantation embryo development is a critical stage, in which several development and stress-response related transcription factors (TF) are involved. Exposing embryos to environmental insults alter some of these stress response-related TF. However, their expression pattern in male and female embryos and their release via exosomes is still unclear. Here, we aimed to investigate the effect of culture-induced oxidative stress on development and expression pattern of stress-related TF in male and female embryos and in respective spent media coupled with exosomes. For this, bovine male and female zygotes were in vitro produced using sexed semen and cultured under 5% and 20% oxygen in exosome-depleted SOFaa medium (SOF with amino acids). Blastocysts were subjected to total RNA isolation followed by quantitative RT-PCR analysis of the selected TF (Nrf2, KLF4, NOTCH1, SREBF2, E2F1, CAT1, SOD1, and OCT4), as well as protein abundance analysis using immunofluorescence and related phenotypes analysis, including reactive oxygen species (ROS) level and total cell count. Furthermore, the spent embryo culture media were collected for exosomes isolation and expression analysis of candidate TF. The data were statistically analysed using one-way ANOVA followed by multiple pair-wise comparisons using the Tukey post hoc test. Results showed that the blastocyst rates of both male (29.9% v. 34.9%) and female (16.7% v. 26.5%) bovine embryos were significantly lower in 20% than in 5% oxygen level. Female blastocysts subjected to the higher oxygen level showed increased ROS level (37.66±1.70v. 45.32±2.05 in male and 29.42±1.44v. 45.51±2.06 in female) and significantly reduced total cell count compared with the male embryo counterpart (136.55±7.8v. 112.75±2.9 in male and 138.75±2.0v. 88.25±4.3 in female cultured in 5% and 20% oxygen levels, respectively). Consequently, the expression levels of Nrf2, KLF4, SREBF2, CAT1, SOD1, and OCT4 were significantly increased in male embryos exposed to oxidative stress compared with those cultured under the lower oxygen level. However, NOTCH1 and E2F1 were significantly increased in female embryos exposed to oxidative stress compared with the male counterparts. The mRNA level of SREBF2 was significantly increased in male embryos cultured under both 5% and 20% O2 compared with female embryos. The protein expression level of Nrf2 and KLF4 was higher in embryos cultured at 20% v. 5% O2 with greater Nrf2 abundance in male embryos. Consequently, the male embryos produced at 20% O2 released a higher number of exosomes enriched with Nrf2, SOD1, and NOTCH1 mRNA than the other groups. Interestingly, the exosomal mRNA expression level of E2F1 tended to be higher in female embryos exposed to oxidative stress than their male counterparts. Taken together, the male embryos were more tolerant to oxidative stress than female embryos via the activation Nrf2-mediated oxidative stress response and development related TF. The release of these TF via exosomes could enhance cellular homeostasis maintenance under oxidative stress.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos.

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P&lt;0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P&lt;0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

Karyomapping for simultaneous genomic evaluation and aneuploidy screening of preimplantation bovine embryos: The first live-born calves

In cattle breeding, the development of genomic selection strategies based on single nucleotide polymorphism (SNP) interrogation has led to improved rates of genetic gain. Additionally, the application of genomic selection to in-vitro produced (IVP) embryos is expected to bring further benefits thanks to the ability to test a greater number of individuals before establishing a pregnancy and to ensure only carriers of desirable traits are born. However, aneuploidy, a leading cause of developmental arrest, is known to be common in IVP embryos. Karyomapping is a comprehensive screening test based on SNP typing that can be used for simultaneous genomic selection and aneuploidy detection, offering the potential to maximize pregnancy rates. Moreover, Karyomapping can be used to characterize the frequency and parental origin of aneuploidy in bovine IVP embryos, which have remained underexplored to date. Here, we report the use of Karyomapping to characterize the frequency and parental origin of aneuploidy in IVP bovine embryos in order to establish an estimate of total aneuploidy rates in each parental germline. We report an estimate of genome wide recombination rate in cattle and demonstrate, for the first time, a proof of principle for the application of Karyomapping to cattle breeding, with the birth of five calves after screening. This combined genomic selection and aneuploidy screening approach was highly reliable, with calves showing 98% concordance with their respective embryo biopsies for SNP typing and 100% concordance with their respective biopsies for aneuploidy screening. This approach has the potential to simultaneously improve pregnancy rates following embryo transfer and the rate of genetic gain in cattle breeding, and is applicable to basic research to investigate meiosis and aneuploidy.

Cellular and epigenetic changes induced by heat stress in bovine preimplantation embryos.

Exposure of the preimplantation embryo to heat stress triggers a series of cellular, molecular, and adaptive changes preventing a normal embryonic development. Heat stress disrupts the embryo cytoskeleton, intracellular calcium levels, mitochondrial function, and induces apoptosis. Moreover, heat stress can act indirectly through induction of reactive oxygen species (ROS), leading to a variety of cellular damage. Embryonic resistance to heat shock is determined by factors such as genotype, developmental stage, apoptosis, redox status, and regulatory molecules. The early embryo is very susceptible to heat stress; it acquires resistance to elevated temperature as development advances. One of the mechanisms involved in the developmental acquisition of thermotolerance is heat-induced apoptosis, which acts as a quality control mechanism to remove damaged blastomeres allowing the embryo to survive after stress. Although embryos at >8-cell stage can activate the apoptotic cascade as an adaptive response to stress, embryos at the two-cell stage are resistant to proapoptotic signals. This lack of apoptotic response has been associated to mitochondrial resistance to depolarization and epigenetic regulations, such as DNA methylation and histone deacetylation. Even though the cellular mechanisms triggered by heat stress have been studied, very little attention has been paid to the vulnerability of the epigenome to drastic temperature changes during the preimplantation period. Therefore, this review aims to characterize the effects of elevated temperature on the bovine embryo, especially addresissing developmental, cellular, and epigenetic alterations triggered in response to temperature.

The following five articles for this Special Issue were previously published and can be found in their respective issues or online

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos (85:2)https://onlinelibrary.wiley.com/doi/10.1002/mrd.22941Impact of preimplantational oral low‐dose estradiol‐17β exposure on the endometrium: The role of miRNA (85:5)https://onlinelibrary.wiley.com/doi/10.1002/mrd.22975Long term effects of ART: What do animals tell us? (85:4)https://onlinelibrary.wiley.com/doi/10.1002/mrd.22970Testicular development in male lambs prenatally exposed to a high‐starch diet (85:5)https://onlinelibrary.wiley.com/doi/10.1002/mrd.22974Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes (85:5)https://onlinelibrary.wiley.com/doi/10.1002/mrd.22977