During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome ...activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 103 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation i.e., (i) 59%, (ii) 42%, and (iii) 58% was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.
Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst ...formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.
Infection-neutralizing antibody responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 vaccination are an essential component of antiviral ...immunity. Antibody-mediated protection is challenged by the emergence of SARS-CoV-2 variants of concern (VoCs) with immune escape properties, such as omicron (B.1.1.529), which is rapidly spreading worldwide. Here we report neutralizing antibody dynamics in a longitudinal cohort of coronavirus disease 2019 convalescent and infection-naive individuals vaccinated with mRNA BNT162b2 by quantifying SARS-CoV-2 spike protein antibodies and determining their avidity and neutralization capacity in serum. Using live-virus neutralization assays, we show that a superior infection-neutralizing capacity against all VoCs, including omicron, developed after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures resulted in an increasing neutralization capacity per anti-spike antibody unit and were paralleled by stepwise increases in antibody avidity. We conclude that an infection-plus-vaccination-induced hybrid immunity or a triple immunization can induce high-quality antibodies with superior neutralization capacity against VoCs, including omicron.
Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues ...and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.
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► LBR- and lamin-A/C-dependent tethers maintain peripheral heterochromatin ► In their absence, all heterochromatin clusters in the nuclear interior ► During cellular differentiation, the LBR tether precedes the lamin A/C tether ► Lamin A/C promotes, whereas LBR delays myogenic differentiation
The lamin B receptor and lamin A/C have sequential roles in the tethering of peripheral heterochromatin during cellular differentiation, corresponding to their differential effects on heterochromatin positioning and transcription of tissue-specific genes.
•We review studies on bovine embryonic genome activation (EGA).•We provide new insights into EGA by RNA sequencing (RNA-Seq).•A gene ontology analysis of genes activated at the 8-cell, 16-cell, and ...blastocyst stage is provided.•An outlook into potential applications of single cell RNA-Seq is given.
Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of 3Huridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.
The yak is remarkable for its adaptation to high altitude and occupies a central place in the economies of the mountainous regions of Asia. At lower elevations, it is common to hybridize yaks with ...cattle to combine the yak's hardiness with the productivity of cattle. Hybrid males are sterile, however, preventing the establishment of stable hybrid populations, but not a limited introgression after backcrossing several generations of female hybrids to male yaks. Here we inferred bovine haplotypes in the genomes of 76 Mongolian yaks using high-density SNP genotyping and whole-genome sequencing. These yaks inherited ∼1.3% of their genome from bovine ancestors after nearly continuous admixture over at least the last 1,500 years. The introgressed regions are enriched in genes involved in nervous system development and function, and particularly in glutamate metabolism and neurotransmission. We also identified a novel mutation associated with a polled (hornless) phenotype originating from Mongolian Turano cattle. Our results suggest that introgressive hybridization contributed to the improvement of yak management and breeding.
Placozoans are a phylum of nonbilaterian marine animals currently represented by a single described species, Trichoplax adhaerens, Schulze 1883. Placozoans arguably show the simplest animal ...morphology, which is identical among isolates collected worldwide, despite an apparently sizeable genetic diversity within the phylum. Here, we use a comparative genomics approach for a deeper appreciation of the structure and causes of the deeply diverging lineages in the Placozoa. We generated a high-quality draft genome of the genetic lineage H13 isolated from Hong Kong and compared it to the distantly related T. adhaerens. We uncovered substantial structural differences between the two genomes that point to a deep genomic separation and provide support that adaptation by gene duplication is likely a crucial mechanism in placozoan speciation. We further provide genetic evidence for reproductively isolated species and suggest a genus-level difference of H13 to T. adhaerens, justifying the designation of H13 as a new species, Hoilungia hongkongensis nov. gen., nov. spec., now the second described placozoan species and the first in a new genus. Our multilevel comparative genomics approach is, therefore, likely to prove valuable for species distinctions in other cryptic microscopic animal groups that lack diagnostic morphological characters, such as some nematodes, copepods, rotifers, or mites.
Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes ...are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5′-UTR, 3′-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes.
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•Nucleosome preservation in mammalian sperm underlies a universal pattern•Centromeres LINE1 and SINE preserve nucleosomes•Nucleosomes occur largely intergenically, while exons and most promoters are depleted•Nucleosome-rich genes are hypomethylated in promoter and code RNA-processing factors
The study demonstrates concordantly in human and bovine the uniformity of the nucleosome-preservation pattern in sperm in a genome-wide manner. A potential role in preimplantation development is suggested for sperm-derived nucleosomes frequently detected in centromere repeats, LINE1 and SINE, and predominantly in genes relevant for RNA and protein processing.
The molecular consequences of the metabolic stress caused by milk production of dairy cows in the early embryo are largely unknown. The objective was to determine the impact of dam metabolic status ...or in vitro culture during embryonic genome activation (EGA) on the transcriptomic profiles of bovine 16-cell stage embryos. Two days after synchronized oestrus, in vitro produced 2- to 4-cell stage embryos were endoscopically transferred in pools of 50 into the oviduct ipsilateral to the corpus luteum of lactating (LACT, n = 3) or nonlactating (i.e. dried off immediately at calving; DRY, n = 3) dairy cows. On Day 4, the oviducts were flushed to recover the embryos. Pools of five Day-2 embryos (n = 5) and Day-4 16-cell stage embryos obtained in vitro (n = 3) or from LACT or DRY cows were subjected to RNAseq. Temporally differentially expressed genes (DEG; FDR<0.05) between Day-2 and Day-4 embryos were determined considering the differences between the three conditions under which EGA occurred. Also, DEG between Day-4 embryos derived from the three conditions were identified. Functional analysis of the temporal DEG demonstrated that genes involved in ribosome, translation and oxidative phosphorylation in the mitochondria were strongly more expressed in Day-4 than Day-2 embryos. Comparison of Day-4 embryos that underwent EGA in vitro, or in LACT or DRY cows, identified DEG enriching for mitochondrial respiration and protein translation, including the mTOR pathway. In conclusion, exposure of the embryo to an unfavourable maternal metabolic status during EGA influences its transcriptome and potentially the competence for pregnancy establishment.
Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in
(APA-
) ...are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type
(APA-
). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis.
Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry.
We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (
-type 1 or 2). The
-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-
and APA-
. The
-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-
. Metabolites that promote oxidative stress and cell death accumulated in APA-
. In contrast, antioxidant metabolites were abundant in APA-
. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states.
Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.
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