A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment.

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy.

Dormancy is a key feature of stem cell function in adult tissues as well as in embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for preimplantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in the loss of pluripotent cells. Through the integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network, and the perturbation of the network compromises embryo survival in diapause. We further identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic MTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

Changes in the Transcription of Proliferation- and Apoptosis-Related Genes in Embryos in Women of Different Ages under the Influence of Extracellular Vesicles from Donor Follicular Fluid In Vitro.

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×1011 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years.

Oscillatory control of embryonic development.

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.

Heat-Stress Impacts on Developing Bovine Oocytes: Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

Histone Lactylation Is Involved in Mouse Oocyte Maturation and Embryo Development.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.

The Clock and Wavefront Self-Organizing model recreates the dynamics of mouse somitogenesis in vivo and in vitro.

During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro.

Maternal factor Trim75 contributes to zygotic genome activation program in mouse early embryos.

BACKGROUND: Zygotic genome activation (ZGA) is an important event in the early embryo development, and human embryo developmental arrest has been highly correlated with ZGA failure in clinical studies. Although a few studies have linked maternal factors to mammalian ZGA, more studies are needed to fully elucidate the maternal factors that are involved in ZGA. METHODS AND RESULTS: In this study, we utilized published single-cell RNA sequencing data from a Dux-mediated mouse embryonic stem cell to induce a 2-cell-like transition state and selected potential drivers for the transition according to an RNA velocity analysis. CONCLUSIONS: An overlap of potential candidate markers of 2-cell-like-cells identified in this research with markers generated by various data sets suggests that Trim75 is a potential driver of minor ZGA and may recruit EP300 and establish H3K27ac in the gene body of minor ZGA genes, thereby contributing to mammalian preimplantation embryo development.

Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq.

During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.

A logic-incorporated gene regulatory network deciphers principles in cell fate decisions.

Organisms utilize gene regulatory networks (GRN) to make fate decisions, but the regulatory mechanisms of transcription factors (TF) in GRNs are exceedingly intricate. A longstanding question in this field is how these tangled interactions synergistically contribute to decision-making procedures. To comprehensively understand the role of regulatory logic in cell fate decisions, we constructed a logic-incorporated GRN model and examined its behavior under two distinct driving forces (noise-driven and signal-driven). Under the noise-driven mode, we distilled the relationship among fate bias, regulatory logic, and noise profile. Under the signal-driven mode, we bridged regulatory logic and progression-accuracy trade-off, and uncovered distinctive trajectories of reprogramming influenced by logic motifs. In differentiation, we characterized a special logic-dependent priming stage by the solution landscape. Finally, we applied our findings to decipher three biological instances: hematopoiesis, embryogenesis, and trans-differentiation. Orthogonal to the classical analysis of expression profile, we harnessed noise patterns to construct the GRN corresponding to fate transition. Our work presents a generalizable framework for top-down fate-decision studies and a practical approach to the taxonomy of cell fate decisions.

The dynamic landscape of enhancer-derived RNA during mouse early embryo development.

Enhancer-derived RNAs (eRNAs) play critical roles in diverse biological processes by facilitating their target gene expression. However, the abundance and function of eRNAs in early embryos are not clear. Here, we present a comprehensive eRNA atlas by systematically integrating publicly available datasets of mouse early embryos. We characterize the transcriptional and regulatory network of eRNAs and show that different embryo developmental stages have distinct eRNA expression and regulatory profiles. Paternal eRNAs are activated asymmetrically during zygotic genome activation (ZGA). Moreover, we identify an eRNA, MZGAe1, which plays an important function in regulating mouse ZGA and early embryo development. MZGAe1 knockdown leads to a developmental block from 2-cell embryo to blastocyst. We create an online data portal, M2ED2, to query and visualize eRNA expression and regulation. Our study thus provides a systematic landscape of eRNA and reveals the important role of eRNAs in regulating mouse early embryo development.

Amphetamine Exposure during Embryogenesis Alters Expression and Function of Tyrosine Hydroxylase and the Vesicular Monoamine Transporter in Adult C. elegans.

Amphetamines (Amph) are psychostimulants broadly used as physical and cognitive enhancers. However, the long-term effects of prenatal exposure to Amph have been poorly investigated. Here, we show that continuous exposure to Amph during early development induces long-lasting changes in histone methylation at the C. elegans tyrosine hydroxylase (TH) homolog cat-2 and the vesicular monoamine transporter (VMAT) homologue cat-1 genes. These Amph-induced histone modifications are correlated with enhanced expression and function of CAT-2/TH and higher levels of dopamine, but decreased expression of CAT-1/VMAT in adult animals. Moreover, while adult animals pre-exposed to Amph do not show obvious behavioral defects, when challenged with Amph they exhibit Amph hypersensitivity, which is associated with a rapid increase in cat-2/TH mRNA. Because C. elegans has helped reveal neuronal and epigenetic mechanisms that are shared among animals as diverse as roundworms and humans, and because of the evolutionary conservation of the dopaminergic response to psychostimulants, data collected in this study could help us to identify the mechanisms through which Amph induces long-lasting physiological and behavioral changes in mammals.

Pleomorphic adenoma gene1 in reproduction and implication for embryonic survival in cattle: a review.

The pleomorphic adenoma gene1 (PLAG1) encodes a DNA-binding, C2H2 zinc-finger protein which acts as a transcription factor that regulates the expression of diverse genes across different organs and tissues; hence, the name pleomorphic. Rearrangements of the PLAG1 gene, and/or overexpression, are associated with benign tumors and cancers in a variety of tissues. This is best described for pleomorphic adenoma of the salivary glands in humans. The most notable expression of PLAG1 occurs during embryonic and fetal development, with lesser expression after birth. Evidence has accumulated of a role for PLAG1 protein in normal early embryonic development and placentation in mammals. PLAG1 protein influences the expression of the ike growth factor 2 (IGF2) gene and production of IGF2 protein. IGF2 is an important mitogen in ovarian follicles/oocytes, embryos, and fetuses. The PLAG1-IGF2 axis, therefore, provides one pathway whereby PLAG1 protein can influence embryonic survival and pregnancy. PLAG1 also influences over 1,000 other genes in embryos including those associated with ribosomal assembly and proteins. Brahman (Bos indicus) heifers homozygous for the PLAG1 variant, rs109815800 (G > T), show greater fertility than contemporary heifers with either one, or no copy, of the variant. Greater fertility in heifers homozygous for rs109815800 could be the result of early puberty and/or greater embryonic survival. The present review first looks at the broader roles of the PLAG1 gene and PLAG1 protein and then focuses on the emerging role of PLAG1/PLAG1 in embryonic development and pregnancy. A deeper understanding of factors which influence embryonic development is required for the next transformational increase in embryonic survival and successful pregnancy for both in vivo and in vitro derived embryos in cattle.

The pleomorphic adenoma gene1 (PLAG1) produces PLAG1 protein which, by binding to specific regions on DNA, influences the activity of other genes that regulate many body functions. One gene is insulin-like growth factor 2 (IGF2) which controls cell metabolism and growth. The PLAG1 gene is particularly active during embryonic and fetal growth, and through IGF2 determines stature later in life. IGF2 protein is also very important in early embryonic development. This review explores the hypothesis that PLAG1 is an important determinant of embryonic survival and the establishment of pregnancy in mammals.

Single-embryo transcriptomic atlas of oxygen response reveals the critical role of HIF-1α in prompting embryonic zygotic genome activation.

Adaptive response to physiological oxygen levels (physO2; 5% O2) enables embryonic survival in a low-oxygen developmental environment. However, the mechanism underlying the role of physO2 in supporting preimplantation development, remains elusive. Here, we systematically studied oxygen responses of hallmark events in preimplantation development. Focusing on impeded transcriptional upregulation under atmospheric oxygen levels (atmosO2; 20% O2) during the 2-cell stage, we functionally identified a novel role of HIF-1α in promoting major zygotic genome activation by serving as an oxygen-sensitive transcription factor. Moreover, during blastocyst formation, atmosO2 impeded H3K4me3 and H3K27me3 deposition by deregulating histone-lysine methyltransferases, thus impairing X-chromosome inactivation in blastocysts. In addition, we found atmosO2 impedes metabolic shift to glycolysis before blastocyst formation, thus resulting a low-level histone lactylation deposition. Notably, we also reported an increased sex-dimorphic oxygen response of embryos upon preimplantation development. Together, focusing on genetic and epigenetic events that are essential for embryonic survival and development, the present study advances current knowledge of embryonic adaptive responses to physO2, and provides novel insight into mechanism underlying irreversibly impaired developmental potential due to a short-term atmosO2 exposure.

TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Defining a TFAP2C-centered transcription factor network during murine peri-implantation.

Transcription factors (TFs) play important roles in early embryonic development, but factors regulating TF action, relationships in signaling cascade, genome-wide localizations, and impacts on cell fate transitions during this process have not been clearly elucidated. In this study, we used uliCUT&RUN-seq to delineate a TFAP2C-centered regulatory network, showing that it involves promoter-enhancer interactions and regulates TEAD4 and KLF5 function to mediate cell polarization. Notably, we found that maternal retinoic acid metabolism regulates TFAP2C expression and function by inducing the active demethylation of SINEs, indicating that the RARG-TFAP2C-TEAD4/KLF5 axis connects the maternal-to-zygotic transition to polarization. Moreover, we found that both genomic imprinting and SNP-transferred genetic information can influence TF positioning to regulate parental gene expressions in a sophisticated manner. In summary, we propose a ternary model of TF regulation in murine embryonic development with TFAP2C as the core element and metabolic, epigenetic, and genetic information as nodes connecting the pathways.

SRRM2 splicing factor modulates cell fate in early development.

Embryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.

Detection of newly synthesized RNA reveals transcriptional reprogramming during ZGA and a role of Obox3 in totipotency acquisition.

Zygotic genome activation (ZGA) after fertilization enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at initiation, is incompletely understood. Here, we develop a method to capture and sequence newly synthesized RNA in early mouse embryos, providing a view of transcriptional reprogramming during ZGA. Our data demonstrate that major ZGA gene activation begins earlier than previously thought. Furthermore, we identify a set of genes activated during minor ZGA, the promoters of which show enrichment of the Obox factor motif, and find that Obox3 or Obox5 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox factors is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoration of Obox3 expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition.