Frontiers in Cell and Developmental Biology

One of the major cause of oocyte quality deterioration along with aging, chromosome segregation abnormalities occur mainly during meiosis I. However, currently, there is a technical limitation in the introduction of mRNA into premature oocytes without impairing embryonic developmental ability. In this study, we established a low-invasive electroporation (EP) method to introduce mRNA into pre-ovulatory, germinal vesicle (GV) mouse oocytes in an easier manner than the traditional microinjection method. The EP method with an optimized impedance value resulted in the efficient introduction of mRNAs encoding enhanced green fluorescent protein (EGFP) into the GV oocytes surrounded by cumulus cells at a survival rate of 95.0%. Furthermore, the introduction of histone H2B-EGFP mRNA into the GV oocytes labeled most of the oocytes without affecting the blastocyst development rate, indicating the feasibility of the visualization of oocyte chromosomal dynamics that enable us to assay chromosomal integrity in oocyte maturation and cell count in embryonic development. The establishment of this EP method offers extensive assays to select pre-implantation embryos and enables the surveying of essential factors for mammalian oocyte quality determination. Summary blurbThis study introduces a low-invasive electroporation method with high survival rate and developmental ability, offering a potential breakthrough in preimplantation embryo treatment and assessment.

Tjp1 + is considered a crucial protein involved in the stepwise assembly of tight junctions (TJs) between compaction and blastocoel cavitation in early development. In this study, we investigated the specific role of Tjp1 + in TJ formation by employing an alternative splicing-specific knockdown of the Tjp1 + exon. To deplete Tjp1 + expression, we used siRNA targeting RNA-binding protein 47 (Rbm47), which induces the inclusion of the + exon in Tjp1 mRNA. The knockdown resulted in approximately 85% reduction in Rbm47 mRNA levels and 75% reduction in Tjp1 + mRNA levels in blastocysts. Surprisingly, despite this knockdown, blastocyst development and TJ permeability of trophectoderm were unaffected. Additionally, we observed an interaction between Tjp1 - and Ocln in Rbm47 knockdown blastocysts, suggesting a compensatory role of Tjp1 -. Overall, our findings indicate that Tjp1 + is not essential for the stepwise assembly of TJs and the completion of TJ biogenesis during blastocyst development in mice although a minimal amount of remaining Tjp1 + is sufficient for TJs assembly. Summary statementSelective loss of Tjp1 + mediated by Rbm47 knockdown did affect mouse blastocyst development, suggesting that Tjp1 + may not be crucial for stepwise TJs assembly during blastocyst development

Numerous reports show that the epididymis plays a key role in the acquisition of sperm fertilizing ability but less information exists on its contribution to embryo development. Evidence from our laboratory showed that mammalian CRISP (Cysteine-Rich Secretory Proteins), known to be expressed in the epididymis, to regulate calcium (Ca2+) channels and to participate in fertilization, may also be relevant for embryo development. More specifically, we found that males with simultaneous mutations in Crisp1 and Crisp3 genes exhibited normal in vivo fertilization but impaired embryo development. In the present work, aimed to investigate the mechanisms underlying this reproductive phenotype, we observed that embryo development failure was not due to delayed fertilization as no differences in sperm transport within the female tract nor in in vivo fertilization were found shortly after mating. The observation that impaired embryo development was also found in eggs fertilized by epididymal sperm either after uterine insemination or in vitro fertilization, revealed that the defects were already present at epididymal level. Of note, eggs fertilized in vitro by mutant sperm exhibited impaired meiotic resumption not due to defects in Ca2+oscillations during egg activation, prompting us to examine potential sperm DNA defects. Interestingly, DNA fragmentation was found in cauda but not caput epididymal mutant sperm revealing that DNA integrity defects appear during epididymal maturation. Moreover, exposure of control sperm to mutant epididymal fluid significantly increased DNA fragmentation, indicating the relevance of the luminal environment for sperm DNA integrity. The finding that incubation of sperm with control epididymal fluid in the presence of Ca2+ also increased DNA fragmentation together with the higher intracellular Ca2+ levels detected in mutant sperm supports a dysregulation of Ca2+ homeostasis as the main responsible for DNA fragmentation and subsequent early development failure of mutant males. Together, our results support the contribution of the epididymis beyond fertilization, identifying CRISP1 and CRISP3 as novel male factors relevant for DNA integrity and early embryo development. Given the existence of human functional homologues of CRISP and the incidence of DNA fragmentation in infertile men, we believe these findings not only provide relevant information on the impact of epididymal factors on embryonic development but will also contribute to a better understanding, diagnosis and treatment of human infertility.

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts from the embryos, begins in the S phase of the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with the major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligos targeting Myc or inhibitors specific for MYC from the one-cell stage. In order to identify when MYC inhibition affected development, we applied time-limited inhibitor treatment, and found that inhibition of MYC at the two-cell, four-cell, and morula stages had no effect on preimplantation development, whereas treatment with the inhibitor at the early two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that Myc is essential for the induction of major ZGA and its subsequent development. Revealing the function of Myc in preimplantation development is expected to contribute to advances in assisted reproductive technology.

Epiboly is the first morphogenetic cell movement that occurs at the onset of gastrulation in zebrafish. During epiboly, the blastoderm thins out and spreads cells over the massive yolk cell. Epiboly progression is controlled by a complex regulatory network that involves diverse molecular effectors. Previously, we reported that reactive oxygen species (ROS) derived from NADPH oxidases (Nox) are required for normal epiboly progression, embryo survival, and early development. We also found that the inhibition of Nox activity during gastrulation downregulates E-cadherin abundance at the enveloping layer (EVL) cell margins. Since the dynamic localization of E-cadherin at the plasma membrane is highly regulated by endocytosis and vesicular trafficking during epiboly, in the present study, we investigated the effects of Nox inhibition and hydrogen peroxide (H2O2) on endocytosis and in the localization of different proteins important for endosomal trafficking in zebrafish embryos. We show that the simultaneous treatment with the Nox inhibitor VAS2870 and the dynamin 2 (Dnm2) inhibitor dynasore rescues the effects of VAS2870 on epiboly delay, embryo mortality and E-cadherin abundance at EVL cell margins. Furthermore, we found that H2O2 impacts the endocytic rate of fluorescent fluid-phase markers at the EVL, as well as the localization and abundance of Rab11, a small GTPase protein involved in recycling endosomes. Our results suggest that Nox-derived ROS participate in the regulation of the initial steps of endocytosis and in the endosomal trafficking required for epiboly progression during early zebrafish development. HIGHLIGHTS- NADPH oxidase (Nox) activity is required for the epiboly and localization of E- cadherin. - Dynamin inhibition rescues the developmental defects produced by the loss of Nox activity. - Nox-derived reactive oxygen species (ROS) participate in the regulation of endosome and E-cadherin trafficking, which is required for epiboly. - Nox inhibition increases the rate of fluorescent fluid-phase markers of endocytosis in EVL cells. - H2O2 decreases fluid-phase internalization in EVL cells. - H2O2 regulates Rab11 localization

Nuclear structure and nucleocytoplasmic interaction are closely linked to the regulation of cellular and higher-order biological processes. An alternative pathway for nucleocytoplasmic transport involving perinuclear vesicle formation and release, termed nuclear envelope budding (NEB), has been observed in diverse species and cell types. NEB-like events have also been reported in mammalian zygotes, including mice. However, their molecular basis remains unclear. Recent results suggest that protein kinase C (PKC) signaling regulates perinuclear vesicle biogenesis during NEB. While multiple PKC isoforms are expressed in mouse oocytes, their functions in zygotes are not fully understood. We investigated the effects of pharmacological PKC inhibition on zygotic development and NEB-like perinuclear vesicle formation. Our results suggest that NEB-like events in mouse zygotes may involve PKC-dependent mechanisms and that PKC activity might be critical for the 1-to 2-cell transition.

Transforming growth factor-beta (TGF-{beta}) supports the in vitro maintenance of embryonic and trophoblast stem cells. Here, we demonstrated that, in a sheep embryo model, the transition from morula to blastocyst is positively regulated by TGF-{beta}3, primarily through its promotion of trophoblast development. Our results indicate that morulae treated with TGF-{beta}3 develop at a higher rate into blastocysts, characterized by an expanded trophoblast layer marked by CDX-2 expression. In blastocysts, TGF-{beta}3 mediates transcriptional activation of genes involved in cell adhesion and lipid metabolism pathways, leading to remarkable in vitro outgrowth expansion and a substantial increase in trophoblast lipid droplet content. Functional analysis reveal that the positive effects of TGF-{beta}3 are mitigated by inhibition of Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2), a key enzyme upregulated by TGF-{beta}3 and a promoter of de novo lipgenensis. These findings suggest that TGF-{beta}3 modulates lipid metabolism during blastocyst formation and may play a potential role in regulating implantation and placental development.

Vocal production involves intricate neural coordination across various brain regions. Stuttering, a common speech disorder, has genetic underpinnings, including mutations in lysosomal-targeting pathway genes. Using a Gnptab-mutant mouse model linked to stuttering, we examined neuron and glial cell morphology in vocal production circuits. Our findings revealed altered astrocyte and microglia processes in these circuits in Gnptab-mutant mice, while control regions remained unaffected. Our results shed light on the potential role of glial cells in stuttering pathophysiology and highlight their relevance in modulating vocal production behaviors.

Differentiation and fusion are two intricate processes involved in skeletal muscle development. The close association of differentiation and fusion makes it difficult to address the process of fusion independently of differentiation. Using the fusion marker myomaker, named TMEM8C in chicken, we found that both TMEM8C transcripts and the differentiated and fusion-competent MYOG+ cells are preferentially regionalized in the central regions of limb foetal muscles in chicken embryos. Because the NOTCH signalling pathway is a potent inhibitor of muscle differentiation during developmental myogenesis, NOTCH function in myoblast fusion was not addressed so far. We analysed the consequences of NOTCH inhibition for myoblast fusion and TMEM8C expression during foetal myogenesis using in vitro and in vivo chicken systems. NOTCH inhibition following chicken embryo immobilisation or in myoblast cultures increased TMEM8C expression and myoblast fusion. Moreover, we showed that NOTCH inhibition induced the un-binding of the HEYL transcriptional repressor from the TMEM8C regulatory regions in limb muscles and myoblast cultures. These results identify a molecular mechanism underlying the fusion-promoting effect of NOTCH-inhibition during foetal myogenesis.

While lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains still poorly understood. Here we develop a time lapse imaging strategy to measure the four phases of the cell cycle in single neural progenitor cells in their endogenous environment. Our results show that neural progenitors possess a great heterogeneity of the cell cycle length. This duration variability is distributed over all phases of the cell cycle, with the G1 phase being the one contributing primarily to cell cycle variability. Within one cell cycle, each phase duration appears stochastic and independent except for a surprising correlation between S and M phase. Lineage analysis indicates that the majority of daughter cells display longer G1 phase than their mothers suggesting that at each cell cycle a mechanism lengthens the G1 phase. We identify an actor of the core cell cycle machinery, the CDC25B phosphatase known to regulate G2/M transition, as an indirect regulator of the duration of the G1 phase. We propose that CDC25B acts via a cell to cell increase in G1 phase heterogeneity revealing a novel mechanism of G1 lengthening associated with tissue development.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy, and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower compared to that of wild-type embryos, while exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in both nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness for normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Na+,K+-ATPase 4 is a unique cell plasma membrane Na+ and K+ transporter of spermatozoa, which is essential for male fertility. Previous studies have shown that Na+,K+-ATPase 4 is highly expressed in the sperm flagellum; however, the spatial arrangement of Na+,K+-ATPase 4 at the subcellular level and its relationship to the functional state of the cells are unknown. We studied this here using stimulated emission depletion (STED) super-resolution microscopy. We show that, under non-capacitated conditions, Na+,K+-ATPase 4 is distributed in a trilinear pattern along the midpiece and as a scattered single line along the principal piece segment of the sperm flagellum. Under capacitated conditions, Na+,K+-ATPase 4 pattern undergoes remodelling and its distribution shifts into a single line along the entire length of the flagellum. On the other hand, Na+,K+-ATPase 1 the somatic isoform of Na+,K+-ATPase also present in sperm, exhibits a similar trilaminar localization at the flagellar midpiece but a bilinear pattern in the principal piece. This distribution, unlike that of Na+,K+-ATPase 4, does not change during sperm capacitation. These differences in the localization pattern and spatial dynamics of Na+,K+- ATPase isoform expression highlights the dissimilarities in the roles of both ion transporters. The specific modulation of Na+,K+-ATPase 4 distribution, combined with the unique role that it has in sperm function, stresses the importance of Na+,K+-ATPase 4 for male fertility. Significance statementThis is the first demonstration of the highly structured nature of Na+,K+-ATPase in the plasma membrane of sperm, including the sperm specific Na+,K+-ATPase 4 isoform, which is key for male fertility, and the somatic Na+,K+-ATPase 1, which is present in all cells. Utilizing stimulated emission depletion (STED) super resolution microscopy, we discovered that Na+,K+- ATPase 4 and Na+,K+-ATPase 1 have different distributions along the sperm flagellum. Moreover, only Na+,K+-ATPase 4 undergoes remodelling during sperm capacitation. These specific patterns of localization that are dependent on the sperm functional state in combination with the different function and regulation of Na+,K+-ATPase isoforms highlights the sophisticated mechanisms that cells have evolved to fulfil their unique function.

Many invertebrates have obliquely striated muscles, in which neighboring thin and thick filaments are staggered and aligned in an oblique manner. This type of muscle allows force production over a wide range of lengths and is beneficial for soft-bodied animals. Unlike cross-striated muscles of vertebrates, most of obliquely striated muscles lack distinct Z-lines and, instead, have dense bodies. Because the dense bodies are located in the middle of the I-bands and contain -actinin, the dogma is that dense bodies are equivalent to the Z-lines anchoring the actin barbed ends. However, we present evidence that the barbed ends of sarcomeric actin filaments in the nematode Caenorhabditis elegans body wall muscle are aligned in a linear Z-line-like arrangement without converging at the dense bodies. Colocalization of F-actin and ATN-1/-actinin was minimal. Furthermore, CAP-1, an -subunit of capping protein/CapZ, was linearly aligned in the middle of the I-bands without concentration at the dense bodies. This linear CAP-1 alignment was maintained in the absence of ATN-1. These results demonstrate that the actin barbed ends are not directly anchored to the dense bodies. Depletion of the capping protein subunit, CAP-1 or CAP-2, caused embryonic or larval lethality with severe actin disorganization in the body wall muscle, indicating that barbed-end regulation by capping protein is essential for sarcomere assembly. These results contradict the current view of the sarcomere organization in C. elegans muscle and suggest a new model of a linear Z-line-like arrangement of actin barbed ends. Significance StatementO_LIWithout clear evidence, there has been a notion that actin filaments are directly anchored to the dense bodies in C. elegans striated muscle. C_LIO_LICapping protein localizes in a linear Z-line-like alignment in C. elegans muscle without concentrating at the dense bodies, indicating that the actin barbed ends are not directly anchored at the dense bodies. C_LIO_LIDepletion of capping protein causes severe sarcomere defects in embryos and larvae indicating a critical role of capping protein in sarcomere assembly. C_LI

Many studies have focused on the mechanisms of stem cell maintenance via their interaction with a particular niche or microenvironment in adult tissues, but how formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila melanogaster ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and underlying Cap Cells (CCs) and Escort Cells (ECs), which are in direct contact with GSCs. In the adult, the Engrailed (En) transcription factor is specifically expressed in niche cells where it directly controls expression of the decapentaplegic gene (dpp) encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In late third instar larval ovaries, in response to BMP signaling from newly-formed niches, adjacent primordial germ cells become GSCs. The bric-a-brac paralogs (bab1 and bab2) encode BTB/POZ-domain containing transcription factors, that are also expressed in developing GSCs niches where they are required for TF formation. Here, we demonstrate that Bab1 and Bab2 display redundant cell autonomous function for TF morphogenesis and we identify a new function for these genes in GSC establishment. Moreover, we show that Bab proteins control dpp expression in otherwise correctly specified CCs, independently of En and its paralog Invected (Inv). In fact, our results also indicate that en/inv function in larval stages are neither essential for TF formation, nor GSC establishment. Finally, when bab2 was overexpressed in ovarian somatic cells outside of the niche, where en/inv were not expressed, ectopic BMP signaling activation was induced in adjacent germ cells of adult ovaries, which formed GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling for acquisition of GSC status.

Both HIV infection and antiretroviral therapy contribute to dyslipidemia and abnormal body fat distribution in people living with HIV (PLWH). Exercise training is an effective intervention to protect against these metabolic changes. However, little is known about the mechanisms underlying the impact of exercise training on lipid metabolism in PLWH. To better understand lipid alterations, this study aimed to comparatively evaluate the effect of high-intensity-functional circuit training for 8 weeks on the plasma lipidome of PLWH (n=13) and HIV-negative subjects (control group; n=14). Anthropometric and biochemical parameters revealed lower levels of leptin, HDL-C, body fat %, and BMI combined with elevated Aspartate Transaminase (AST) and Homeostasis Model Assessment of {beta}-cell function (HOMA_beta) in PLWH when compared to control subjects that persisted from baseline to post-exercise training. Nonetheless, contrasting levels of adiponectin, fasting insulin, HOMA_IR and Adipo_IR, together with phosphatidylcholine-containing lipids observed at baseline were equalized after training in PLWH. In control subjects, significant reductions in concentrations of triglycerides alongside phosphatidylinositol and glycosylated ceramides were observed post exercise training. By contrast, PWLH displayed a decrease in concentrations of free fatty acids, cholesteryl esters, and glycosylated ceramides together with increased levels of diglycerides, acylcarnitines, and free cholesterol after exercise training. In addition to specific lipidome alterations in each group, this study showed concomitant modulation of several glycerophospholipids and sphingolipids suggesting health-promoting effects of short-term high-intensity exercise training. Collectively, these modulated lipid species represent interesting targets for future lipidomic-based studies evaluating not only the effects of exercise training, but also the molecular mechanisms resulting in a healthier plasma lipidome profile.

Axial elongation and sequential segmentation are developmental processes that occur simultaneously and are highly conserved in vertebrates and most arthropods. These features rely on the dynamic expression of a genetic network that establishes the segmented patterning and regulates various cellular behaviors, including tissue rearrangements and cell divisions. In vertebrates, Wnt and Fgf signaling are essential for these processes. While some studies in arthropods have linked these pathways to segmentation, there is still much to discuss regarding their regulatory role in cellular processes. In this study, we pharmacologically inhibited Wnt and Fgf signaling pathways by exposing developing Tribolium castaneum embryos to IWP-3 and SU5402, respectively. We observed that both treatments resulted in a shortening of the embryos and a decrease in the number of cell divisions during a period characterized by high proliferation rates. Although the segmented patterning was not disrupted, the segments were smaller in the embryos treated with the Fgf inhibitor than in the controls. Additionally, time-lapse imaging revealed that cell movement along the anteroposterior axis was affected in the IWP-3-treated embryos. In contrast, Fgf inhibition primarily altered the direction of cell movements at the posterior end of the embryo. Our findings provide insight into the roles of Wnt and Fgf signaling pathways in regulating significant cellular behaviors during the posterior growth of Tribolium and possibly other arthropods.

The production of an adequate number of gametes in both sexes is necessary for normal reproduction, for which the regulation of proliferation from early gonadal development to adulthood is key. Cystic proliferation of embryonic stem germ cells prior the onset of gametogenesis is an especially important step prior to the beginning of meiosis. However, in vertebrates, the molecular regulators of cystic proliferation remain unknown. Here, we report that ndrg1b, a member of the N-myc downstream regulated family, is an important regulator of cystic proliferation in medaka. We generated mutants of ndrg1b that led to a disruption of proliferation type II, independently of the TGF-{beta} signaling pathway. This loss of cystic proliferation was observed from embryogenic to adult stages, impacting the success of gamete production and reproductive parameters such as spawning and fertilization. Interestingly, the depletion of cystic proliferation of the ndrg1b mutant also impacted male sexual behavior, with a decrease of mating vigor. These data illustrate why it is also necessary to consider gamete production capacity in order to analyze reproductive behavior. HIGHLIGHTSO_LINdrg1b is involved in the regulation of cystic proliferation in gonad from embryo to adulthood. C_LIO_LIThe cystic proliferation is independently of the TGF-{beta} signaling pathway. C_LIO_LIDecrease of production of gametes declines reproductive success for both sexes. C_LIO_LIReduction of cystic proliferation declines male sexual behavior, with a decrease of mating vigor. C_LI

Y box-binding protein 1 (YB-1; Ybx1/ybx1) regulates transcription and translation of targeted genes through DNA/RNA-binding. Our research in zebrafish has revealed a high abundance of Ybx1 in the primary growth (PG) follicles in the ovary, which decreases precipitously as the follicles enter the secondary growth (SG) phase. To understand the function of Ybx1 in folliculogenesis, we created an ybx1 mutant using TALEN and observed a disruption in folliculogenesis in the mutant (ybx1-/-) during the transition from previtellogenic (PV) to early vitellogenic (EV) stage of the SG phase, resulting in underdeveloped ovaries and reduced female fertility. Transcriptome and Western blot analyses identified several differentially expressed genes between mutant (ybx1-/-) and control (ybx1+/-) ovaries. Notably, the expression of cdkn1a (p21), a cell cycle inhibitor, increased dramatically in ybx1-/- follicles. Disrupting cdkn1a gene with CRISPR/Cas9 resulted in embryonic lethality. In p21 heterozygote (cdkn1a+/-), however, follicle activation and maturation in the ovary were both advanced, contrasting with the ybx1-/- mutant. Interestingly, partial loss of p21 could alleviate the phenotype of ybx1-/-. Folliculogenesis resumed in ybx1-/-;p21+/- females with normal follicle activation (PG-PV transition) and vitellogenic growth (PV-EV transition). Interestingly, the follicle cells from the ybx1-/- mutant displayed a poor proliferative activity both in vivo and in vitro; however, the cells from the ybx1-/-;p21+/- follicles resumed normal proliferation. In conclusion, our study suggests that Ybx1 serves a pivotal role in controlling early folliculogenesis in zebrafish, and its acts, at least partly, by repressing the expression of cdkn1a, a cell cycle inhibitor.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by qRT-PCR of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathway. To evaluate the role of WNT on hAE competence, we exposed early day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental features of bovine hAE. Summary statementThis study shows the importance of the WNT pathway on bovine haploid androgenetic development by walking through transcriptomics and pluripotency markers associated with cell fate determination during early development.

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.