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In Brief: During zygotic genome activation, thousands of genes are activated, and those pre-mRNAs must be accurately spliced to support the production of functional proteins. This study shows that SON is necessary for proper nuclear speckle organization, pre-mRNA splicing, transcriptome establishment, and histone methylation in mouse preimplantation embryos., Abstract: Thousands of genes are activated in late two-cell embryos, which means that numerous pre-mRNAs are generated during this time. These pre-mRNAs must be accurately spliced to ensure that the mature mRNAs are translated into functional proteins. However, little is known about the roles of pre-mRNA splicing and the cellular factors modulating pre-mRNA splicing during early embryonic development. Here, we report that downregulation of SON, a large Ser/Arg (SR)-related protein, reduced embryonic development and caused deficient blastomere cleavage. These embryonic developmental defects result from dysregulated nuclear speckle organization and pre-mRNA splicing of a set of cell cycle-related genes. Furthermore, SON downregulation disrupted the transcriptome (2128 upregulated and 1399 downregulated) in four-cell embryos. Increased H3K4me3, H3K9me3, and H3K27me3 levels were detected in four-cell embryos after SON downregulation. Taken together, these results demonstrate that accurate pre-mRNA splicing is essential for early embryonic development and that SON plays important roles in nuclear speckle organization, pre-mRNA splicing, transcriptome establishment, and histone methylation reprogramming during early embryonic development.

In Brief: Bovine embryos lacking SMC2 (a core component of condensins I and II) are unable to survive maternal recognition of pregnancy. SMC2 KO embryos are able to form blastocysts, exhibiting a reduced cell proliferation ability, and arrest their development shortly after hatching., Abstract: Condensins are large protein complexes required for chromosome assembly and segregation during mitosis and meiosis. Mouse or bovine embryos lacking SMC2 (a core component of condensins I and II) do not complete development to term, but it is unknown when they arrest their development. Herein, we have assessed the developmental ability of bovine embryos lacking SMC2 due to a naturally occurring mutation termed HH3 (Holstein Haplotype 3) or by CRISPR-mediated gene ablation. To determine if embryos homozygous for the HH3 allele survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. Mendelian inheritance of the HH3 allele was observed at E14 conceptuses but conceptuses homozygous for HH3 failed to achieve elongation and lacked an embryonic disc. To assess the consequence of the ablation of condensins I and II at earlier developmental stages, SMC2 KO bovine embryos were generated in vitro using CRISPR technology. SMC2 KO embryos were able to form blastocysts but exhibited reduced cell proliferation as evidenced by a significantly lower number of total, trophectoderm (CDX2+), and inner cell mass (SOX2+) cells at Day (D) 8 post-fertilization compared to their WT counterparts and were unable to survive to D12 in vitro. SMC2 ablation did not alter relative telomere length at D8, D12, or E14. In conclusion, condensins I and II are required for blastomere mitosis during early development, and embryos lacking those complexes arrest their development shortly after blastocyst hatching.

In Brief: In the present study the sustainable effect of L-carnitine during the culture period on the post-transfer development was investigated. Taken together, we uncovered direct effects of L-carnitine on the bioenergetic profile of day 7 blastocysts along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos., Abstract: L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid-free conditions, one group with LC (LC embryos) and the control group without LC (control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates as regards early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analyses via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP-dependent activity, cellular stress, and respiration. Pathways like oxidative phosphorylation and thermogenesis, extracellular matrix receptor signaling, PI3K-Akt, and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in LC embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC embryos. In summary, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

In Brief: Proper early embryonic development in mammals relies on precise cellular signaling pathways. This study reveals that NSUN5 is crucial for the regulation of the Hippo pathway, ensuring normal proliferation and differentiation in mouse preimplantation embryos., Abstract: NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the two-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C's important role in stabilizing rRNA and mRNA and the Hippo signaling pathway's critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.

In Brief: A ketogenic diet (KD) elevates blood β-hydroxybutyrate to concentrations that are known to perturb the development, metabolism, histone acetylation and viability of preimplantation mouse embryos in culture. This study shows that a maternal KD changes available nutrient levels in the oviduct, leading to altered embryo development and epigenetic state in vivo., Abstract: A ketogenic diet elevates blood β-hydroxybutyrate to concentrations that perturb the development, metabolism, histone acetylation (H3K27ac) and viability of preimplantation mouse embryos in vitro. However, whether a ketogenic diet alters β-hydroxybutyrate concentrations within female reproductive fluid is unknown. This study aimed to quantify glucose and β-hydroxybutyrate within mouse blood and oviduct fluid following standard diet and ketogenic diet consumption and to assess whether a maternal periconceptional ketogenic diet impacts in vivo embryo development and blastocyst H3K27ac. Female C57BL/6 × CBA mice were fed a standard or ketogenic diet (n = 24 each) for 24-27 days. Glucose and β-hydroxybutyrate were quantified in blood via an electronic monitoring system and in oviduct fluid via ultramicrofluorescence. The developmental grade of flushed blastocysts was recorded, and blastocyst cell number and H3K27ac were assessed via immunofluorescence. A maternal ketogenic diet elevated β-hydroxybutyrate in day 24 blood (P < 0.001) and oviduct fluid (P < 0.05) compared with a standard diet, whereas glucose was unchanged. A periconceptional ketogenic diet did not impact blastocyst cell number; however, it significantly delayed blastocyst development (P < 0.05) and reduced trophectoderm-specific H3K27ac (P < 0.05) compared with standard diet-derived embryos. Maternal ketogenic diet consumption is, therefore, associated with reproductive tract nutrient changes and altered embryonic development and epigenetics in vivo. Future studies to assess whether periconceptional/gestational ketogenic diet consumption impacts human preimplantation, fetal, and long-term offspring development and health are warranted.

In Brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching., Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

In Brief: HSP90AA1 is a ubiquitous molecular chaperone that can resist cellular stress, such as oxidative stress and apoptosis, and mediate the efficacy and protein folding of normal cells during heat stress, as well as many other functions. This study further reveals the role of HSP90AA1 in bovine oocyte maturation and early embryonic development., Abstract: HSP90AA1, a highly abundant and ubiquitous molecular chaperone, plays important roles in various cellular processes including cell cycle control, cell survival, and hormone signaling pathways. In this study, we investigated the functions of HSP90AA1 in bovine oocyte and early embryo development. We found that HSP90AA1 was expressed at all stages of development, but was mainly located in the cytoplasm, with a small amount distributed in the nucleus. We then evaluated the effect of HSP90AA1 on the in vitro maturation of bovine oocytes using tanespimycin (17-AAG), a highly selective inhibitor of HSP90AA1. The results showed that inhibition of HSP90AA1 decreased nuclear and cytoplasmic maturation of oocytes, disrupted spindle assembly and chromosome distribution, significantly increased acetylation levels of α-tubulin in oocytes and affected epigenetic modifications (H3K27me3 and H3K27ac). In addition, H3K9me3 was increased at various stages during early embryo development. Finally, the impact of HSP90AA1 on early embryo development was explored. The results showed that inhibition of HSP90AA1 reduced the cleavage and blastocyst formation rates, while increasing the fragmentation rate and decreasing blastocyst quality. In conclusion, HSP90AA1 plays a crucial role in bovine oocyte maturation as well as early embryo development.

In Brief: Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle., Abstract: Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

In Brief: Peroxisome proliferator-activated receptor gamma (PPARG) is a critical regulator of placental function, but earlier roles in preimplantation embryo development and embryonic origins of placental formation have not been established. Results herein demonstrate that PPARG responds to pharmacologic stimulation in the bovine preimplantation embryo and influences blastocyst development, cell lineage specification, and transcripts important for placental function., Abstract: Peroxisome proliferator-activated receptor gamma (PPARG) is a key regulator of metabolism with conserved roles that are indispensable for placental function, suggesting previously unidentified and important roles in preimplantation embryo development. Herein, we report the functional characterization of bovine PPARG to reveal expression beginning on D6 of development with nuclear and ubiquitous patterns. Day 6 PPARG+ embryos have fewer total cells and a lower proportion of trophectoderm cells compared to PPARG- embryos (P < 0.05). Coculture with a PPARG agonist, rosiglitazone (Ros), or antagonist GW9662 (GW), decreases blastocyst development (P < 0.01). Day 7.5 (D7.5) developmentally delayed embryos exposed to Ros express lower transcript abundance of key genes important for placental development and cell lineage formation (CDX2, RXRB, SP1, TFAP2C, SIRT1, and PTEN). In contrast, Ros does not alter transcript abundance in D7.5 blastocysts, but GW treatment lowers RXRA, RXRB, SP1, and NFKB1 expression. Knockout of embryonic PPARG does not alter blastocyst formation and hatching ability but decreases total cell number in D7.5 blastocysts. The decreased embryo development response and affected pathways following targeted pharmacological perturbation vs embryonic knockout of PPARG suggest roles of both maternal and embryonic origins. These data reveal regulatory contributions of PPARG in preimplantation embryo development, cell lineage formation, and regulation of transcripts associated with placental function.

In Brief: It is not known when a functional circadian clock is established in the developing embryo. Lack of expression of key genes involved in the clock mechanism is indicative that a functional circadian clock mechanism is absent in the mammalian preimplantation embryo through the blastocyst stage of development., Abstract: An embryonic circadian clock could conceivably organize cellular and developmental events temporally and in synchrony with other circadian rhythms in the mother. The hypothesis that a functional molecular clock exists in the preimplantation bovine, pig, human, and mouse embryo was tested by using publicly available RNAseq datasets to examine developmental changes in expression of the core genes responsible for the circadian clock - CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2. In general, the transcript abundance of each gene decreased as development advanced to the blastocyst stage. The most notable exception was for CRY2, where transcript abundance was low and constant from the two-cell or four-cell to the blastocyst stage. Developmental patterns were generally the same for all species although there were some species-specific patterns such as an absence of PER1 expression in the pig, an increase in ARNTL expression at the four-cell stage in human, and an increase in expression of Clock and Per1 from the zygote to two-cell stage in the mouse. Analysis of intronic reads (indicative of embryonic transcription) for bovine embryos indicated an absence of embryonic transcription. Immunoreactive CRY1 was not detected in the bovine blastocyst. Results indicate that the preimplantation mammalian embryo lacks a functional intrinsic clock although specific components of the clock mechanism could conceivably play a role in other functions in the embryo.

In Brief: Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos., Abstract: Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.

In Brief: Several factors affect the reprogramming efficiency of nuclear transfer embryos. This study shows that inhibiting 18S rRNA m6A methyltransferase METTL5 during nuclear transfer can improve the developmental rate of nuclear transfer embryos., Abstract: N6-methyladenosine (m6A) is one of the most important epigenetic modifications in eukaryotic RNAs, which regulates development and diseases. It is identified by several proteins. Methyltransferase-like 5 (METTL5), an enzyme that methylates 18S rRNA m6A, controls the translation of proteins and regulates pluripotency in embryonic stem cells. However, the functions of METTL5 in embryonic development have not been explored. Here, we found that Mettl5 was upregulated in somatic cell nuclear transfer (SCNT) embryos compared with normal fertilized embryos. Therefore, we hypothesized that METTL5 knockdown during the early stage of SCNT would improve the developmental rate of SCNT embryos. Notably, injection of Mettl5 siRNA (si-Mettl5) into enucleated oocytes during nuclear transfer increased the rate of development and the number of cells in blastocysts. Moreover, inhibition of METTL5 reduced the activity of phosphorylated ribosomal protein S6, decreased the levels of the repressive histone modification H3K27me3 and increased the expression of activating histone modifications H3K27ac and H3K4me3 and mRNA levels of some 2-cell-specific genes. These results expand our understanding of the role of METTL5 in early embryonic development and provide a novel idea for improving the efficiency of nuclear transfer cloning.

In Brief: Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation., Abstract: The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes.

Cryopreservation is a process in which the intact living cells, tissues, or embryos are preserved at subzero temperatures for preservation. The cryopreservation process highly impacts the survival and quality of the in vitro-produced (IVP) embryos. Some studies have highlighted the use of oviduct extracellular vesicles (EVs) to improve the cryotolerance of IVP embryos but the mechanism has not been well studied. The present study unravels the role of in vitro cultured bovine oviduct epithelial cells-derived EVs in improving the re-expansion and hatching potential of thawed blastocysts (BLs). The comparison of cryotolerance between synthetic oviduct fluid (SOF) and SOF + EVs-supplemented day-7 cryopreserved BLs revealed that the embryo's ability to re-expand critically depends on the intact paracellular sealing which facilitates increased fluid accumulation during cavity expansion after shrinkage. Our results demonstrated that BLs cultured in the SOF + EVs group had remarkably higher re-expansion (67.5 ± 4.2%) and hatching rate (84.8 ± 1.4%) compared to the SOF group (53.4 ± 3.4% and 63.9 ± 0.9%, respectively). Interestingly, EVs-supplemented BLs exhibited greater influence on the expression of core genes involved in trophectoderm (TE) maintenance, formation of tight junction (TJ) assembly, H2O channel proteins (aquaporins), and Na+/K+ ATPase alpha 1. The EVs improved the fluid flux and allowed the transport of H2O into an actively re-expanded cavity in EVs-cultured cryo-survived BLs relative to control BLs. Our findings explored the function of EVs in restoring the TE integrity, improved the cell junctional contacts and H2O movement which helps the blastocoel re-expansion after thawing the cryopreserved BLs.

As obese and overweight patients commonly display hyperlipidemia and are increasingly accessing fertility clinics for their conception needs, our studies are directed at understanding the effects of hyperlipidemia on early pregnancy. We have focused on investigating palmitic acid (PA) and oleic acid (OA) treatment alone and in combination from the mouse two-cell stage embryos as a model for understanding their effects on the mammalian preimplantation embryo. We recently reported that PA exerts a negative effect on mouse two-cell progression to the blastocyst stage, whereas OA co-treatment reverses that negative effect. In the present study, we hypothesized that PA treatment of mouse embryos would disrupt proper localization of cell fate determining and blastocyst formation gene products and that co-treatment with OA would reverse these effects. Our results demonstrate that PA treatment significantly (P < 0.05) reduces blastocyst development and cell number but did not prevent nuclear localization of YAP in outer cells. PA treatment significantly reduced the number of OCT4+ and CDX2+ nuclei. PA-treated embryos had lower expression of blastocyst formation proteins (E-cadherin, ZO-1 and Na/K-ATPase alpha1 subunit). Importantly, co-treatment of embryos with OA reversed PA-induced effects on blastocyst development and increased inner cell mass (ICM) and trophectoderm (TE) cell numbers and expression of blastocyst formation proteins. Our findings demonstrate that PA treatment does not impede cell fate gene localization but does disrupt proper blastocyst formation gene localization during mouse preimplantation development. OA treatment is protective and reverses PA's detrimental effects. The results advance our understanding of the impact of FFA exposure on mammalian preimplantation development.

Lipid droplets (LDs) consist of a core of neutral lipids such as triacylglycerols and cholesteryl esters covered by a phospholipid monolayer. Recent studies have shown that LDs not only store neutral lipids but are also associated with various physiological functions. LDs are found in most eukaryotic cells and vary in size and quantity. It has long been known that mammalian oocytes contain LDs. Porcine and bovine oocytes contain substantial amounts of LDs, which cause their cytoplasm to darken, whereas mouse and human oocytes are translucent due to their low LD content. A sufficient amount of LDs in mammalian oocytes has been thought to be associated with oocyte maturation and early embryonic development, but the necessity of LDs has been questioned because embryonic development proceeds normally even when LDs are removed. However, recent studies have revealed that LDs play a crucial role during implantation and that maintaining an appropriate amount of LDs is important for early embryonic development, even in mammalian species with low amounts of LDs in their oocytes. This suggests that a fine-tuned balance of LD content is essential for successful mammalian embryonic development. In this review, we discuss the physiological importance of LDs in mammalian oocytes and preimplantation embryos based on recent findings on LD biology.

The mouse preimplantation embryo is sensitive to its environment, including maternal dietary protein restriction, which can alter the developmental programme and affect lifetime health. Previously, we have shown maternal low-protein diet (LPD) causes a reduction in blastocyst mTORC1 signalling coinciding with reduced availability of branched-chain amino acids (BCAAs) in surrounding uterine fluid. BCAA deficiency leads to increased endocytosis and lysosome biogenesis in blastocyst trophectoderm (TE), a response to promote compensatory histotrophic nutrition. Here, we first investigated the induction mechanism by individual variation in BCAA deficiency in an in vitro quantitative model of TE responsiveness. We found isoleucine (ILE) deficiency as the most effective activator of TE endocytosis and lysosome biogenesis, with less potent roles for other BCAAs and insulin; cell volume was also influential. TE response to low ILE included upregulation of vesicles comprising megalin receptor and cathepsin-B, and the response was activated from blastocyst formation. Secondly, we identified the transcription factor TFEB as mediating the histotrophic response by translocation from cytoplasm to nucleus during ILE deficiency and in response to mTORC1 inhibition. Lastly, we investigated whether a similar mechanism responsive to maternal nutritional status was found in human blastocysts. Blastocysts from women with high body-mass index, but not the method of fertilisation, revealed stimulated lysosome biogenesis and TFEB nuclear migration. We propose TE lysosomal phenotype as an early biomarker of environmental nutrient stress that may associate with long-term health outcomes.

Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

Oocyte vitrification has significantly improved the survival rate and become the mainstream method for cryopreserving oocytes. Previous studies have demonstrated that the ultrastructure, mitochondrial function, DNA methylation, and histone modification exhibit an irreversible effect after oocyte vitrification. However, little is known about the effects of oocyte vitrification on glucose transport and metabolism. This study aims to determine whether mouse oocyte vitrification causes abnormal glucose metabolism and identify a strategy to correct abnormal glucose metabolism. Furthermore, this study further investigates the effects of oocyte vitrification on glucose uptake, and glucose metabolism, and energy levels. The results indicated that vitrification significantly reduced the glucose transport activity, NADPH, glutathione, and ATP levels, and increased reactive oxygen species levels in oocytes (P < 0.01). Vitrification also reduced the expression of glucose transporter isoform 1 (GLUT1) (P < 0.01). Adding a GLUT1 inhibitor reduced the glucose uptake capacity of oocytes. Furthermore, the inclusion of vitamin C into thawing and culture solutions restored abnormal glucose transportation and metabolism and improved the survival, two-cell embryo, and blastocyst rates of the vitrified groups via parthenogenesis (P < 0.05). Overall, this method may improve the quality and efficiency of oocyte vitrification.

Pre-implantation embryos undergo genome-wide DNA demethylation, however certain regions, like imprinted loci remain methylated. Further, the mechanisms ensuring demethylation resistance by TRIM28 in epigenetic reprogramming remain poorly understood. Here, TRIM28 was knocked down in oocytes, and its effects on porcine somatic cell nuclear transfer (SCNT) embryo development was examined. Our results showed that SCNT embryos constructed from TRIM28 knockdown oocytes had significantly lower cleavage (53.9 ± 3.4% vs 64.8 ± 2.7%) and blastocyst rates (12.1 ± 4.3% vs 19.8 ± 1.9%) than control-SCNT embryos. The DNA methylation levels at the promoter regions of the imprinting gene IGF2 and H19 were significantly decreased in the 4-cell stage, and the transcript abundance of other imprinting gene was substantially increased. We also identified an aberrant two-fold decrease in the expression of CXXC1and H3K4me3 methyltransferase (ASH2L and MLL2), and the signal intensity of H3K4me3 had a transient drop in SCNT 2-cell embryos. Our results indicated that maternal TRIM28 knockdown disrupted the genome imprints and caused epigenetic variability in H3K4me3 levels, which blocked the transcription activity of zygote genes and affected the normal developmental progression of porcine SCNT embryos.

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics - Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV-) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV- spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa's DNA integrity, additional damage mechanisms are involved.

Until recently, it has been difficult to derive and maintain stable embryonic stem cells lines from livestock species. Sheep ESCs with characteristics similar to those described for rodents and primates have not been produced. We report the derivation of sheep ESCs under a chemically defined culture system containing fibroblast growth factor 2 (FGF2) and a tankyrase/Wnt inhibitor (IWR1). We also show that several culture conditions used for stabilizing naïve and intermediate pluripotency states in humans and mice were unsuitable to maintain ovine pluripotency in vitro. Sheep ESCs display a smooth dome-shaped colony morphology, and maintain an euploid karyotype and stable expression of pluripotency markers after more than 40 passages. We further demonstrate that IWR1 and FGF2 are essential for the maintenance of an undifferentiated state in de novo derived sheep ESCs. The derivation of stable pluripotent cell lines from sheep blastocysts represents a step forward toward understanding pluripotency regulation in livestock species and developing novel biomedical and agricultural applications.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I arginine methyltransferase that methylates the arginine residues of histone and nonhistone. Carm1 regulates various cellular processes, including transcriptional regulation, mRNA processing, cellular proliferation, and differentiation. Blastomeres with high Carm1 expression levels show cleavage tendency to inner cell mass (ICM) in mouse embryos. However, details about the factors for CARM1 distribution in mouse early embryos and the role of Carm1 in blastocyst development remain unclear. Here, the endonuclear distribution of CARM1 protein was heterogeneous between blastomeres from the late four-cell stage to the blastocyst stage. The heterogeneity of CARM1 distribution in blastomeres at the late four-cell stage was randomly obtained from two-cell stage embryos. From the four-cell stage to morula, CARM1 in individual blastomere remained heterogeneous. In the blastocyst stage, CARM1 protein level in ICM was much higher than that in trophoblast. We found that microRNA (miRNA) miR-181a is an important regulator for Carm1 distribution at the late four-cell stage. The ratio of heterogeneous embryos was reduced in all the embryos when miR-181a was inhibited. CARM1 inhibition reduced the level of symmetrical histone H3 arginine-26 dimethylation and impaired blastocyst development. Silencing Carm1 reduced cell number and increased cell apoptosis at the blastocyst stage. These results show a CARM1 heterogeneous distribution from the four-cell embryos to the blastocysts. miR-181a regulates the control of CARM1 heterogeneous distribution in the four-cell-stage embryos, and CARM1 is an important protein in regulating blastocyst development.

Oviduct fluid is essential for the fertilization and subsequent preimplantation development. Glycine is abundant in oviduct fluid and is reported to be critical for preimplantation development of fertilized eggs in mammals. However, the mechanism by which glycine exerts its action on fertilized eggs is yet to be understood. Here we show that glycine regulates the preimplantation development of mouse fertilized eggs via glycine receptors. Among them, the alpha-4 subunit (Glra4) and the β subunit are expressed in mouse fertilized eggs, and lacking Glra4 inhibits embryonic development to the blastocyst stage, decreases the number of cells in the blastocysts and the litter size. Thus, we identify a novel function of the glycine receptor, which is considered to act mainly as a neurotransmitter receptor, as a regulator of embryonic development and our data provide new insights into the interactions between oviduct milieu and mammalian fertilized egg.

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86-91%) and snoRNA (9-14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

Heat stress compromises bovine oocyte developmental competence, but the effects of high temperature during oocyte maturation on embryo chromatin organization is unknown. In this study bovine oocytes were exposed to heat shock (41°C) for 12 h during in vitro maturation and then submitted to in vitro fertilization. The heat shock did not affect (P > 0.05) the cleavage but reduced (P < 0.01) the blastocyst rate on Day 7 and Day 8. No effect (P > 0.05) on total cell number was found, but the heat shock increased (P < 0.05) the proportion of apoptotic cells in blastocysts at Day 8. Immunofluorescence analysis of H3K9me3 and HP1 was performed in embryos at 52 h post in vitro fertilization. An accumulation of H3K9me3 in the nuclei of embryos derived from heat-shocked oocytes at four-cell and eight-cell stages was found. Also, a non-expected higher proportion (P < 0.05) of four-cell stage embryos displaying nuclei with increased HP1 fluorescence was observed, suggesting an abnormal chromatin compaction in embryos from heat-shocked oocytes. Embryos at eight-cell stage derived from heat-shocked oocytes displayed lower (P < 0.05) relative amount of HSP40 transcripts than control ones. In conclusion, heat shock before fertilization has an effect on embryo chromatin, influencing the accumulation of H3K9me3 and HP1 in early embryos as well as further development.

The uterine microenvironment during pre-implantation presents a pro-survival milieu and is essential for embryo elongation in ruminants. The European roe deer (Careolus capreolus) pre-implantation embryo development is characterised by a 4-month period of reduced development, embryonic diapause, after which the embryo rapidly elongates and implants. We investigated the uterine fluid proteome by label-free liquid chromatography tandem mass spectrometry at four defined stages covering the phase of reduced developmental pace (early diapause, mid-diapause and late diapause) and embryo elongation. We hypothesised that embryo development during diapause is halted by the lack of signals that support progression past the blastocyst stage. Three clusters of differentially abundant proteins were identified by a self-organising tree algorithm: (1) gradual reduction over development; (2) stable abundance during diapause, followed by a sharp rise at elongation; and (3) gradual increase over development. Proteins in the different clusters were subjected to gene ontology analysis. 'Cellular detoxification' in cluster 1 was represented by alcohol dehydrogenase, glutathione S-transferase and peroxiredoxin-2. ATP-citrate synthase, nucleolin, lamin A/C, and purine phosphorylase as cell proliferation regulators were found in cluster 2 and 'cortical cytoskeleton', 'regulation of substrate adhesion-dependent cell spreading' and 'melanosome' were present in cluster 3. Cell cycle promoters were higher abundant at elongation than during diapause, and polyamines presence indicates their role in diapause regulation. This study provides a comprehensive overview of proteins in the roe deer uterine fluid during diapause and forms a basis for studies aiming at understanding the impact of the lack of cell cycle promoters during diapause.

Mitochondria, originally of bacterial origin, are highly dynamic organelles that have evolved a symbiotic relationship within eukaryotic cells. Mitochondria undergo dynamic, stage-specific restructuring and redistribution during oocyte maturation and preimplantation embryo development, necessary to support key developmental events. Mitochondria also fulfil a wide range of functions beyond ATP synthesis, including the production of intracellular reactive oxygen species and calcium regulation, and are active participants in the regulation of signal transduction pathways. Communication between not only mitochondria and the nucleus, but also with other organelles, is emerging as a critical function which regulates preimplantation development. Significantly, perturbations and deficits in mitochondrial function manifest not only as reduced quality and/or poor oocyte and embryo development but contribute to post-implantation failure, long-term cell function and adult disease. A growing body of evidence indicates that altered availability of metabolic co-factors modulate the activity of epigenetic modifiers, such that oocyte and embryo mitochondrial activity and dynamics have the capacity to establish long-lasting alterations to the epigenetic landscape. It is proposed that preimplantation embryo development may represent a sensitive window during which epigenetic regulation by mitochondria is likely to have significant short- and long-term effects on embryo, and offspring, health. Hence, mitochondrial integrity, communication and metabolism are critical links between the environment, the epigenome and the regulation of embryo development.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 h. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific enzyme-linked immunosorbent assay (ELISA). The biological activity was assessed by quantifying the response of interferon-stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT-specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

The POU5F1 gene encodes one of the 'core' transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with POU5F1 expression and function in in vitro -derived cell lines. Also, its permanent inactivation in differentiated cells depends on de novo methylation of its promoter. However, little is known about the epigenetic regulation of POU5F1 expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the POU5F1 5' upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation and the gradual repression of POU5F1 Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development., (© 2018 Society for Reproduction and Fertility.)

Initial stages of implantation involve bi-directional molecular crosstalk between the blastocyst and endometrium. This study investigated an association between infertility etiologies, specifically advanced maternal age (AMA) and endometriosis, on the embryo-endometrial molecular dialogue prior to implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts ( n  = 41 ≥ Grade 3BB) donated from patients presenting with AMA or endometriosis, compared to fertile donor oocyte controls. Extracellular vesicles isolated from co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Specifically, AMA resulted in 16 miRNAs with increased expression ( P  ≤ 0.05) and strong evidence for negative regulation toward 206 target genes. VEGFA , a known activator of cell adhesion, displayed decreased expression ( P  ≤ 0.05), validating negative regulation by 4 of these increased miRNAs: miR-126; 150; 29a; 29b ( P  ≤ 0.05). In endometriosis patients, a total of 10 significantly altered miRNAs displayed increased expression compared to controls (miR-7b; 9; 24; 34b; 106a; 191; 200b; 200c; 342-3p; 484) ( P  ≤ 0.05), targeting 1014 strong evidence-based genes. Three target genes of miR-106a ( CDKN1A , E2F1 and RUNX1 ) were independently validated. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation ( P  ≤ 0.05). These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue and may be prospective biomarkers of implantation success. One of the limitations of this study is that it was a stimulated, in vitro model and therefore may not accurately reflect the in-vivo environment., (© 2018 Society for Reproduction and Fertility.)

For many years, modifications of the uterine extracellular matrix (ECM) during gestation have not been considered as critical for successful canine ( Canis lupus familiaris ) pregnancy. However, previous reports indicated an effect of free-floating blastocysts on the composition of the uterine ECM. Here, the expression of selected genes involved in structural functions, cell-to-cell communication and inhibition of matrix metalloproteinases were targeted utilizing qPCR and immunohistochemistry. We found that canine free-floating embryos affect gene expression of FN1 , ECM1 and TIMP4 This seems to be associated with modulation of trophoblast invasion, and proliferative and adhesive functions of the uterus. Although not modulated at the beginning of pregnancy, the decrease of structural ECM components (i.e. COL1 , -3 , -4 and LAMA 2 ) from pre-implantation toward post-implantation at placentation sites appears to be associated with softening of the tissue in preparation for trophoblast invasion. The further decrease of these components at placentation sites at the time of prepartum luteolysis seems to be associated with preparation for the release of fetal membranes. Reflecting a high degree of communication, intercellular cell adhesion molecules are induced following placentation ( Cx26 ) or increase gradually toward prepartum luteolysis ( Cx43 ). The spatio-temporal expression of TIMPs suggests their active involvement in modulating fetal invasiveness, and together with ECM1 , they appear to protect deeper endometrial structures from trophoblast invasion. With this, the dog appears to be an interesting model for investigating placental functions in other species, e.g. in humans in which Placenta accreta appears to share several similarities with canine subinvolution of placental sites (SIPS). In summary, the canine uterine ECM is only moderately modified in early pregnancy, but undergoes vigorous reorganization processes in the uterus and placenta following implantation., (© 2018 Society for Reproduction and Fertility.)

Embryo implantation is a complex process involving synchronised crosstalk between a receptive endometrium and functional blastocysts. Apoptosis plays an important role in this process as well as in the maintenance of pregnancy. In this study, we analysed the expression pattern of programmed cell death 4 ( Pdcd4 ), a gene associated with apoptosis in the mouse endometrium, during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, in situ hybridisation, Western blotting and immunohistochemistry. The results showed that Pdcd4 was increased along with days of pregnancy and significantly reduced at implantation sites (IS) from day 5 of pregnancy (D5). The level of Pdcd4 at IS was substantially lower than that at interimplantation sites (IIS) on D6 and D7. In addition, Pdcd4 expression in the endometrium was reduced in response to artificially induced decidualisation in vivo and in vitro Downregulation of Pdcd4 gene expression in cultured primary stromal cells promoted decidualisation, while upregulation inhibited the decidualisation process by increasing apoptosis. These results demonstrate that Pdcd4 is involved in stromal cell decidualisation by mediating apoptosis and therefore plays a role in embryo implantation in mice., (© 2018 Society for Reproduction and Fertility.)

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O 2 , has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O 2 , and SOF without feeders at 5% or 20% O 2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O 2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O 2 , BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O 2 ., (© 2017 Society for Reproduction and Fertility.)

The first two differentiation events in the embryo result in three cell types - epiblast, trophectoderm (TE) and hypoblast. The purpose here was to identify molecular markers for each cell type in the bovine and evaluate the differences in gene expression among individual cells of each lineage. The cDNA from 67 individual cells of dissociated blastocysts was used to determine transcript abundance for 93 genes implicated as cell lineage markers in other species or potentially involved in developmental processes. Clustering analysis indicated that the cells belonged to two major populations (clades A and B) with two subpopulations of clade A and four of clade B. Use of lineage-specific markers from other species indicated that the two subpopulations of clade A represented epiblast and hypoblast respectively while the four subpopulations of clade B were TE. Among the genes upregulated in epiblast were AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2 Genes overexpressed in hypoblast included ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17 , while genes overexpressed in all four TE populations were ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN The subpopulations of TE varied among each other for multiple genes including the prototypical TE marker IFNT. New markers for each cell type in the bovine blastocyst were identified. Results also indicate heterogeneity in gene expression among TE cells. Further studies are needed to confirm whether subpopulations of TE cells represent different stages in the development of a committed TE phenotype., (© 2017 Society for Reproduction and Fertility.)

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro , the ovarian steroids 17β-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization., (© 2017 Society for Reproduction and Fertility.)

Vitrification of embryos is a routine procedure in IVF ( in vitro fertilization) laboratories. In the present study, we aimed to investigate the effect of vitrification on mouse preimplantation embryo development in vitro , and effect on the epigenetic status of imprinted gene Grb10 in mouse embryos. The blastocyst formation rate for vitrified 8-cell embryos was similar to the non-vitrified 8-cell embryos, whereas the blastocyst hatching rate was lower than that of the non-vitrified group. The expression level of Grb10 major-type transcript decreased significantly in vitrified blastocysts compared with non-vitrified and in vivo blastocysts. Moreover, the global DNA methylation level in 8-cell embryos and blastocysts, and the DNA methylation at CpG island 1 (CGI1) of Grb10 in blastocysts were also significantly decreased after vitrification. In vitro culture condition had no adverse effect, except for on the DNA methylation in Grb10 CGI1. These results suggest that vitrification may reduce the in vitro development of mouse 8-cell embryos and affect the expression and DNA methylation of imprinted gene Grb10 ., (© 2017 Society for Reproduction and Fertility.)

This study examines the impacts of the urokinase-type plasminogen activator (uPA) on the in vitro maturation (IVM) of bovine oocytes. Cumulus-oocyte complexes in IVM medium were treated with uPA, amiloride (an uPA inhibitor), dimethyl sulfoxide (DMSO) or left untreated (control group). After 24 h of IVM, oocytes were recovered for testing or were in vitro fertilized and cultured to the blastocyst stage. The factors examined in all groups were: (i) oocyte nuclear maturation (Hoëscht staining); (ii) oocyte cytoplasmic maturation (cortical granules, CGs, distribution assessed by LCA-FITC); (iii) oocyte and cumulus cell (CC) gene expression (RT-qPCR); and (iv) embryo development (cleavage rate and blastocyst yield). Oocytes subjected to uPA treatment showed rates of nuclear maturation and CG distribution patterns similar to controls ( P  > 0.05), whereas lower rates of oocyte maturation were recorded in the amiloride group ( P  < 0.05). Both in oocytes and CC, treatment with uPA did not affect the transcription of genes related to apoptosis, cell junctions, cell cycle or serpin protease inhibitors. In contrast, amiloride altered the expression of genes associated with cell junctions, cell cycle, oxidative stress and CC serpins. No differences were observed between the control and uPA group in cleavage rate or in blastocyst yield recorded on Days 7, 8 or 9 post-insemination. However, amiloride led to drastically reduced cleavage rate (28.5% vs 83.2%) and Day 9 embryo production (6.0% vs 21.0%) over the rates recorded for DMSO. These results indicate that the proteolytic activity of uPA is needed for successful oocyte maturation in bovine., (© 2017 Society for Reproduction and Fertility.)

During the transit through the oviduct, the early embryo initiates an extensive DNA methylation reprogramming of its genome. Given that these epigenetic modifications are susceptible to environmental factors, components present in the oviductal milieu could affect the DNA methylation marks of the developing embryo. The aim of this study was to examine if culture of bovine embryos with oviductal fluid (OF) can induce DNA methylation changes at specific genomic regions in the resulting blastocysts. In vitro produced zygotes were cultured in medium with 3 mg/mL bovine serum albumin (BSA) or 1.25% OF added at the one- to 16-cell stage (OF1-16), one- to 8-cell stage (OF1-8) or 8- to 16-cell stage (OF8-16), and then were cultured until Day 8 in medium with 3 mg/mL BSA. Genomic regions in four developmentally important genes ( MTERF2 , ABCA7 , OLFM1 , GMDS ) and within LINE-1 retrotransposons were selected for methylation analysis by bisulfite sequencing on Day 7-8 blastocysts. Blastocysts derived from OF1-16 group showed lower CpG methylation levels in MTERF2 and ABCA7 compared with the BSA group. However, CpG sites within MTERF2 , ABCA7 and OLFM1 showed higher methylation levels in groups OF1-8 and OF8-16 than in OF1-16. For LINE-1 elements, higher CpG methylation levels were observed in blastocysts from the OF1-16 group than in the other experimental groups. In correlation with the methylation changes observed, mRNA expression level of MTERF2 was increased, while LINE-1 showed a decreased expression in blastocysts from OF1-16 group. Our results suggest that embryos show transient sensitivity to OF at early stages, which is reflected by specific methylation changes at the blastocyst stage., (© 2017 Society for Reproduction and Fertility.)

A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus-oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane ( P  < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling., (© 2017 Society for Reproduction and Fertility.)

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro -cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 10 5  EV/mL from ampullary or isthmic OF at either 1 × 10 4  g (10 K) or 1 × 10 5  g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P  < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced., (© 2017 Society for Reproduction and Fertility.)

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine., (© 2016 Society for Reproduction and Fertility.)

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming., (© 2016 Society for Reproduction and Fertility.)

DNA methylation and histone modification play important roles in the development of mammalian embryos. Cytochalasin B (CB) is an actin polymerization inhibitor that can significantly affect cell activity and is often used in studies concerning cytology. In recent years, CB is also commonly being used in in vitro experiments on mammalian embryos, but few studies have addressed the effect of CB on the epigenetic modification of embryonic development, and the mechanism underlying this process is also unknown. This study was conducted to investigate the effects of CB on DNA methylation and histone modification in the development of parthenogenetically activated porcine embryos. Treatment with 5 μg/mL CB for 4 h significantly increased the cleavage rate, blastocyst rate and total cell number of blastocysts. However, the percentage of apoptotic cells and the expression levels of the apoptosis-related genes BCL-XL, BAX and CASP3 were significantly decreased. Treatment with CB significantly decreased the expression levels of DNMT1, DNMT3a, DNMT3b, HAT1 and HDAC1 at the pronuclear stage and promoted the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). After CB treatment, the level of AcH3K9 was upregulated and the level of H3K9me3 was downregulated. When combined with Scriptaid and 5-Aza-Cdr, CB further improved the embryonic development competence and decreased the expression of BCL-XL, BAX and CASP3 In conclusion, these results suggest that CB could improve embryonic development and the quality of the blastocyst by improving the epigenetic modification during the development of parthenogenetically activated embryos., (© 2016 Society for Reproduction and Fertility.)