Cell Stage Research Articles

Effects of cumulus cells on the in vitro cytoplasmic maturation of immature oocytes in pigs

The exposure of cumulus cells to nuclear matured oocytes can be regulated through the forced delay of nuclear maturation or the alteration of in vitro maturation (IVM) time in cumulus-oocyte complexes (COCs). However, to date, no evidence has been presented for the enhancement of cytoplasmic maturation by them, indicating irrelevance of cumulus cells in cytoplasmic maturation. Therefore, in order to identify the requirement of cumulus cells in achieving the cytoplasmic maturation of immature oocytes, this study investigated the effects of cumulus cells on the in vitro cytoplasmic maturation of oocytes within COCs derived from porcine medium antral follicles (MAFs) post-the completion of nuclear maturation. For these, with IVM of COCs for 44 h (control), cumulus cell-free oocytes with completed nuclear maturation were in-vitro-matured additionally for 0, 6, or 12 h, and then a variety of factors representing the cytoplasmic maturation of oocytes were analyzed and compared. As the results, the IVM of COCs for 32 h showed complete nuclear maturation and incomplete cytoplasmic maturation. Moreover, after the removal of cumulus cells from COCs with the completion of nuclear maturation, IVM for an additional 6 or 12 h resulted in significant increases in the size of the perivitelline space, the proportion of oocytes with a normal intracellular mitochondrial distribution and a normal round first polar body, and the preimplantation development into the 2-cell and blastocyst stages after parthenogenetic activation. Simultaneously, they showed significant reduction in the level of intracellular reactive oxygen species and no significant differences in the total number of blastocysts. Furthermore, oocytes obtained by this approach did not significantly differ from control oocytes produced by IVM of COCs for 44 h. Our results demonstrate that the cumulus cells enclosing COCs derived from porcine MAFs are not essential for the completion of cytoplasmic maturation after complete nuclear maturation by COCs.

Transcriptome analysis of thermomorphogenesis in ovules and during early seed development in Brassica napus

BackgroundPlant sexual reproduction is highly sensitive to elevated ambient temperatures, impacting seed development and production. We previously phenotyped this effect on three rapeseed cultivars (DH12075, Topas DH4079, and Westar). This work describes the transcriptional response associated with the phenotypic changes induced by heat stress during early seed development in Brassica napus.ResultsWe compared the differential transcriptional response in unfertilized ovules and seeds bearing embryos at 8-cell and globular developmental stages of the three cultivars exposed to high temperatures. We identified that all tissues and cultivars shared a common transcriptional response with the upregulation of genes linked to heat stress, protein folding and binding to heat shock proteins, and the downregulation of cell metabolism. The comparative analysis identified an enrichment for a response to reactive oxygen species (ROS) in the heat-tolerant cultivar Topas, correlating with the phenotypic changes. The highest heat-induced transcriptional response in Topas seeds was detected for genes encoding various peroxidases, temperature-induced lipocalin (TIL1), or protein SAG21/LEA5. On the contrary, the transcriptional response in the two heat-sensitive cultivars, DH12075 and Westar, was characterized by heat-induced cellular damages with the upregulation of genes involved in the photosynthesis and plant hormone signaling pathways. Particularly, the TIFY/JAZ genes involved in jasmonate signaling were induced by stress, specifically in ovules of heat-sensitive cultivars. Using a weighted gene co-expression network analysis (WGCNA), we identified key modules and hub genes involved in the heat stress response in studied tissues of either heat-tolerant or sensitive cultivars.ConclusionsOur transcriptional analysis complements a previous phenotyping analysis by characterizing the growth response to elevated temperatures during early seed development and reveals the molecular mechanisms underlying the phenotypic response. The results demonstrated that response to ROS, seed photosynthesis, and hormonal regulation might be the critical factors for stress tolerance in oilseed rape.

Humidified atmosphere in a time-lapse embryo culture system does not improve ongoing pregnancy rate: a retrospective propensity score model study derived from 496 first ICSI cycles.

To investigate whether high relative humidity conditions (HC), when using a time-lapse system (TLS) with sequential culture media, are beneficial to embryo culture, improving ongoing pregnancy rates. We included patients undergoing their first ICSI cycle treatment from April 2021 to May 2022. Patients assigned to dry conditions (DC) or HC were 278 and 218, respectively. We used a GERI TLS, three chambers configured in humidity conditions and three in dry conditions. The effect of HC on ongoing pregnancy rate was assessed by the propensity matched sample, to reduce potential differences between women undergoing either HC or DC and reduce biased estimation of treatment effect. After adjusting for several confounding variables and applying the propensity score (PS), no significant differences were observed in the rates of normal (2PN) and abnormal (1PN and 3PN) fertilization, blastulation, top-quality blastocysts, frozen blastocysts, ongoing pregnancies, and miscarriages. The 2-cell (t2) and 4-cell (t4) stages and cell divisions between such stages occurred earlier and were more synchronous in the in DC. These results suggest that HC conditions do not improve the rate of ongoing pregnancy and several embryological outcomes, under the conditions used in this study based on a time-lapse system and sequential culture with day 3 medium change-over.

Human oocyte area is associated with preimplantation embryo usage and early embryo development: the Rotterdam Periconception Cohort

PurposeTo investigate the association between oocyte area and fertilization rate, embryo usage, and preimplantation embryo development in order to establish if oocyte area can be a marker for optimal early embryo development.MethodsFrom 2017 to 2020, 378 couples with an indication for IVF (n = 124) or ICSI (n = 254) were included preconceptionally in the Rotterdam Periconception Cohort. Resulting oocytes (n = 2810) were fertilized and submitted to time-lapse embryo culture. Oocyte area was measured at the moment of fertilization (t0), pronuclear appearance (tPNa), and fading (tPNf). Fertilization rate, embryo usage and quality, and embryo morphokinetics from 2-cell stage to expanded blastocyst stage (t2-tEB) were used as outcome measures in association with oocyte area. Oocytes were termed “used” if they were fertilized and embryo development resulted in transfer or cryopreservation, and otherwise termed “discarded”. Analyses were adjusted for relevant confounders.ResultsOocyte area decreased from t0 to tPNf after IVF and ICSI, and oocytes with larger area shrank faster (β − 12.6 µm2/h, 95%CI − 14.6; − 10.5, p < 0.001). Oocytes that resulted in a used embryo were larger at all time-points and reached tPNf faster than oocytes that fertilized but were discarded (oocyte area at tPNf in used 9864 ± 595 µm2 versus discarded 9679 ± 673 µm2, p < 0.001, tPNf in used 23.6 ± 3.2 h versus discarded 25.6 ± 5.9 h, p < 0.001). Larger oocytes had higher odds of being used (oocyte area at tPNf ORused 1.669, 95%CI 1.336; 2.085, p < 0.001), were associated with faster embryo development up to the morula stage (e.g., t9 β − 0.131 min, 95%CI − 0.237; − 0.025, p = 0.016) and higher ICM quality.ConclusionOocyte area is an informative marker for the preimplantation development of the embryo, as a larger oocyte area is associated with higher quality, faster developing embryos, and higher chance of being used. Identifying determinants associated with oocyte and embryo viability and quality could contribute to improved preconception care and subsequently healthy pregnancies.

Construction of fluorescent transgenic zebrafish Tg (ttn.2: EGFP)

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.

Knock-down of YME1L1 induces mitochondrial dysfunction during early porcine embryonic development.

YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome c, a pro-apoptotic signal, was released from the mitochondria after the knock-down of YME1L1. Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.

Mitochondrial activity and redox status in oocytes from old mice: The interplay between maternal and postovulatory aging

Maternal aging has been reported to reduce oocyte quality and, in turn, lower the developmental potential of the resulting embryos. Here, we show that maternally aged oocytes display two strikingly different phenotypes: some have normal morphology, whereas others have significantly shrunk cytoplasm. The latter phenotype usually prevails in aged females. Our objective was to characterize both types of maternally aged oocytes and investigate the origins of this diversity. Importantly, our experiments indicate that shrunk maternally aged oocytes are severely compromised in terms of mitochondrial functionality as compared to their young or morphologically normal maternally aged counterparts: they display significantly decreased mitochondrial activity and lower amounts of ROS. In contrast, morphologically normal maternally aged oocytes had the same mitochondrial activity as young ones, while their ROS levels were higher. Surprisingly, the shrunk phenotype was completely absent in maternally aged oocytes that matured in vitro, suggesting that it is not caused inherently by maternal aging, but may be related to other factors, like postovulatory aging. Indeed, an additional culture of in vitro matured young and old oocytes (i.e., in vitro postovulatory aging) significantly decreased their mitochondrial activity and led to cytoplasm shrinkage. In vivo postovulatory aging had a similar effect on oocytes from both young and old females. Finally, we examined the developmental potential of oocytes obtained from aged females. Shrunk (i.e., most likely postovulatory aged) oocytes failed to become fertilized, whereas morphologically normal ones (i.e., most likely not subjected to postovulatory aging) underwent fertilization and subsequent cleavage divisions, although they achieved the 2-cell stage less frequently than morphologically normal oocytes from young females. Importantly, the quality of blastocysts as well as the live birth rate for morphologically normal oocytes from old and young females were similar. In summary, our data clearly indicate that two pools of oocytes present in oviducts of aged females differ significantly in their quality and developmental potential and that the more severely affected phenotype results most likely from a synergistic action of maternal and postovulatory aging.

Zygotic Genome Activation by the Totipotency Pioneer Factor Nr5a2

ABSTRACT Oocyte cytoplasm is able to reprogram somatic nuclei to totipotency, suggesting that maternally provided factors reprogram and trigger zygotic genome activation (ZGA) in the embryo. The transcription factors that initiate ZGA are poorly conserved between species and largely unknown in mammals. Pioneer factors bind nucleosomal DNA and are recruited to closed chromatin in vivo, eliciting chromatin opening and ZGA in fruit flies and zebrafish, although it is unknown whether these act in mammals as well. This study aimed to search for orphan nuclear receptors that may function as pioneer factors for ZGA in mice. Transcription profiles of 2 mouse strains during the oocyte-to-embryo transition were studied. A total of 2508 extended ZGA genes were upregulated in 2-cell embryos of the 2 strains, with 985 genes common to both (core ZGA genes). A consensus sequence was identified that was enriched within 8 kb upstream of ~70% of core and extended ZGA gene and contained a sequence correlating to higher chromatin accessibility and histone acetylation during major ZGA. The orphan nuclear receptor Nr5a2 was contained in the CA version of this motif. The Nr5a2 sequence was maternally deposited before ZGA as transcripts in metaphase II eggs and zygotes, and was also strongly expressed in 2-cell embryos. To test its function, Nr5a2 was inhibited by a chemical compound (SR1848) and embryos failed to form blastocysts and fragmented or died at 108 hours postfertilization. This suggested the function of Nr5a2 may be to maintain naive pluripotency in mouse embryonic stem and may be required for pluripotency establishment in vivo. Administration of SR1848 on Nr5a2 after the 2-cell stage resulted in a less severe phenotype. Using single-molecule fluorescence in situ hybridization (ZGA-FISH), it was observed that treatment of zygotes with SR1848 resulted in a dose-dependent reduction of nascent ZGA transcripts, suggesting that Nr5a2 regulates ZGA. Single-embryo RNA sequencing (RNA-seq) of 2-cell embryos resulted in a strong decrease in Nr5a2 abundance in embryos treated with SR1848. In addition, Nr5a2 inhibition by SR1848 resulted in downregulation of several orphan nuclear receptors including its own gene, suggesting that Nr5a2 is required for their expression during ZGA. To establish whether Nr5a2 is required for chromatic accessibility in 2-cell embryos, a microscopy approach was used to quantify open chromatin in single cells. Knockdown and inhibition with siRNA and SR1848, respectively, of Nr5a2 revealed chromatin accessibility was reduced following knockdown compared with controls. Using SeEN-seq (selected engagement on nucleosome sequencing), it was determined that Nr5a2 bound at the entry-exit sites on the nucleosome, very similarly to how the pioneer factors Oct4-Sox2 and GATA3 bind to nucleosomal DNA. Taken together, the results of this study suggest that the orphan nuclear receptor Nr5a2 activates up to 72% of major ZGA genes in mouse embryos and exhibits many properties consistent with pioneer factor activity in vivo and in vitro.

MP01-08 THE TESTIS AND EPIDIDYMIS-SPECIFIC WFDC LOCUS GENES ARE REQUIRED FOR MALE FERTILITY

You have accessJournal of UrologyCME1 Apr 2023MP01-08 THE TESTIS AND EPIDIDYMIS-SPECIFIC WFDC LOCUS GENES ARE REQUIRED FOR MALE FERTILITY Katarzyna Kent, Kimia Bakhshandeh, Kanchan Yadav, Martin Matzuk, Larry Lipshultz, and Thomas Garcia Katarzyna KentKatarzyna Kent More articles by this author , Kimia BakhshandehKimia Bakhshandeh More articles by this author , Kanchan YadavKanchan Yadav More articles by this author , Martin MatzukMartin Matzuk More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Thomas GarciaThomas Garcia More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite 96 million years of evolution separating humans and rodents, 11 closely-related reproductive tract-specific genes in human—SPINT3, WFDC6, EPPIN, WFDC8, WFDC9, WFDC10A, WFDC11, WFDC10B, WFDC13, SPINT4, and WFDC3—and the 13 reproductive tract-specific orthologs in mouse (listed below), form highly conserved syntenic gene clusters indicative of a conserved, combined critical functions. Further, despite significant progress towards a non-hormonal male contraceptive targeting the protein encoded by one of these genes, EPPIN, and associations found between gene mutations in EPPIN and an increased risk of idiopathic male infertility, neither EPPIN nor any of the closely-related genes within the WFDC locus have been explored functionally through genetically manipulated mouse models. To clarify the involvement of these genes in male fertility, we generated and studied mice lacking a combined total of 13, 10, 5, or 4 genes within the conserved gene cluster. METHODS: Custom gRNAs were assembled into ribonucleoprotein complexes with Cas9 nuclease and electroporated into zygotes harvested from superovulated B6D2F1 females. Embryos were cultured overnight to the 2-cell stage before being transferred into the oviducts of pseudopregnant ICR mice. Founder mutations in pups born were identified by PCR and Sanger sequencing. RESULTS: Founder mouse containing a -173.2 kb deletion, corresponding to deletion of all 13 genes (Spint3, Wfdc6a, Eppin, Wfdc8, Wfdc6b, Wfdc16, Wfdc9, Wfdc10, Wfdc11, Wfdc13, Spint4, Spint5, Wfdc3) were expanded for further study. Likewise, founder mice containing either a -115.4 kb deletion (10 genes deleted from Spint3 to Wfdc13), a -46.7 kb deletion (5 genes deleted from Wfdc16 to Wfdc13), and a -28.8 kb deletion (4 genes deleted from Wfdc6a to Wfdc6b) were expanded for further study. We also generated and expanded a single knockout of Wfdc3. The 13 gene, 10 gene, and 4 gene knockouts were all infertile due to a block of spermatogenesis and azoospermia. The 5 gene knockouts were infertile due to asthenozoospermia, while the single knockout of Wfdc3 was fertile with no obvious reproductive phenotype. CONCLUSIONS: Our knockout mouse studies, the first of its kind to study an entire locus of closely related genes, have at last clarified the functional requirement of WFDC locus genes in male fertility, which has implications towards improved clinical diagnosis of men with infertility. Source of Funding: This study has been supported by National Institutes of Health R01 HD106056 and R01 HD095341 (to T.X.G.) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e4 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katarzyna Kent More articles by this author Kimia Bakhshandeh More articles by this author Kanchan Yadav More articles by this author Martin Matzuk More articles by this author Larry Lipshultz More articles by this author Thomas Garcia More articles by this author Expand All Advertisement PDF downloadLoading ...

Sperm-contributed centrioles segregate stochastically into blastomeres of 4-cell stage Caenorhabditis elegans embryos.

Whereas both sperm and egg contribute nuclear genetic material to the zygote in metazoan organisms, the inheritance of other cellular constituents is unequal between the 2 gametes. Thus, 2 copies of the centriole are contributed solely by the sperm to the zygote in most species. Centrioles can have a stereotyped distribution in some asymmetric divisions, but whether sperm-contributed centrioles are distributed in a stereotyped manner in the resulting embryo is not known. Here, we address this question in Caenorhabditis elegans using marked mating experiments, whereby the presence of the 2 sperm-contributed centrioles is monitored in the embryo using the stable centriolar component SAS-4::GFP, as well as GFP::SAS-7. Our analysis demonstrates that the distribution of sperm-contributed centrioles is stochastic in 4-cell stage embryos. Moreover, using sperm from zyg-1 mutant males that harbor a single centriole, we show that the older sperm-contributed centriole is likewise distributed stochastically in the resulting embryo. Overall, we conclude that, in contrast to the situation during some asymmetric cell divisions, centrioles contributed by the male germ line are distributed stochastically in embryos of C. elegans.

Self-transfecting GMO-PMO chimera targeting Nanog enable gene silencing in vitro and suppresses tumor growth in 4T1 allografts in mouse

Phosphorodiamidate morpholino oligonucleotide (PMO)-based antisense reagents cannot enter cells without the help of a delivery technique, which limits their clinical applications. To overcome this problem, self-transfecting guanidinium-linked morpholino (GMO)-PMO or PMO-GMO chimeras have been explored as antisense agents. GMO facilitates cellular internalization and participates in Watson-Crick base pairing. Targeting NANOG in MCF7 cells resulted in decline of the whole epithelial to mesenchymal transition (EMT) and stemness pathway, evident through its phenotypic manifestations, all of which were promulgated in combination with Taxol due to downregulation of MDR1 and ABCG2. GMO-PMO-mediated knockdown of no tail gene resulted in desired phenotypes in zebrafish even upon delivery after 16-cell stages. In BALB/c mice, 4T1 allografts were found to regress via intra-tumoral administration of NANOG GMO-PMO antisense oligonucleotides (ASOs), which was associated with occurrence of necrotic regions. GMO-PMO-mediated tumor regression restored histopathological damage in liver, kidney, and spleen caused by 4T1 mammary carcinoma. Serum parameters of systemic toxicity indicated that GMO-PMO chimeras are safe. To the best of our knowledge, self-transfecting antisense reagent is the first report since the discovery of guanidinium-linked DNA (DNG), which could be useful as a combination cancer therapy and, in principle, can render inhibition of any target gene without using any delivery vehicle.

Evaluate the developmental competence of human 8-cell embryos by single-cell RNA sequencing.

The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes. Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Preimplantation Genetic Testing for Aneuploidy (PGT-A) Reveals High Levels of Chromosomal Errors in In Vivo-Derived Pig Embryos, with an Increased Incidence When Produced In Vitro.

Preimplantation genetic testing for aneuploidy (PGT-A) is widespread, but controversial, in humans and improves pregnancy and live birth rates in cattle. In pigs, it presents a possible solution to improve in vitro embryo production (IVP), however, the incidence and origin of chromosomal errors remains under-explored. To address this, we used single nucleotide polymorphism (SNP)-based PGT-A algorithms in 101 in vivo-derived (IVD) and 64 IVP porcine embryos. More errors were observed in IVP vs. IVD blastocysts (79.7% vs. 13.6% p < 0.001). In IVD embryos, fewer errors were found at blastocyst stage compared to cleavage (4-cell) stage (13.6% vs. 40%, p = 0.056). One androgenetic and two parthenogenetic embryos were also identified. Triploidy was the most common error in IVD embryos (15.8%), but only observed at cleavage, not blastocyst stage, followed by whole chromosome aneuploidy (9.9%). In IVP blastocysts, 32.8% were parthenogenetic, 25.0% (hypo-)triploid, 12.5% aneuploid, and 9.4% haploid. Parthenogenetic blastocysts arose from just three out of ten sows, suggesting a possible donor effect. The high incidence of chromosomal abnormalities in general, but in IVP embryos in particular, suggests an explanation for the low success of porcine IVP. The approaches described provide a means of monitoring technical improvements and suggest future application of PGT-A might improve embryo transfer success.

Mitigating the detrimental developmental impact of early fetal alcohol exposure using a maternal methyl donor-enriched diet.

Fetal alcohol exposure at any stage of pregnancy can lead to fetal alcohol spectrum disorder (FASD), a group of life-long conditions characterized by congenital malformations, as well as cognitive, behavioral, and emotional impairments. The teratogenic effects of alcohol have long been publicized; yet fetal alcohol exposure is one of the most common preventable causes of birth defects. Currently, alcohol abstinence during pregnancy is the best and only way to prevent FASD. However, alcohol consumption remains astoundingly prevalent among pregnant women; therefore, additional measures need to be made available to help protect the developing embryo before irreparable damage is done. Maternal nutritional interventions using methyl donors have been investigated as potential preventative measures to mitigate the adverse effects of fetal alcohol exposure. Here, we show that a single acute preimplantation (E2.5; 8-cell stage) fetal alcohol exposure (2 × 2.5 g/kg ethanol with a 2h interval) in mice leads to long-term FASD-like morphological phenotypes (e.g. growth restriction, brain malformations, skeletal delays) in late-gestation embryos (E18.5) and demonstrate that supplementing the maternal diet with a combination of four methyl donor nutrients, folic acid, choline, betaine, and vitamin B12, prior to conception and throughout gestation effectively reduces the incidence and severity of alcohol-induced morphological defects without altering DNA methylation status of imprinting control regions and regulation of associated imprinted genes. This study clearly supports that preimplantation embryos are vulnerable to the teratogenic effects of alcohol, emphasizes the dangers of maternal alcohol consumption during early gestation, and provides a potential proactive maternal nutritional intervention to minimize FASD progression, reinforcing the importance of adequate preconception and prenatal nutrition.

Pregastrular Development of Amphibians: Ontogenetic Diversity and Eco-Devo

Comparative and ecological aspects of the reorganizations of early development in the class Amphibia are analyzed. We used data on the developmental diversity in a number of families belonging to the orders Anura and Caudata, in which many species had lost their connection with the aquatic environment. Model representatives of the class Amphibia (Ambystoma mexicanum, Rana temporaria, and Xenopus laevis) have small eggs (no more than 2.5 mm in diameter). In these species, the slowdown in the rate of cell divisions and the loss of synchrony occur at the midblastula stage. However, phylogenetically basal amphibian species (Ascaphus truei, Cryptobranchus alleganiensis) are characterized by the large (4–6 mm in diameter) yolky eggs and a short series of synchronous blastomere divisions (the synchrony is already lost at the 8-cell stage of cleavage). They do not have a “midblastula transition”, which is characteristic of the above model species. On the other hand, many evolutionarily advanced non-model species of caudate and anuran amphibians (for example, Desmognathus fuscus, Gastrotheca riobambae, Philoria sphagnicolus), as well as the basal species, are characterized by the large, yolk-rich eggs and the early loss of cell division synchrony. Phylogenetic analysis suggests that the cleavage pattern of the most extensively studied amphibians, the Mexican axolotl (Caudata) and the African clawed frog (Anura), represents a homoplasy. The midblastula transition, which is characteristic of these two species, might have evolved convergently in these two orders of amphibians as an embryonic adaptation to development in lentic water.

P66Shc in sheep preimplantation embryos: Expression and regulation of oxidative stress through the manganese superoxide dismutase-reactive oxygen species metabolic pathway.

p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4- to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

The phase field model can accurately simulate the evolution of microstructures with complex morphologies, and it has been widely used for cell modeling in the last two decades. However, compared to other cellular models such as the coarse-grained model and the vertex model, its high computational cost caused by three-dimensional spatial discretization hampered its application and scalability, especially for multicellular organisms. Recently, we built a phase field model coupled with in vivo imaging data to accurately reconstruct the embryonic morphogenesis of Caenorhabditis elegans from 1- to 8-cell stages. In this work, we propose an improved phase field model by using the stabilized numerical scheme and modified volume constriction. Then we present a scalable phase-field framework, MorphoSim, which is 100 times more efficient than the previous one and can simulate over 100 mechanically interacting cells. Finally, we demonstrate how MorphoSim can be successfully applied to reproduce the assembly, self-repairing, and dissociation of a synthetic artificial multicellular system - the synNotch system.

Chrysoeriol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress and Autophagy

Chrysoeriol (CHE) is a flavonoid substance that exists in many plants. It has various physiological and pharmacological effects, including anti-inflammatory, antioxidant, anti-tumor, and protective activity, especially for the cardiovascular system and liver. Among common livestock embryos, porcine embryos are often considered high-quality objects for studying the antioxidant mechanisms of oocytes. Because porcine embryos contain high levels of lipids, they are more vulnerable to external stimuli, which affect development. Our study explored the influence of CHE supplementation on oxidative stress in porcine oocytes and its possible mechanisms. Different concentrations of CHE (0, 0.1, 1, and 3 µM) were supplemented in the in vitro culture medium of the porcine oocytes. The results showed that supplementation with 1 µM CHE significantly increased the blastocyst rate and total cell number of embryos in vitro. After finding the beneficial effects of CHE, we measured reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (MMP) when the oocytes reached the 4-cell stage of development and determined the levels of apoptosis, cell proliferation, and autophagy at the blastocyst stage of development. The expression levels of some related genes were preliminarily detected by qRT-PCR. The results showed that the apoptosis of blastocysts in the CHE-treated culture also decreased compared with the untreated culture. Furthermore, CHE downregulated intracellular ROS and increased GSH in the embryos. CHE was also shown to improve the activity of mitochondria and inhibit the occurrence of autophagy. In addition, antioxidant-related genes (SOD1, SOD2, and CAT) and cell pluripotency-related genes (SOX2, OCT4, and NANOG) were upregulated. At the same time, apoptosis-related (Caspase 3) and autophagy-related (LC3B) genes showed a downward trend after supplementation with CHE. These results indicate that CHE improved the development of porcine embryos in vitro by reducing oxidative stress and autophagy levels.

Comprehensive characterization of the embryonic factor LEUTX

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

Beneficial Effect of Polysaccharide Gel Made of Xanthan Gum and Locust Bean Gum on Bovine Oocytes.

The present study examined the effect of polysaccharides gels made of xanthan gum and locust bean gum (gel culture system) on oocyte maturation and explored the molecules causing the beneficial effect of the gel culture system. Oocytes and cumulus cells complexes were collected from slaughterhouse-derived ovaries and cultured on a plastic plate or gel. The gel culture system improved the rate of development to the blastocyst stage. The oocytes that matured on the gel contained high lipid contents and F-actin formation, and the resultant 8-cell stage embryos had low DNA methylation levels compared to their plate counterparts. RNA sequencing of the oocytes and embryos revealed the differentially expressed genes between the gel and plate culture systems, and upstream regulator analysis revealed estradiol and TGFB1 as top activated upstream molecules. The medium of the gel culture system contained higher concentrations of estradiol and TGFB1 than that of the plate cultures system. Supplementation of the maturation medium with either estradiol or TGFB1 resulted in high lipid content in oocytes. In addition, TGFB1 improved the developmental ability of the oocytes and increased F-actin content while reducing DNA methylation levels in the 8-cell stage embryos. In conclusion, the gel culture system is useful for embryo production, potentially through the upregulation of TGFB1.