Stages Of Embryo Development Research Articles

Dead-End (dnd) Gene Cloning and Gonad-Specific Expression Pattern in Starry Flounder (Platichthys stellatus).

Simple SummaryThe dead end (dnd) gene encodes an RNA-binding protein that plays a role for migration of primordial germ cells (PGCs) to the gonadal region during embryogenesis in vertebrates. Here, the starry flounder (Platichthys stellatus) dead end (psdnd) gene was characterized and its expression patterns were analyzed. Full-length psdnd mRNA was 1495 bp long, encoding 395 amino acids. psdnd was only expressed in gonadal tissues, we detected no psdnd expression in somatic cells. Furthermore, psdnd was strongly expressed during early embryogenesis. Our findings suggest that psdnd expression is gonad-specific and could therefore be used as a germ cell marker in starry flounder.dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

Abstract 42: Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer

Abstract Chemo-persistent residual tumors are a major barrier for curative cancer therapy and provide a reservoir of cancer cells for eventual relapse. This clinically critical tumor cell population is poorly understood and lacks faithful in vitro models. We compared the transcriptional profiles of paired samples from patient with solid tumors or hematological neoplasias at the stage of post-treatment residual tumors or minimal residual disease vs. at their respective baselines. For a large proportion of patients, post-treatment tumor cells had a molecular signature that resembled that of embryonic diapause, a dormant stage of suspended development in early mammalian embryos triggered by stress and induced by suppression of Myc activity and overall biosynthesis. This Myc-inactivated molecular program was acquired during treatment and not pre-existing at baseline. Remarkably, baseline tumors with multiple MYC copy numbers maintained their MYC amplification in the post-treatment persistent tumor cells but suppressed Myc activity. We simulated this cancer cell state using preclinical models of breast and prostate cancer and multiple myeloma, including patient-derived cancer organoids and PDX models. Parallel approaches of genome sequencing and DNA barcode-mediated clonal tracking in vitro and in vivo independently indicated that the residual cancer cell subpopulations that persisted after treatment with common chemotherapeutic classes were not driven by rare pre-existing clones or de novo genetic events. Transcriptional analysis of the treatment-persistent cancer cells in 3D organoid cultures and in xenograft/PDX models indicated a diapause-like transcriptional adaptation with inactivated Myc, similar to that in patients. We functionally examined the role of Myc suppression in in vitro models of breast cancer and multiple myeloma using genetic (CRISPR/Cas9 editing and interference) and pharmacological (inhibition of Myc transcriptional co-activator Brd4) approaches. Suppression of Myc in cancer cells induced an embryonic diapause-like molecular profile and attenuated the acute chemotherapeutic cytotoxicity. Myc-inactivated cancer cells had reduced apoptotic priming and maintained low redox stress even in the presence of cytotoxic agents, which may contribute to cell survival during treatment. High-throughput screening of chemo-persistent cells revealed that inhibitors of CDK9 could revert the biosynthetic pause, reactivate Myc, attenuate the diapause-like state, and re-sensitize the residual cancer cells to chemotherapy. Overall, our study shows that cancer cells can co-opt the stress survival mechanism of embryonic diapause by dynamically suppressing Myc to enter transient drug-refractory dormancy. Citation Format: Eugen Dhimolea, Ricardo De Matos Simoes, Dhvanir Kansara, Caroline Vilas, Aziz Al'Khafaji, Juliette Bouyssou, Aedin Culhane, Constantine S. Mitsiades. Treatment-induced embryonic diapause-like adaptation through suppression of Myc activity as mediator of drug persistence in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 42.

Characterizing the Low-Dose Effects of Methylmercury on the Early Stages of Embryo Development Using Cultured Human Embryonic Stem Cells.

Background:Global concerns of methylmercury (MeHg) exposure have been raised, especially on its effects on pregnant women. Recent epidemiological studies have revealed associations between maternal blood/hair MeHg concentrations, adverse pregnancy outcomes, and developmental deficits. However, the underlying mechanisms remain unclear.Objectives:In this study, we characterized the effects of MeHg exposure on undifferentiated human embryonic stem cells (hESCs) and extrapolated the effects to human embryonic development.Methods:hESCs were exposed to 0, 1, 5, 10, 50, 100 or MeHg for 24 h or 6 d. Cell adherence and colony formation and expansion were examined under the microscope. Cell attachment, viability/proliferation, apoptosis, stress response, cell cycle, autophagy, and expression of cell lineage marker genes and proteins were measured at the end of exposures.Results:Our results indicated that exposure to nanomolar concentrations of MeHg was associated with a) higher levels of reactive oxygen species (ROS) and hemeoxygenase-1 (HO-1), suggesting increased stress and adaptive responses; b) lower cellular adhesion, viability/proliferation, and colony formation and expansion; c) higher levels of apoptosis, reflected by higher cleaved caspase-3 expression and Annexin V binding; d) higher levels of cytoskeleton protein expression; e) higher rates of G1/S phase cell cycle arrest; and f) autophagy inhibition, as shown by a lower LC3BII/LC3BI ratio and accumulation of SQSTM1 (p62). These outcomes were accompanied by higher expressions of self-renewal genes or proteins or both, including OCT4, SOX2, NANOG, and cytokine receptor IL6ST, as well as pluripotency and the cell fate regulator cyclin D1.Discussion:These results revealed that under the selection pressure of exposure to low doses of MeHg, some hESCs underwent apoptosis, whereas others adapted and survived with enhanced self-renewal gene expression and specific morphological phenotypes. Findings from the present study provide in vitro evidence that low doses of MeHg adversely affect hESCs when exposed during a period of time that models embryonic pre-, during, and early postimplantation stages. https://doi.org/10.1289/EHP7349

Loss of Ahi-1 Impairs the Self-Renewal Activity of Normal and BCR-ABL+ Stem Cells in Vitro and in Vivo

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have identified AHI-1 (Abelson helper integration site-1) oncogene as a potential therapeutic target in CML. It encodes a scaffold protein with a SH3 domain, multiple proline rich motifs and several WD40 repeats. Its expression is highly increased in BCR-ABL+ LSCs. Overexpression of Ahi-1 in primitive hematopoietic cells enhances cell proliferation in vitro and induces leukemia in vivo, and these effects can be enhanced by BCR-ABL. We have also demonstrated that AHI-1 interacts with multiple kinases, including BCR-ABL, β-catenin and JAK2 to mediate LSC activities and TKI resistance in vitro and in vivo . Interestingly, mouse Ahi-1 is highly expressed in hematopoietic stem cells (HSCs, CD45+EPCR+CD48-CD150+ (E-SLAM)) and progenitors (lin-Sca-1+c-kit+ (LSK)), but expressed at low levels in differentiated cells. In addition, Ahi-1 transcripts are found to be expressed in human embryonic stem cells (ES) and at all stages of mouse embryo development, with increasing expression just prior to birth, suggesting that Ahi-1 expression is developmentally regulated. To investigate the precise role of Ahi-1 in regulating normal and LSC self-renewal and differentiation, we have developed a new conditional Ahi-1 knockout mouse model. Ahi-1 deficient mice (AKO) were viable and fertile, but displayed around 30% reduction of bone marrow (BM) and thymus cellularity and altered lineage differentiation as compared to control wild-type mice (AWT). Interestingly, cell cycle analysis showed that loss of Ahi-1 reduced quiescent G0 cells by about 17% in HSC (lin-Sca-1+c-kit+CD48-) as compared to AWT cells. Ahi-1 deficient LSK cells did not exhibit differences in output of colony forming cells (CFCs) but did have dramatically reduced CFC secondary replating efficiency as compared to control cells. In vivo limited dilution assays (LDA), measured by bone marrow transplantation, demonstrated that Ahi-1 loss did not result in significant changes in the frequency of HSC numbers in primary transplanted mice (AWT vs AKO: 1/85000 vs 1/74000), but greatly reduced the reconstitution ability of these cells in serial transplantation assays, since no detectable donor cells were found in tertiary transplanted mice with AKO cells, while engrafted donor cells were still detected in tertiary transplanted mice with AWT cells. These results suggest that Ahi-1 plays a key role in regulating long-term repopulation activity of normal HSCs.The effects of Ahi-1 on LSC growth and maintenance were more profound when BCR-ABL was expressed in Ahi-1-deficient LSCs. Primitive Ahi-1-deficient BM cells transduced with BCR-ABL retroviruses (AKOBA) displayed reduced cell proliferation in the presence of growth factors, especially 14 days after in vtiro culture, compared to BCR-ABL-expressing BM cells from AWT mice (AWTBA). The FACS analysis for these cells after 7 days in culture showed reduced LSK numbers (30%) in AKOBA cells compared to AWTBA control cells. CFC assays revealed a 40% reduction in colonies in AKOBA as compared to AWTBA cells, but the effect was strikingly enhanced in the second and third replating assays (~70% reduction) in AKOBA cells. Most interestingly, we demonstrated that loss of Ahi-1 significantly enhanced survival of leukemic mice generated by crossing of Ahi-1 conditional KO mice with BCR-ABL-transgenic mice as compared to Ahi-1 wild-type/BCR-ABL control mice (65 days vs. 14 days, P=0.008). These results demonstrate that Ahi-1 plays a critical role in regulating HSC functionality and that its deletion perturbs self-renewal and leukemogenesis of BCR-ABL+ LSCs. DisclosuresNo relevant conflicts of interest to declare.

Alternariol exerts embryotoxic and immunotoxic effects on mouse blastocysts through ROS-mediated apoptotic processes.

Alternariol (AOH), a mycotoxin belonging to the genus Alternaria, has been shown to induce cytotoxicity, including apoptosis and cell cycle arrest, in several mammalian cell types. However, its effects on early-stage embryonic development require further investigation. Here, we have shown that AOH exerts embryotoxic effects on mouse blastocyst-stage embryos and long-term adverse effects on immunity in one-day-old newborn mice of the next generation. Significant apoptosis and decrease in total cell number, predominantly through loss of inner cell mass (ICM), and to a minor extent, trophectoderm (TE) cells, were observed in AOH-treated blastocysts. Moreover, AOH exerted detrimental effects on pre- and post-implantation embryo development potential and induced a decrease in fetal weight in in vitro development and embryo transfer assays. Injection of pregnant mice with AOH (1, 3 and 5mg/kg body weight/day) for 4days resulted in apoptosis of blastocyst-stage embryos and injurious effects on embryonic development from the zygote to blastocyst stage or embryo degradation and a further decrease in fetal weight. Furthermore, AOH exerted a long-term impact on the next generation, triggering a significant increase in total oxidative stress content and expression of genes encoding antioxidant proteins. Lower expression of CXCL1, IL-1β and IL-8 related to innate immunity was detected in liver tissue extracts obtained from one-day-old newborns of AOH-injected pregnant mice (5mg/kg body weight/day) relative to their non-treated counterparts. In addition, ROS served as an upstream regulator of AOH-triggered apoptotic processes and impairment of embryonic development. Our collective results highlight the potential of AOH as an embryotoxic and immunotoxic risk factor during embryo and infant development stages in mice.

Human embryo research, stem cell-derived embryo models and invitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

SummaryThe ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport.

Using self-organizing human models of gastrulation, we previously showed that (1) BMP4 initiates the cascade of events leading to gastrulation, (2) BMP4 signal reception is restricted to the basolateral domain, and (3) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically presented NOGGIN could inhibit basally delivered BMP4. Apically imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

Carryover effects of long-term high water temperatures on fitness-related traits of the offspring of the sea urchin Strongylocentrotus intermedius

It is important to study the fitness of marine invertebrates in exposure to high water temperature. We studied whether the long-term high temperatures work on the fitness-related traits (righting behavior, covering behavior, foraging behavior, Aristotle's lantern reflex, body size) of S. intermedius whose parents (males and females) were exposed to ambient or high temperatures (~3 °C higher than the ambient) for a long period of time. The present study found that test diameter, wet body weight and test weight of offspring were not significantly different between temperature treatments, indicating that the parental sea urchins in exposure to high temperatures develop no carryover effects on the body size of the offspring sea urchins. We found no significant difference in foraging behavior, Aristotle's lantern reflex, lantern length and lantern weight of sea urchins after their parents had experienced long-term high temperatures. In addition, no significant change was found in the righting and covering behaviors of sea urchins whose parents were at long-term high temperatures. These results indicate that no significant lasting effects exhibited in the fitness-related behaviors and tissue size after their parents were exposed to high temperatures for a long time. The crushing force of test and test thickness showed no significant difference in the offspring of S. intermedius, no matter whether their parents were exposed to long-term high temperatures or not. The current results enrich our understanding that the parental sea urchin experiencing long-term high temperatures probably develop no carryover effects on the test of their offspring. We found that sea urchins whose parents were exposed to long-term elevated temperatures showed a significantly higher lantern length/test diameter and a significantly lower test height/test diameter in offspring sea urchins due to the thermal experience of their parents, showing the plasticity of lantern and test of offspring sea urchins in response to the thermal experience of their parents. Together with our previous investigation, the present study indicates that small sea urchins are less susceptible to the carryover effects of high temperatures in comparison with the developmental stages of embryos and larvae.

Features of growth and development of egg cross chicken embryos «Lomann Brown»

Purpose:to study morphometric parameters of absolute values of linear and weight body sizes, specific growth rate and relative (allometric) growth of chicken embryos of the «Lohmann Brown» egg cross at different stages of embryogenesis.Materials and methods.The absolute values of linear and weight body sizes of chicken embryos were estimated using morphometric methods. The formula of I. I. Schmalhausen and S. Brody was calculated the specific growth rate of length and body weight of chicken embryos by the formula simple allometry — relative (allometric) growth of body length from body mass.Results.This is manifested in the increase in the specific growth rate of body length of the embryo at 5 days of the late-fetal stage, 8th, 10th, 12th day of the early-fetal stage and specific growth rate of body mass for 6 days of the late-fetal stage of the late-fetal stage, 10-th and 12-th day of the early-fetal stage. At all stages of embryo development, there is a negative allometry of the relative growth rate of the embryo body length, except for 14 days of the mid-fetal stage, where negative isometry was observed (b=-1,000). Higher values of the power coefficient reflecting the slower growth of the embryo in length relative to their body weight, observed in late-fetal stage at 5-6 days (b=0,913-0,995), in early-fetal stage — 10-e (b=0,960) and 12 days (b=0,928), in mid-fetal stage — 13-th (b=0,821) and 15 days (b=0,981) and late-fetal stage — 20 days (b=0,836).Conclusion.New knowledge derived from this study can be applied not only in research, but in the poultry industry to assess the impact of preincubation processing of eggs on the development of embryos and embryonic mortality at different stages of embryogenesis, the definition of normal and abnormal development of embryos, as well as to assess the impact of other factors, artificial incubation on embryo development, hatchability of eggs and safety of poultry.

From flowers to seeds: how the metabolism of flowers frames plant reproduction

Flowers are characterized by a plenitude of primary and secondary metabolites and flower-specific biosynthetic pathways that all concur to promote plant reproduction and the initial stages of embryo development. The floral secondary metabolites of flowers contribute to scent and colour, which are used by flowers to attract pollinators. Besides, many metabolites responsible for the conferral of colour also serve as photo-protectants towards the damaging effects of UV solar radiation. The whole metabolism of flowers is sustained by a network of primary metabolites that provide metabolic precursors for the biosynthesis of secondary metabolites and support flower development. Moreover, many primary metabolites are channelled into nectar, the food of pollinators. However, this complex metabolic network is susceptible to environmental constraints such as heat and drought, which can hamper plant reproduction by destabilizing the whole metabolism of flowers. Here, we provide a short overview of the different metabolic pathways of flowers and how they support pollination and fertilization.

Co-encapsulation of tertinoin and resveratrol by solid lipid nanocarrier (SLN) improves mice in vitro matured oocyte/ morula-compact stage embryo development

As a promising strategy in overcoming drug resistance, the nano drug co-delivery system (NDCDS) can transport two or more drugs into the cell. In this study, we sought to compare the dual and single drug-delivery system, to deliver the optimal dose of Resveratrol (RES) and Tretinoin (TTN) into the in vitro matured oocyte and morula-compact stage embryonic cells. The formation of single (RES/TTN) and dual-drug (RES + TTN)-SLN were confirmed by Uv–vis spectrophotometery, dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) technologies. In two experiments, the oocytes/presumptive zygotes were cultured under various concentrations of the single (RES/TTN) and dual-drug (RES + TTN)-SLN. In vitro toxicity studies, including nuclear staining (Aceto-orcein and Hoechst 33342), H2DCFDA fluorescent staining, chemiluminescence assay, and quantitative reverse transcription-PCR (qRT-PCR) techniques, indicated an excellent oocyte/embryo internalization of RES and TTN. Moreover, when oocytes/embryos were treated with the lowest concentration of RES + TTN-SLN, antioxidants-related genes were upregulated, apoptotic-related genes were downregulated, and intra/extracellular ROS production was reduced. In vitro cytotoxicity studies also demonstrated that single/dual-encapsulation of RES or TTN were safe even at the highest concentration (10 and 5 μM) compared to the control group. To sum it up, both delivery systems of RES and TTN by SLN (dual or single encapsulation) can deliver the optimal dose of RES and TTN into the oocyte/embryo. Where the dual-delivery of RES and TTN even at the lowest concentration (0.25 μM + 0.1 μm) showed a synergistic anti-oxidative effect in oocyte/embryo with a better inhibition of intra/extra-cellular ROS production by an enhanced/controlled intracellular penetration.

The RNA content of human sperm reflects prior events in spermatogenesis and potential post-fertilization effects.

Transcriptome analyses using high-throughput methodologies allow a deeper understanding of biological functions in different cell types/tissues. The present study provides an RNA-seq profiling of human sperm mRNAs and lncRNAs (messenger and long non-coding RNAs) in a well-characterized population of fertile individuals. Sperm RNA was extracted from twelve ejaculate samples under strict quality controls. Poly(A)-transcripts were sequenced and aligned to the human genome. mRNAs and lncRNAs were classified according to their mean expression values (FPKM: Fragments Per Kilobase of transcript per Million mapped reads) and integrity. Gene Ontology analysis of the Expressed and Highly Expressed mRNAs showed an involvement in diverse reproduction processes, while the Ubiquitously Expressed and Highly Stable mRNAs were mainly involved in spermatogenesis. Transcription factor enrichment analyses revealed that the Highly Expressed and Ubiquitously Expressed sperm mRNAs were primarily regulated by zinc-fingers and spermatogenesis-related proteins. Regarding the Expressed lncRNAs, only one-third of their potential targets corresponded to Expressed mRNAs and were enriched in cell-cycle regulation processes. The remaining two-thirds were absent in sperm and were enriched in embryogenesis-related processes. A significant amount of post-testicular sperm mRNAs and lncRNAs was also detected. Even though our study is solely directed to the poly-A fraction of sperm transcripts, results indicate that both sperm mRNAs and lncRNAs constitute a footprint of previous spermatogenesis events and are configured to affect the first stages of embryo development.

Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi.

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

Comparative transcriptome analysis during developmental stages of direct somatic embryogenesis in Tilia amurensis Rupr

Tilia species are valuable woody species due to their beautiful shape and role as honey trees. Somatic embryogenesis can be an alternative method for mass propagation of T. amurensis. However, the molecular mechanisms of T. amurensis somatic embryogenesis are yet to be known. Here, we conducted comparative transcriptional analysis during somatic embryogenesis of T. amurensis. RNA-Seq identified 1505 differentially expressed genes, including developmental regulatory genes. Auxin related genes such as YUC, AUX/IAA and ARF and signal transduction pathway related genes including LEA and SERK were differentially regulated during somatic embryogenesis. Also, B3 domain family (LEC2, FUS3), VAL and PKL, the regulatory transcription factors, were differentially expressed by somatic embryo developmental stages. Our results could provide plausible pathway of signaling somatic embryogenesis of T. amurensis, and serve an important resource for further studies in direct somatic embryogenesis in woody plants.

Circular RNA circMYBPC1 promotes skeletal muscle differentiation by targeting MyHC

Skeletal muscle development is a complex and highly orchestrated biological process mediated by a series of myogenesis regulatory factors. Numerous studies have demonstrated that circular RNAs (circRNAs) are involved in muscle differentiation, but the exact molecular mechanisms involved remain unclear. Here, we analyzed the expression of circRNAs at the adult and embryo development stages of cattle musculus longissimus. A stringent set of 1,318 circRNAs candidates were identified, and we found that 495 circRNAs were differentially expressed between embryonic and adult tissue libraries. We subsequently focused on one of the most downregulated circRNAs (using the adult stage expression as control), and this was named muscle differentiation-associated circular RNA (circMYBPC1). With RNA binding protein immunoprecipitation (RIP) and RNA pull-down assays, circMYBPC1 was identified to promote myoblast differentiation by directly binding miR-23a to relieve its inhibition on myosin heavy chain (MyHC). In addition, RIP assays demonstrated that circMYBPC1 could directly bind MyHC protein. In vivo observations also suggested that circMYBPC1 may stimulate skeletal muscle regeneration after muscle damage. These results revealed that the novel non-coding circRNA circMYBPC1 promotes differentiation of myoblasts and may promote skeletal muscle regeneration. Our results provided a basis for in-depth analysis of the role of circRNA in myogenesis and muscle diseases.

Incidence and risk factors for velamentous umbilical cord insertion in singleton pregnancies after assisted reproductive technology.

Assisted reproductive technology (ART) is gaining popularity worldwide. However, it is associated with increased incidence of velamentous umbilical cord insertion (VCI) in the placenta, resulting in adverse perinatal outcomes. This study aimed to identify the risk factors that might affect the incidence of VCI in pregnancies after ART treatment. We retrospectively analyzed the records of 906 singleton pregnancies via ART; all women delivered in our facility. Three ART-related variables and infant sex were examined: (1) fertilization method (conventional in vitro fertilization or intracytoplasmic sperm injection), (2) type of embryo at the time of transfer (fresh or frozen-thawed), (3) developmental stage of embryo at the time of transfer (cleavage stage or blastocyst), and (4) infant sex (male or female). Logistic regression analysis was used to assess the impact of these variables on the incidence of VCI. Of 906 cases, 55 had VCI (incidence rate, 6.1%). After adjusting for potential confounders, blastocyst stage of development (adjusted odds ratio [aOR]: 4.3, 95% confidence interval [CI]: 1.9-12.7) and female sex (aOR: 2.2, 95% CI: 1.2-3.9) emerged as independent risk factors for the development of VCI. The fertilization method and type of embryo at the time of transfer did not affect the incidence of VCI. Blastocyst stage of development and female sex pose a higher risk for developing VCI. Thus, more attention should be paid to pregnancies achieved by blastocyst and with a female fetus to detect VCI proactively and safeguard the health of both mother and fetus/neonate.

The number of previous failed embryo transfer cycles is an independent factor affecting implantation rate in women undergoing IVF/ICSI treatment: A retrospective cohort study.

The implantation rate (IR) in assisted reproductive technologies such as in vitro fertilization (IVF) and intracytoplasmic sperm injection is affected by many different factors such as age, quality of embryo, and stage of embryo development. This study aimed to investigate to what extent the number of previous failed embryo transfer cycles is an independent factor affecting IR.This was a single-center, retrospective cohort study of a consecutive series of 6376 day-3 embryo transfer (ET) cycles following IVF between January 2012 and August 2018. None of the subjects underwent endometrial scratch/injury prior to the treatment cycle, or received intravenous immunoglobulin, steroid, dehydroepiandrosterone, intralipid or heparin during the treatment with the aim of improving implantation rates.Multiple regression analysis showed that the 3 most important independent factors affecting the IR, in decreasing of importance: age, frozen or fresh embryo transfer and the number of previous ET cycles. Having controlled for 2 of the more important confounding variables including maternal age and the type of embryo, the IR in women who had 0, 1, 2, and 3 or more previous failed ET cycles were 45.8%, 35.9%, 31.2%, 21.0%, respectively (P < .001).Repeated implantation failure is a significant independent factor affecting the IR. The number of previous failed ET cycles should be considered in counselling women regarding the prognosis of a further IVF-ET treatment cycle.

THE HISTORY, OPPORTUNITIES AND PROSPECTS OF TIME-LAPSE TECHNOLOGIES IN THE STUDY OF EARLY HUMAN EMBRYONIC DEVELOPMENT

Despite advances in assisted reproductive technologies, the high failure rate of existing stimulation protocols remains a key industry challenge. One of the leading reasons for this is the limited ability to assess the biological potential of the embryo and its chances of implantation. Over the past ten years, the focus of attention in reproductive technologies has significantly shifted from the patient to the embryo, since the need to improve their effectiveness stimulates the need to understand the deep processes of early development of the embryo. In order to increase the effectiveness of in vitro fertilization procedures in clinical embryology, high-tech methods of culturing and evaluating embryos are being introduced and improved. The purpose of the review is to demonstrate the history, possibilities and prospects in the study of early human embryonic development of time-lapse imaging technology. The active study and use of the capabilities of the time-lapse slow-motion technology allowed not only to expand the understanding of the processes of early development of the embryo, but also at the current moment allows us to assess its potential from the point of view of both biological and clinical perspectives. The main advantages of this method are the possibility of morphological assessment during the continuous cultivation of embryos in closed-type incubators without their extraction, as well as the determination of the exact time intervals of key events of the stages of embryo development with special attention to those moments that are not available for observation and fixation under conditions of traditional cultivation. clinical practice. The main point of growth for the development of time-lapse imaging technology was the creation and validation of the so-called morphokinetic criteria and algorithms for assessing the quality of developing embryos. The key perspective of the method is its use in combination with elements of artificial intelligence in order to predict the most potential embryo for transfer into the uterine cavity. Modern directions of research using the method of time-lapse shooting are the continuation of the development of morphokinetic algorithms and their effective criteria, the introduction of the technology of self-learning computer programs and the adaptation of these tools in clinical practice, the search and assessment of possible factors influencing the morphokinetics of embryos, quality control of the work of embryological laboratories. The future development of such technologies is presented in combination not only with the capabilities of artificial intelligence, but also in combination with the use of non-invasive genetic screening, the assessment of metabolomics and proteomics of developing embryos.

The Embryotoxic Effects of in Ovo Administered Sunset Yellow FCF in Chick Embryos.

Sunset yellow (SY) at prescribed concentrations has been approved by regulatory authorities in several countries as an additive dye in the food, beverage, cosmetic, and pharmaceutical industries. However, there are some reports that it may cause several health problems. The aim of this study is to evaluate embryotoxic effects of SY on liver and kidney in chick embryos. Babcock white Leghorn eggs were randomly divided into four groups. Non-treated eggs served as control group. The eggs in groups SY200, SY1000, and SY2000 were treated with a single injection of 200, 1000, and 2000 ng SY into the air sac just before incubation. The developmental stages of embryos were determined on the 10th, 13th, 16th, and 21st days of incubation. Samples of the liver and kidney were taken and routine histological procedures were performed. The highest relative embryo weight was seen in all SY treated groups on the 16th day of incubation. Necrosis of some hepatocytes and cytoplasmic degenerations were observed in all SY groups in the liver. There were degenerated or destructed renal cortex structures and necrosis in the kidney. The cell’s nuclear areas and diameters of renal cortex structures were different in all SY groups compared to the control group (p < 0.05). It was concluded that in ovo administered SY has many unfavorable effects on liver and kidney in chick embryos. The results obtained in this study suggest that it may be advisable to re-assess safety levels of SY in many industries.

Molecular Response in Intestinal and Immune Tissues to in Ovo Administration of Inulin and the Combination of Inulin and Lactobacillus lactis Subsp. cremoris.

Intestinal microbiota are a key factor in maintaining good health and production results in chickens. They play an important role in the stimulation of immune responses, as well as in metabolic processes and nutrient digestion. Bioactive substances such as prebiotics, probiotics, or a combination of the two (synbiotic) can effectively stimulate intestinal microbiota and therefore replace antibiotic growth promoters. Intestinal microbiota might be stimulated at the early stage of embryo development in ovo. The aim of the study was to analyze the expression of genes related to energy metabolism and immune response after the administration of inulin and a synbiotic, in which lactic acid bacteria were combined with inulin in the intestines and immune tissues of chicken broilers. The experiment was performed on male broiler chickens. Eggs were incubated for 21 days in a commercial hatchery. On day 12 of egg incubation, inulin as a prebiotic and inulin with Lactobacillus lactis subsp. cremoris as a synbiotic were delivered to the egg chamber. The control group was injected with physiological saline. On day 35 post-hatching, birds from each group were randomly selected and sacrificed. Tissues (spleen, cecal tonsils, and large intestine) were collected and intended for RNA isolation. The gene panel (ABCG8, HNF4A, ACOX2, APBB1IP, BRSK2, APOA1, and IRS2) was selected based on the microarray dataset and biological functions of genes related to the energy metabolism and immune responses. Isolated RNA was analyzed using the RT-qPCR method, and the relative gene expression was calculated. In our experiment, distinct effects of prebiotics and synbiotics following in ovo delivery were manifested in all analyzed tissues, with the lowest number of genes with altered expression shown in the large intestines of broilers. The results demonstrated that prebiotics or synbiotics provide a potent stimulation of gene expression in the spleen and cecal tonsils of broiler chickens. The overall number of gene expression levels and the magnitude of their changes in the spleen and cecal tonsils were higher in the group of synbiotic chickens compared to the prebiotic group.