Biology subjects

Lundin, K.

Publications and source records attributed to Lundin, K..

4 recordsLinked to original sources

bioRxiv2024

Oxygen level alters energy metabolism in bovine preimplantation embryos

STUDY QUESTIONWhat is the effect of different oxygen (O2) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWEREmbryos grown in hypoxia (6% O2) from zygotes until the blastocyst stage had the highest blastocyst formation rate, whereas normoxia (20% O2) delayed transcriptomic reprogramming and embryonic genome activation, and induced changes in energy metabolism gene expression. WHAT IS KNOWN ALREADYMammalian preimplantation embryo development is a complex sequence of events where, within a week, the zygote is reprogrammed to totipotency and subsequently diverges to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is sensitive to oxygen levels that can affect various cellular processes. STUDY DESIGN, SIZE, DURATIONIn this study, we used triplicate bovine embryos as a model for human embryogenesis to compare the influence of O2 levels on preimplantation embryonic development by culturing embryos either in normoxic (20% O2) or physiological hypoxic (6% O2) conditions, or sequential hypoxia until 16-cell stage and then switching to ultrahypoxic culture (2% O2). PARTICIPANTS/MATERIALS, SETTING, METHODSAs the readout for varied O2 effects, we performed RNA sequencing using 5 targeted STRT-N method on single embryos. We compared zygotes, 4-, 8-, 16-cell and blastocyst stage embryos grown in either normoxic or hypoxic condition, adding ultrahypoxia for blastocyst stage embryos as the third condition. MAIN RESULTS AND THE ROLE OF CHANCEWe found that the initial cleavage rate was not affected by O2 levels but there was a clear difference in blastocyst formation rate. In hypoxia, 36% of embryos reached blastocyst stage while in normoxia the blastocyst formation rate was 13%. In final ultrahypoxia condition only 4.6% of embryos reached blastocyst stage. Transcriptomic profiles showed that normoxic conditions slowed down oocyte transcript degradation and embryonic genome activation. Key metabolic enzyme genes were also altered between hypoxic and normoxic conditions at the blastocyst stage. Both hypoxic and ultrahypoxic conditions induced energy production by upregulating genes involved in glycolysis and lipid metabolism typical to in vivo embryos. In contrast, normoxic conditions failed to upregulate glycolysis genes and only depended on primitive oxidative phosphorylation metabolism. We conclude that constant hypoxia culture of in vitro embryos provided the highest blastocyst formation rate and appropriate energy metabolism. Normoxia altered the energy metabolism and decreased the blastocyst formation rate. Even though ultrahypoxia at blastocyst stage resulted in a drop of blastocyst formation, the transcriptional profile of surviving embryos was normal. LARGE SCALE DATAThe raw data (BCL files) are available at Zenodo: XXXXX FASTQ files generated are available in the EMBLs European Bioinformatics Institute (EMBL-EBI)-BioStudies with accession number X-XXXX. LIMITATIONS, REASONS FOR CAUTIONThe limitation of this study is the use of bovine as an animal model instead of human embryos. Due to this, the direct translation of the results to human should be taken with caution. WIDER IMPLICATIONS OF THE FINDINGSThis study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. In addition, we provide new insights on why embryos grown in normoxia do not have the same success rate as embryos grown in hypoxia. We did not observe any benefits of lowering the oxygen levels to 2%, calling for caution of switching to this culture system. STUDY FUNDING/COMPETING INTEREST(S)This project has received funding from the European Unions Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 813707.; Work in the JK laboratory is supported by Jane and Aatos Erkko Foundation, Sigrid Juselius Foundation, Liv och Halsa (Finland), Swedish Brain Foundation and Swedish Research Council. The study was also supported by the Estonian Research Council (grant no. PRG1076) and the Horizon Europe NESTOR project (grant no. 101120075). What this means for patients?During assisted reproduction treatment (ART) oocytes are fertilized in vitro by sperm to form an embryo. The embryos are then exposed to environmental effects during the 5 days of culture before implantation. Embryo culture takes place inside the incubator, where different oxygen levels can be used. In this study, we compared 3 different oxygen concentrations for embryo culture: atmospheric or normoxia (20%), physiologic or hypoxia (6%) and combined low and ultra-low concentration with initial culture at 6% oxygen until day 3 followed by 2% (ultrahypoxia) until day 5 of culture. We conducted this study because many ART clinics use different oxygen concentrations for embryo culture, and we aimed to provide insight on how these different concentrations impact embryo development by monitoring gene expression. As using human embryos for this type of experiments is not commonly approved, we used bovine embryos whose early development resembles that of humans. We found that culturing embryos for 5 days at constant low oxygen concentration (hypoxia) gives the highest number of embryos that reach the blastocyst stage (day 5) and have expected gene expression that provides the physiological path of embryo development. Culturing embryos at atmospheric oxygen concentration showed that only 13% of fertilised eggs reached the blastocyst stage at day 5, while culturing at the final ultra-low oxygen concentration had the lowest percentage of matured embryos, and we did not observe any benefits that would justify the use of such a culture system. We suggest that in vitro culturing of embryos at constant 5-6% oxygen gives the best results.

developmental biology↗

bioRxiv2021

Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

DUX4 has recently been recognized as a key regulator in human embryonic genome activation (EGA). The exact role of DUX4 in human embryo is still elusive, partly due to the cytotoxicity of persistent DUX4 expression in cellular models. We report here that a transient DUX4 expression in human embryonic stem cells (hESCs) retains cell viability while inducing an EGA-like expression program in a subpopulation of the cells. These cells showed resemblance to 8-cell stage blastomeres and were thus named induced blastomere-like (iBM) cells. Trajectory inference from the single-cell RNA-seq data suggested that the expression profile of these cells progressed in a manner similar to the morula to blastocyst transition in human embryo. Finally, viable iBM cells could be enriched using an antibody against NaPi2b (SLC34A2), paving the way for further experimental approaches. The iBM cells can become a powerful tool to model transcriptional dynamics and regulation during early human embryogenesis.

cell biology↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗

bioRxiv2019

Complement in human pre-implantation embryos: attack and defense

It is essential for early human life that immunological responses to developing embryos are tightly regulated. An imbalance in the activation and regulation of the human complement system occurs in pregnancy complications, such as pre-eclampsia and recurrent miscarriage. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation and deposition takes place on developing human embryos already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products such as C1q, C3 and C5 on healthy developing embryos, which highlights the need for strict complement regulation. The early embryos express the complement membrane inhibitors CD46, CD55 and CD59 and bind the soluble regulators C4bp and factor H. These findings show that complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing of embryos at oocyte, zygote, 4-cell and 8-cell stages showed expression of complement genes, e.g. C1s, C2, C3, C5, factor B and factor D. This shows that the embryonic cells themselves have the capacity to express C3 and C5, which may become activated and function as mediators of cellular signaling. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and may impact cellular homeostasis and differentiation at the earliest stage of human life.\n\nStatement of significanceWhile canonical functions of the complement system relate to pathogen-defence, it is known to drive certain immune pathologies. The work here described shows, for the first time, expression and localization of a full range of complement molecules in human pre-implantation embryos. We demonstrate complement attack against early embryos, and show presence of embryonic defence mechanisms. Furthermore, we reveal early embryonic production of complement activators, suggesting non-canonical roles in cell signalling and development. Our findings thus reveal a fundamental role for complement at the earliest stages of human embryogenesis. Our data opens up for future studies into the role of complement, both in relation to infertility and pregnancy complications, as well as basic cellular processes during early human development.

developmental biology↗