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Medical Microbiology and Immunology Aug 2020It is known that tetraspanin proteins are involved in many physiological somatic cell mechanisms. Additionally, research has indicated they also have a role in various... (Review)
Review
It is known that tetraspanin proteins are involved in many physiological somatic cell mechanisms. Additionally, research has indicated they also have a role in various infectious diseases and cancers. This review focuses on the molecular interactions underlying the tetraspanin web formation in gametes. Primarily, tetraspanins act in the reproductive tract as organizers of membrane complexes, which include the proteins involved in the contact and association of sperm and oocyte membranes. In addition, recent data shows that tetraspanins are likely to be involved in these processes in a complex way. In mammalian fertilization, an important role is attributed to CD molecules belonging to the tetraspanin superfamily, particularly CD9, CD81, CD151, and also CD63; mostly as part of extracellular vesicles, the significance of which and their potential in reproduction is being intensively investigated. In this article, we reviewed the existing knowledge regarding the expression of tetraspanins CD9, CD81, CD151, and CD63 in mammalian spermatozoa, oocytes, and embryos and their involvement in reproductive processes, including pathological events.
Topics: Animals; Embryonic Development; Female; Humans; Male; Mammals; Oocytes; Reproduction; Spermatozoa; Tetraspanins; Zygote
PubMed: 32424440
DOI: 10.1007/s00430-020-00676-0 -
Cryobiology Dec 2020The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages....
The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.
Topics: Animals; Blastocyst; Cryopreservation; Embryonic Development; Female; Pregnancy; Swine; Vitrification; Zygote
PubMed: 33065107
DOI: 10.1016/j.cryobiol.2020.10.009 -
Methods in Molecular Biology (Clifton,... 2023Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing...
Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.
Topics: Rats; Animals; Gene Editing; CRISPR-Cas Systems; Zygote; Electroporation; Electroporation Therapies
PubMed: 36995672
DOI: 10.1007/978-1-0716-2990-1_11 -
Current Opinion in Plant Biology Feb 2020In the last two decades, work on auxin signaling has helped to understand many aspects of the fundamental process underlying the specification of tissue types in the... (Review)
Review
In the last two decades, work on auxin signaling has helped to understand many aspects of the fundamental process underlying the specification of tissue types in the plant embryo. However, the immediate steps after fertilization including the polarization of the zygote and the initial body axis formation remained poorly understood. Valuable insight into these enigmatic processes has been gained by studying fertilization in grasses. Recent technical advances in transcriptomics of developing embryos with high spatial and temporal resolution give an emerging picture of the rapid changes of the zygotic developmental program. Together with the use of live imaging of novel fluorescent marker lines, these data are now the basis of unraveling the very first steps of the embryonic patterning process.
Topics: Embryonic Development; Magnoliopsida; Seeds; Signal Transduction; Zygote
PubMed: 31727540
DOI: 10.1016/j.pbi.2019.10.002 -
Current Topics in Developmental Biology 2020The perpetuation and preservation of distinct species rely on mechanisms that ensure that only interactions between gametes of the same species can give rise to viable...
The perpetuation and preservation of distinct species rely on mechanisms that ensure that only interactions between gametes of the same species can give rise to viable and fertile offspring. Species-specificity can act at various stages, ranging from physical/behavioral pre-copulatory mechanisms, to pre-zygotic incompatibility during fertilization, to post-zygotic hybrid incompatibility. Herein, we focus on our current knowledge of the molecular mechanisms responsible for species-specificity during fertilization. While still poorly understood, decades of research have led to the discovery of molecules implicated in species-specific gamete interactions, starting from initial sperm-egg attraction to the binding of sperm and egg. While many of these molecules have been described as species-specific in their mode of action, relatively few have been demonstrated as such with definitive evidence. Thus, we also raise remaining questions that need to be addressed in order to characterize gamete interaction molecules as species-specific.
Topics: Acrosome; Animals; Cell Adhesion; Cell Fusion; Female; Fertilization; Male; Ovum; Sea Urchins; Species Specificity; Sperm-Ovum Interactions; Spermatozoa; Xenopus laevis; Zygote
PubMed: 32591072
DOI: 10.1016/bs.ctdb.2019.10.005 -
Methods in Molecular Biology (Clifton,... 2023Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells,...
Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome-editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-strand oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.
Topics: Mice; Animals; Gene Editing; Gene Targeting; Embryonic Stem Cells; Oligodeoxyribonucleotides; CRISPR-Cas Systems; Gene Knock-In Techniques; Zygote
PubMed: 36773141
DOI: 10.1007/978-1-0716-3016-7_8 -
Current Opinion in Genetics &... Feb 2022Proper higher-order chromatin folding can profoundly impact gene expression. In early animal development, chromatin undergoes dramatic reorganization to convert... (Review)
Review
Proper higher-order chromatin folding can profoundly impact gene expression. In early animal development, chromatin undergoes dramatic reorganization to convert terminally differentiated gametes to early embryos. The recent rapid development of ultra-sensitive chromatin analysis technologies has revealed a drastic reconstruction of chromatin architecture, which includes a surprising relaxation followed by de novo and slow establishment of 3D genome organization in early embryos. Such progress adds another fascinating dimension to epigenetic reprogramming in early development that also features degradation of maternal RNA and activation of the zygotic genome during the maternal to zygotic transition (MZT). Nevertheless, the role of higher-order chromatin architecture in this critical developmental time window is yet to be understood. In this article, we review the latest findings from a variety of species (with a primary focus on mammals) on the establishment of 3D chromatin structure in gametogenesis and early development. These data shed light on how chromatin organization is regulated, and how it coordinates with MZT and other crucial events in early development. Finally, we discuss the crucial questions that remain to be answered in the future.
Topics: Animals; Chromatin; Embryonic Development; Epigenomics; Gene Expression Regulation, Developmental; Genome; Mammals; Zygote
PubMed: 34896808
DOI: 10.1016/j.gde.2021.11.002 -
Cell Proliferation Mar 2023Pre-replication complex (pre-RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre-RC, and proper regulation of CDT1 and MCM2 are...
Pre-replication complex (pre-RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre-RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re-replication and thus DNA damage check-point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre-RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its' degradation is essential for zygotes to prevent from DNA re-replication in G2 stage.
Topics: Zygote; Cell Cycle Proteins; DNA Replication; Cell Cycle; DNA
PubMed: 36479743
DOI: 10.1111/cpr.13377 -
Plant Reproduction Jun 2020Recent understandings ofArabidopsiszygote. Body axis formation is essential for the proper development of multicellular organisms. The apical-basal axis in Arabidopsis... (Review)
Review
Recent understandings ofArabidopsiszygote. Body axis formation is essential for the proper development of multicellular organisms. The apical-basal axis in Arabidopsis thaliana is determined by the asymmetric division of the zygote, following its cellular polarization. However, the regulatory mechanism of zygote polarization is unclear due to technical issues. The zygote is located deep in the seed (ovule) in flowers, which prevents the living dynamics of zygotes from being observed. In addition, elucidation of molecular pathways by conventional forward genetic screens was not enough because of high gene redundancy in early development. Here, we present a review introducing two new methods, which have been developed to overcome these problems. Method 1: the two-photon live-cell imaging method provides a new system to visualize the dynamics of intracellular structures in Arabidopsis zygotes, such as cytoskeletons and vacuoles. Microtubules form transverse rings and control zygote elongation, while vacuoles dynamically change their shapes along longitudinal actin filaments and support polar nuclear migration. Method 2: the transcriptome method uses isolated Arabidopsis zygotes and egg cells to reveal the gene expression profiles before and after fertilization. This approach revealed that de novo transcription occurs extensively and immediately after fertilization. Moreover, inhibition of the de novo transcription was shown to sufficiently block the zygotic division, thus indicating a strong possibility that yet unidentified zygote regulators can be found using this transcriptome approach. These new strategies in Arabidopsis will help to further our understanding of the fundamental principles regarding the proper formation of plant bodies from unicellular zygotes.
Topics: Arabidopsis; Cell Division; Ovule; Seeds; Zygote
PubMed: 32322957
DOI: 10.1007/s00497-020-00389-7 -
Current Opinion in Plant Biology Feb 2022The maternal-to-zygotic transition (MZT) is a major developmental transition in the life cycles of animals. It consists of two associated processes: maternal transcript... (Review)
Review
The maternal-to-zygotic transition (MZT) is a major developmental transition in the life cycles of animals. It consists of two associated processes: maternal transcript clearance and zygotic genome activation (ZGA). The concept of MZT has been controversially discussed in plants. In this short review, we summarize recent advances in understanding the timing of ZGA and the similarities and differences between ZGA in eudicots and monocots. We discuss the parental contributions to the transcriptome of the proembryo and parental control of early embryogenesis, and we examine distinct differences in the ZGA between animals and plants, update relevant concepts on MZT, and highlight outstanding questions in this field.
Topics: Animals; Embryonic Development; Gene Expression Regulation, Developmental; Plants; Seeds; Transcriptome; Zygote
PubMed: 34823206
DOI: 10.1016/j.pbi.2021.102144