Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD. Two piglets were obtained after embryo transfer, one of piglets was identified as DMD-modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD-modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD-modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5'-NNNVRYAC sequence as a protospacer-adjacent motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals.

The deleted in azoospermia like (DAZL) is required for germ cells development and maintenance. In chickens, the mRNA and protein of DAZL, a representative maternally inherited germ plasm factor, are detected in the germ plasm of oocyte, zygote, and all stages of the intrauterine embryos. However, it is still insufficient to explain the origin and specification process of chicken germ cells, because the stage at which the zygotic transcription of DAZL occurs and the stage at which the maternal DAZL RNA/protein clears have not yet been fully identified. Moreover, a comprehensive understanding of the expression of DAZL interacting genes during the germ cells specification and development and zygotic genome activation (ZGA) is lacking in chickens. In this study, we identified a set of DAZL interacting genes in chickens using in silico prediction method. Then, we analyzed the whole-transcriptome sequencing (WTS)-based expression of DAZL and its interacting genes in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage embryos (EGK.I to EGK.X). In the results, DAZL transcripts are increased in the zygote (onset of transcription), maintained the increased level until EGK.VI, and decreased from EGK.VIII (possible clearance of maternal RNAs). Among the DAZL interacting genes, most of them are increased either at 1st ZGA or 2nd ZGA, indicating their involvement in germ cells specification and development.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (-1369~+28 nt), Δ2-pGzmg (-939~+28 nt), Δ3-pGzmg (-711~+28 nt) and Δ4-pGzmg (-417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the -417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.

Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system-a modern genome editing technology-AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed "ex vivo handling of embryos", requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken β-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells.

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

Double fertilization is a key determinant of grain yield, and the failure of fertilization during hybridization is one important reason for reproductive isolation. Therefore, fertilization has a very important role in the production of high-yield and well-quality hybrid of rice. Here, we used RNA sequencing technology to study the change of the transcriptome during double fertilization with the help of the mutant fertilization barrier (feb) that failed to finish fertilization process and led to seed abortion. The results showed that 1669 genes were related to the guided growth of pollen tubes, 332 genes were involved in the recognition and fusion of the male-female gametes, and 430 genes were associated with zygote formation and early free endosperm nuclear division. Among them, the genes related to carbohydrate metabolism; signal transduction pathways were enriched in the guided growth of pollen tubes, the genes involved in the photosynthesis; fatty acid synthesis pathways were activated by the recognition and fusion of the male-female gametes; and the cell cycle-related genes might play an essential role in zygote formation and early endosperm nuclear division. Furthermore, among the 1669 pollen tube-related genes, it was found that 7 arabinogalactan proteins (AGPs), 1 cysteine-rich peptide (CRP), and 15 receptor-like kinases (RLKs) were specifically expressed in anther, while 2 AGPs, 7 CRPs, and 5 RLKs in pistil, showing obvious unequal distribution which implied they might play different roles in anther and pistil during fertilization. These studies laid a solid foundation for revealing double fertilization mechanism of rice and for the follow-up investigation.

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.

The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of Drosophila melanogaster, the full development cycle of which (from zygote to sexually mature adults) passed under simulated microgravity by a random positioning machine. The rate of cellular respiration was studied by polarography as a parameter reflecting the functional state of mitochondria. The content of cytoskeletal proteins and histones was determined using Western blotting. The relative content of mRNA was determined using qRT-PCR. The results obtained indicated an increase in the rate of cellular respiration under simulated microgravity conditions during the full cycle of gametogenesis in Drosophila melanogaster due to complex I of the respiratory chain. In addition, an increase in the contents of actin cytoskeleton components was observed against the background of an increase in the mRNA content of the cytoskeleton's encoding genes. Moreover, we observed an increase in the relative content of histone H3 acetylated at Lys9 and Lys27, which may explain the increase in the expression of cytoskeletal genes. In conclusion, the formation of an adaptive pattern of functioning of the Drosophila melanogaster ovaries that developed under simulated microgravity includes structural and functional changes and epigenetic regulation.

Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called "in vivo somatic cell genome editing". This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.

Echiura is one of the most intriguing major subgroups of phylum Annelida because, unlike most other annelids, echiuran adults lack metameric body segmentation. Urechis unicinctus lives in U-shape burrows of soft sediments. Little is known about the molecular mechanisms underlying the development of U. unicinctus. Herein, we overviewed the developmental process from zygote to juvenile U. unicinctus using immunohistochemistry and F-actin staining for the nervous and muscular systems, respectively. Through F-actin staining, we found that muscle fibers began to form in the trochophore phase and that muscles for feeding were produced first. Subsequently, in the segmentation larval stage, the transversal muscle was formed in the shape of a ring in an anterior-to-posterior direction with segment formation, as well as a ventromedian muscle for the formation of a ventral nerve cord. After that, many muscle fibers were produced along the entire body and formed the worm-shaped larva. Finally, we investigated the spatiotemporal expression of Uun_st-mhc, Uun_troponin I, Uun_calponin, and Uun_twist genes found in U. unicinctus. During embryonic development, the striated and smooth muscle genes were co-expressed in the same region. However, the adult body wall muscles showed differential gene expression of each muscle layer. The results of this study will provide the basis for the understanding of muscle differentiation in Echiura.

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells and embryos. A previous investigation by our group revealed potentially hazardous effects of CTN on mouse oocyte maturation and pre- and post-implantation embryo development via the induction of apoptosis. The present study showed that CTN induces apoptosis and inhibits cell proliferation in the inner cell mass of mouse blastocysts. Notably, we observed for the first time that both these effects are suppressed by liquiritigenin (LQ). LQ is a type of flavonoid isolated from Glycyrrhiza radix with several biochemical and pharmacological activities, including antioxidant and anti-inflammatory properties. The preincubation of blastocysts with LQ clearly prevented CTN-induced disruption of pre- and post-implantation embryonic development and fetal weight loss, both in vitro and in vivo. CTN-induced damage processes directly promoted reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP) and activation of caspase-9 and caspase-3, which were effectively blocked by LQ. Moreover, in an animal model, intravenous injection of dams with CTN (3 mg/kg/day) triggered apoptosis of blastocysts, disruption of embryonic development from the zygote to the blastocyst stage and a decrease in fetal weight. Pre-injection with LQ (5 mg/kg/day) effectively reduced apoptosis and impaired the cytotoxic effects of CTN on development. Our in vivo findings further confirm that CTN exposure via injection has the potential to impair pre- and post-implantation development, leading to apoptosis and the suppression of sequent embryonic development, which can be effectively prevented by LQ.

Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.