Current Approaches to and the Application of Intracytoplasmic Sperm Injection (ICSI) for Avian Genome Editing.

Poultry are one of the most valuable resources for human society. They are also recognized as a powerful experimental animal for basic research on embryogenesis. Demands for the supply of low-allergen eggs and bioreactors have increased with the development of programmable genome editing technology. The CRISPR/Cas9 system has recently been used to produce transgenic animals and various animals in the agricultural industry and has also been successfully adopted for the modification of chicken and quail genomes. In this review, we describe the successful establishment of genome-edited lines combined with germline chimera production systems mediated by primordial germ cells and by viral infection in poultry. The avian intracytoplasmic sperm injection (ICSI) system that we previously established and recent advances in ICSI for genome editing are also summarized.

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.

Myogenetic oligodeoxynucleotide complexed with berberine promotes differentiation of chicken myoblasts.

Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myotubes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulation of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

Transcription of Endogenous Retrovirus Group K Members and Their Neighboring Genes in Chicken Skeletal Muscle Myoblasts.

Skeletal muscle myoblasts are myogenic precursor cells that generate myofibers during muscle development and growth. We recently reported that broiler myoblasts, compared to layer myoblasts, proliferate and differentiate more actively and promptly into myocytes, which corresponds well with the muscle phenotype of broilers. Furthermore, RNA sequencing (RNA-seq) revealed that numerous genes are differentially expressed between layer and broiler myoblasts during myogenic differentiation. Based on the RNA-seq data, we herein report that chicken myoblasts transcribe endogenous retrovirus group K member (ERVK) genes. In total, 16 ERVKs were highly expressed in layer myoblasts and two (termed BrK1 and BrK2) were significantly induced in broiler myoblasts. These transcribed ERVKs had a total of 182 neighboring genes within ±100 kb on the chromosomes, of which 40% were concentrated within ±10 kb of the ERVKs. We further investigated whether the transcription of ERVKs affects the expression of their neighboring genes. BrK1 had two neighboring genes; LOC107052719 was overlapping with BrK1 and downregulated in the broiler myoblasts, and FAM19A2 was upregulated in the broiler myoblasts as well as BrK1. BrK2 had 14 neighboring genes, and only one gene, LOC772243, was differentially expressed between layer and broiler myoblasts. LOC772243 was overlapping with BrK2 and suppressed in the broiler myoblasts. These data indicate that the transcription of ERVKs may impact the expression of their neighboring genes in chicken myoblasts.

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail.

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

Distinct cell proliferation, myogenic differentiation, and gene expression in skeletal muscle myoblasts of layer and broiler chickens.

Myoblasts play a central role during skeletal muscle formation and growth. Precise understanding of myoblast properties is thus indispensable for meat production. Herein, we report the cellular characteristics and gene expression profiles of primary-cultured myoblasts of layer and broiler chickens. Broiler myoblasts actively proliferated and promptly differentiated into myotubes compared to layer myoblasts, which corresponds well with the muscle phenotype of broilers. Transcriptomes of layer and broiler myoblasts during differentiation were quantified by RNA sequencing. Ontology analyses of the differentially expressed genes (DEGs) provided a series of extracellular proteins as putative markers for characterization of chicken myogenic cells. Another ontology analyses demonstrated that broiler myogenic cells are rich in cell cycle factors and muscle components. Independent of these semantic studies, principal component analysis (PCA) statistically defined two gene sets: one governing myogenic differentiation and the other segregating layers and broilers. Thirteen candidate genes were identified with a combined study of the DEGs and PCA that potentially contribute to proliferation or differentiation of chicken myoblasts. We experimentally proved that one of the candidates, enkephalin, an opioid peptide, suppresses myoblast growth. Our results present a new perspective that the opioids present in feeds may influence muscle development of domestic animals.

Toll-like receptor ligand-dependent inflammatory responses in chick skeletal muscle myoblasts.

Toll-like receptors (TLRs) are a group of sensory receptors which are capable of recognizing a microbial invasion and activating innate immune system responses, including inflammatory responses, in both immune and non-immune cells. However, TLR functions in chick myoblasts, which are myogenic precursor cells contributing to skeletal muscle development and growth, have not been studied. Here, we report the expression patterns of TLR genes as well as TLR ligand-dependent transcriptions of interleukin (IL) genes in primary-cultured chick myoblasts. Almost TLR genes were expressed both in layer and broiler myoblasts but TLR1A was detected only in embryonic layer chick myoblasts. Chick TLR1/2 ligands, Pam3CSK4 and FSL-1, induced inflammatory ILs in both layer and broiler myoblasts but a TLR4 ligand, lipopolysaccharide, scarcely promoted. This is the first report on TLR ligand-dependent inflammatory responses in chick myoblasts, which may provide useful information to chicken breeding and meat production industries.

Glucagon-like Peptide-1 Receptor Expression in the Pancreatic D Cells of Three Avian Species; White Leghorn Chickens, Northern Bobwhites, and Common Ostriches.

Glucagon-like peptide (GLP)-1 is released from the intestinal L cells in response to food ingestion and stimulates insulin secretion from the pancreatic B cells, by binding to its specific receptor (GLP-1R), which is expressed on the pancreatic B cells in the mammalian pancreas. Previously, we demonstrated that chicken GLP-1R was expressed on the pancreatic D cells by using a specific antibody against chicken GLP-1R. In the present study, we compared the localization of GLP-1R in the pancreases of three avian species; white leghorn chicken, northern bobwhite, and common ostrich, using the double immunofluorescence technique. We found that the types of pancreatic islets in the northern bobwhite pancreas were similar to those found in the chicken pancreas. The ostrich pancreas contained several types of pancreatic islets. GLP-1R-immunoreactive cells were found in all types of pancreatic islets in both northern bobwhite and ostrich and expressed somatostatin immunoreactivity. The present results indicate that the pancreatic D cells are the target cells of GLP-1, and GLP-1 might play a physiological role via somatostatin in the avian species.

The dynamic development of germ cells during chicken embryogenesis.

Appropriate regulation of cell proliferation during embryogenesis is crucial for the maintenance of germness. An in-depth understanding of germ cell developmental processes may yield valuable information on germ cell biology and applied sciences. However, direct evidences about germ cell proliferation and cell cycling during avian embryonic development has not been well-studied. Thus, we explored chicken germ cell dynamics during embryonic development via flow cytometry employing a germ cell-specific anti-cVASA antibody (the chicken VASA homolog is termed CVH) and propidium iodide staining. The numbers of male germ cells increased significantly during early embryonic development, but proliferation was decreased significantly with accumulation at the G0/G1 phase after embryonic d 14 (E.14), indicating initiation of mitotic arrest in the testis. On the other hand, the number of female germ cells increased significantly throughout embryogenesis, and proliferating cells were continuously evident in the ovary to the time of hatching, although gradual accumulation of cells at the G2/M phase was also evident. 5-ethynyl-2΄-deoxyuridine (EdU) incorporation analysis revealed that populations of mitotically active germ cells existed in both sexes during late embryogenesis, indicating either the maintenance of stem cell populations, or asynchronous meiosis. Collectively, these results indicate that chicken germ cells exhibited conserved developmental processes that were clearly sexually dimorphic.

Genome Modification Technologies and Their Applications in Avian Species.

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology.

Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts.

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24 h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP+ /DsRed+ hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products.

Size-dependent isolation of primordial germ cells from avian species.

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species.

Isolation, Characterization, and In Vitro Culturing of Spermatogonial Stem Cells in Japanese Quail (Coturnix japonica).

We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonial stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells (TCs) from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated TCs were 91.00% ± 2.12% and 88.00% ± 1.87% in juvenile and adult testes, respectively, and immunohistochemical evaluation indicated the expression of integrin alpha-6 (ITGA6), GDNF family receptor alpha-1 (GFRA1), and Deleted in azoospermia-like (DAZL) in specific TCs. SSCs were purified by differential plating of TCs and then subjected to quantitative reverse transcription-polymerase chain reaction, which showed differential expression of SSC-specific, and germness and stemness-related genes. Coculture of quail SSCs with mouse embryonic fibroblasts and Sertoli cells as a feeder layer resulted in the generation of stable SSC colonies and short-term cultivation, and the expression of SSC and germ cell markers was maintained during several passages of culture. Collectively, these results demonstrate the efficient isolation and characterization of quail SSCs and the suitability of Sertoli cells as a feeder layer for in vitro culture of quail SSCs. Quail SSCs will facilitate the production of germline chimeras and transgenesis.

The avian-specific small heat shock protein HSP25 is a constitutive protector against environmental stresses during blastoderm dormancy.

Small heat shock proteins (sHSPs) range in size from 12 to 42 kDa and contain an α-crystalline domain. They have been proposed to play roles in the first line of defence against various stresses in an ATP-independent manner. In birds, a newly oviposited blastoderm can survive several weeks in a dormant state in low-temperature storage suggesting that blastoderm cells are basically tolerant of environmental stress. However, sHSPs in the stress-tolerant blastoderm have yet to be investigated. Thus, we characterised the expression and function of sHSPs in the chicken blastoderm. We found that chicken HSP25 was expressed especially in the blastoderm and was highly upregulated during low-temperature storage. Multiple alignments, phylogenetic trees, and expression in the blastoderms of Japanese quail and zebra finch showed homologues of HSP25 were conserved in other avian species. After knockdown of chicken HSP25, the expression of pluripotency marker genes decreased significantly. Furthermore, loss of function studies demonstrated that chicken HSP25 is associated with anti-apoptotic, anti-oxidant, and pro-autophagic effects in chicken blastoderm cells. Collectively, these results suggest avian HSP25 could play an important role in association with the first line of cellular defences against environmental stress and the protection of future embryonic cells in the avian blastoderm.

Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2.

Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70-120min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved-thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

DAZL Expression Explains Origin and Central Formation of Primordial Germ Cells in Chickens.

The timing and biological events associated with germ cell specification in chickens have not been determined yet. In this study, we report the origin of primordial germ cells (PGCs) and germ plasm dynamics through investigation of the expression of the chicken homolog of deleted in azoospermia-like (cDAZL) gene during germ cell specification. Asymmetric localization of germ plasm in the center of oocytes from preovulatory follicle stages leads to PGCs being formed in the center. During cleavage stages, DAZL expression pattern changes from a subcellular localization to a diffuse form before and after zygotic genome activation. Meanwhile, PGCs exhibit transcriptional active status during their specification. In addition, knockdown studies of cDAZL, which result in reduced proliferation, aberrant gene expression profiles, and PGC apoptosis in vitro, suggest its possible roles for PGC formation in chicken. In conclusion, DAZL expression reveals formation and initial positioning of PGCs in chickens.

Production of Interspecific Germline Chimeras via Embryo Replacement.

In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

The birth of quail chicks after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors - phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) - all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca(2+) rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca(2+) oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.