Search

Objective Recently, the application in the field of germplasm resource conservation has become an important application of primordial germ cells (PGCs). However, due to the lack of deep understanding of the biological characteristics of PGCs at different time points, there is no systematic scheme for the selection of PGCs at which time points in practical application, which affects the practical application effect of PGCs. This study aims to clarify the differences in PGCs during development. Methods Here, migration experiment, EdU proliferation assay and cell apoptosis assay were conducted to compare the differences in the migration ability, the proliferation ability and the recovery efficiency among female and male PGCs at E3.5, E4.5, and E5.5, which were explained by the following transcriptome sequencing analysis. Results We found that there were larger differences between female and male PGCs at different embryonic ages, while smaller differences between female and male PGCs at the same embryonic age. Further comparison showed that the cell migration ability of female and male PGCs decreased gradually during development, so female and male PGCs at E3.5 are more suitable for in vitro allotransplantation. At the same time, the proliferation ability of PGCs gradually decreased during development, and cell adhesion and extracellular matrix communication were weakened, indicating that female and male PGCs of E3.5 are more suitable for in vitro long-term culture cell line establishment. Interestingly, female and male PGCs at E5.5 showed strong DNA damage repair ability, thus more suitable for in vitro long-term cryopreservation. Conclusion This study provides a theoretical basis for systematically selecting PGCs at suitable developmental time points as cell materials for efficient utilization by analyzing the characteristics of female and male PGCs at different developmental time points based on transcriptome.

Abstract Objectives Chicken (Gallus gallus), as the most economically important poultry, is a classical and ideal model for studying the mechanism of vertebrate developmental biology and embryology. However, the sex determination and differentiation in chicken is still elusive, which limited the application and slowed down many basic studies in chicken. Data description We applied PacBio Iso-seq to multiple spatiotemporal embryo-gonad tissues in the male and female chicken, which contain the blastoderm (E0, un-differentiation stage), genital ridge (E3.5–6.5, sex-differentiation stage) and gonads (E18.5, full-sex-differentiation stage). We obtained 51,479 and 48,356 full-length transcripts in male and female chicken embryo, respectively. The comprehensive annotated and evaluated these transcripts. The 1,293 and 1,556 candidate lncRNAs, 5,766 and 4,211 AS events in male and female. Collectively, our data constitutes a grand increase in the known number of lncRNA, AS (Alternative splicing) and Poly(A) during chicken embryo sex-differentiation and plays an important role in improving current genome annotation. In the meantime, the data will be enriched the functional studies in other birds.

ABSTRACT: Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

ABSTRACT: In chicken, primordial germ cells (PGC) are crucial for the preservation and manipulation of genetic resources in poultry production. The HiS and FAcs culture systems are two important methods for the in vitro cultivation of chicken PGCs. The purpose of this study was to compare and analyze the two cultivation systems for PGCs (His and FAcs culture systems) to assess their efficacy and applicability in supporting PGC growth, maintaining PGC characteristics, and lineage transmission ability. The study found that both HiS and FAcs culture systems could maintain the basic biological characteristics of chicken PGCs, including the simultaneous expression of pluripotency and reproductive marker genes, as well as the presence of abundant glycogen granules. Subsequently, we identified 2,145 differentially expressed genes (DEG) through RNA sequencing. GO and KEGG analysis revealed a large number of DEGs enriched in the cell adhesion and calcium ion binding pathways, and the analysis found that these genes maintained a higher level in HiS-PGCs. Further personalized analysis found that the regulatory genes for maintaining PGC pluripotency were highly expressed in HiS-PGCs, while germ cell-related genes showed similar expression in both systems. Additionally, through RNA sequencing data and cell proliferation ability, it was found that PGCs in the FAcs system had a higher proliferation rate and a faster cell cycle. Finally, it was discovered that the expression of cell migration-related genes was maintained at a higher level in HiS-PGCs, but the migration efficiency of HiS-PGCs did not show a significant difference compared to FAcs-PGCs. These results suggest that both HiS and FAcs culture systems can maintain the proliferation and basic characteristics of chicken PGCs, but differences exist in cell proliferation, pluripotency regulation, and cell adhesion. These findings provide new information for optimizing PGC cultivation systems and are important for the preservation and genetic improvement of chicken PGCs.

Bone morphogenetic protein 4 (BMP4) can induce the formation of chicken primordial germ cells (PGCs) in vitro; however, its regulatory mechanism in poultry remains unknown. This study aimed to use RNA-seq to analyze PGCs in chicken embryos and iPGCs induced by BMP4 in vitro, clarify the internal regulatory factors of PGCs, analyze the mechanism of the formation of PGCs, and lay a theoretical foundation for the further optimization of PGCs induction systems. Embryonic stem cells (ESCs), PGCs and iPGCs induced by BMP4 in vitro were collected. The transcriptional maps of the three cell types were studied using RNA-seq. The results showed 6,142 genes differentially expressed between PGCs and iPGCs, of which 2,728 were upregulated in iPGCs and 3,414 were downregulated in iPGCs. Compared to that in ESCs, BMP4 was significantly upregulated in PGCs and iPGCs. KEGG results showed that both the TGF-β and Wnt signaling pathways were activated during the formation of PGCs in vitro and in vivo, and the activation was more significant during iPGCs induced by BMP4. The expression of Nodal, an inhibitory factor of TGF-β signaling, was significantly decreased in PGCs and iPGCs, but was not expressed in iPGCs, which further supports our conclusion. Additionally, the Lysosome and PI3K-AKT signaling pathways were significantly enriched in PGCs and iPGCs, respectively. Further, transmission electron microscopy (TEM) results showed that the number of autolysosomes was significantly higher after the addition of BMP4, which is consistent with the KEGG results. Furthermore, the number of PGCs was significantly reduced after ATG14 was interfered in vivo and in vitro. In conclusion, this study screened out the key signaling pathways during the formation of PGCs, aiming to provide help for enriching the mechanism network regulating PGCs formation in chicken and laying a theoretical foundation for further improving the efficiency of inducing PGCs in vitro.

Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock.

The Chinese crested (CC) duck is a unique indigenous waterfowl breed, which has a crest cushion that affects its survival rate. Therefore, the CC duck is an ideal model to investigate the genetic compensation response to maintain genetic stability. In the present study, we first generated a chromosome-level genome of CC ducks. Comparative genomics revealed that genes related to tissue repair, immune function, and tumors were under strong positive selection, indicating that these adaptive changes might enhance cancer resistance and immune response to maintain the genetic stability of CC ducks. We also assembled a Chinese spot-billed (Csp-b) duck genome, and detected the structural variations (SVs) in the genome assemblies of three ducks (i.e., CC duck, Csp-b duck, and Peking duck). Functional analysis revealed that several SVs were related to the immune system of CC ducks, further strongly suggesting that genetic compensation in the anti-tumor and immune systems supports the survival of CC ducks. Moreover, we confirmed that the CC duck originated from the mallard ducks. Finally, we revealed the physiological and genetic basis of crest traits and identified a causative mutation in TAS2R40 that leads to crest formation. Overall, the findings of this study provide new insights into the role of genetic compensation in adaptive evolution.

Developing a pleasant and supportive emotional bond with teachers might help students feel more at home at school. However, research on the relationship between positive teacher–student relationships and students’ feelings of belonging in university settings is scarce. Using data from 426 student surveys, this study estimated the moderated mediation model which is generated based on attachment theory and reciprocity theory. As a result, teacher–student reciprocity increases students’ sense of belonging to the university, and teacher–student attachment moderates this effect. The effects of teacher-student reciprocity and teacher-student attachment on students’ sense of belonging to the institution are positively moderated by social group attachment. This work contributes to the study of the sense of belonging by expanding on the applications of attachment theory and reciprocity theory. This research has a practical application.

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal–Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.

In recent years, inducing pluripotent stem cells to differentiate into functional primordial germ cells (PGCs) in vitro has become an important method of obtaining a large number of PGCs. However, the instability and low induction efficiency of the in vitro PGC induction system restrict the application of PGCs in transgenic animal production, germplasm resource conservation and other fields. In this study, we successfully established a two-step induction model of chicken PGCs in vitro, which significantly improved the formation efficiency of PGC-like cells (PGCLCs). To further improve the PGC formation efficiency in vitro, 5025 differentially expressed genes (DEGs) were obtained between embryonic stem cells (ESCs) and PGCs through RNA-seq. GO and KEGG enrichment analysis revealed that signaling pathways such as BMP4, Wnt and Notch were significantly activated during PGC formation, similar to other species. In addition, we noted that cAMP was activated during PGC formation, while MAPK was suppressed. Based on the results of our analysis, we found that the PGC formation efficiency was significantly improved after activating Wnt and inhibiting MAPK, and was lower than after activating cAMP. To sum up, in this study, we successfully established a two-step induction model of chicken PGCs in vitro with high PGC formation efficiency, which lays a theoretical foundation for further demonstrating the regulatory mechanism of PGCs and realizing their specific applications.

ABSTRACT: Spermatogonia Stem Cells (SSCs) are the basis of spermatogenesis. In the poultry industry, asthenospermia and azoospermia in roosters seriously reduce economic benefits. In this study, we explored SSCs formation mechanisms in detail. TDRD1, which is a downstream target gene of TCF7L2 and is modified by histone methylation, was screened through multiomics analysis. Functionally, RT-qPCR, flow cytometry, immunohistochemistry, and indirect immunofluorescence results showed that H3K4me2 regulated TDRD1 to promote SSCs formation both in vivo and in vitro. Furthermore, ChIP-qPCR and dual luciferase assays showed that H3K4me2 was enriched in the −800 to 0 bp region of the TDRD1 promoter and positively regulated TDRD1 transcription to promote SSCs formation. Interestingly, in mechanistic terms, dual luciferase assays showed that TDRD1 transcription levels were significantly decreased after co-transfection with dCas9-LSD1-P1/P2/P3 and OETCF7L2, while TDRD1 transcript levels were not significantly altered after transfecting dCas9-LSD1-P4 and OETCF7L2. These results suggested that H3K4me2 enrichment in P1, P2, and P3 of the TDRD1 promoter promotes TDRD1 transcription by reducing enrichment of TCF7L2. This study explored the specific regulatory mechanisms involving the Wnt signaling pathway, H3K4me2, and TDRD1, enriched the regulatory network regulating the formation of SSCs, and laid a theoretical foundation for the specific application of SSCs.

Autophagy plays an important role in the pluripotency and differentiation of stem cells. Transcriptome data showed that the autophagy genes MAP1LC3A and MAP1LC3B were significantly upregulated in primordial germ cells (PGCs). The Kyoto Encyclopedia of Genes and Genome (KEGG) results showed that the lysosome signaling pathway, which is related to autophagy, was significantly enriched in PGCs. Quantitative RT-PCR, western blotting, and transmission electron microscopy (TEM) results showed that autophagy was expressed in both embryonic stem cells (ESCs) and PGCs but was significantly activated in PGCs. To explore the role of autophagy in the differentiation of chicken ESCs into PGCs, autophagy was activated and inhibited using rapamycin and bafilomycin A1, respectively. Results of qRT-PCR, flow cytometry, and indirect immunofluorescence showed that the efficiency of PGC formation significantly decreased after autophagy inhibition. Our results showed, for the first time, that autophagy plays an indispensable role in the formation of chicken PGCs, which lays the foundation for studying the mechanism of autophagy in chicken PGCs and in bird gene editing and the rescue of endangered birds.

Fungicides are a type of pesticide used to protect plants and crops from pathogenic fungi. Azoxystrobin (AZO), a natural methoxyacrylate derived from strobilurin, is one of the most widely used fungicides in agriculture. AZO exerts its fungicidal activity by inhibiting mitochondrial respiration, but its cytotoxicity to mammalian oocytes has not been studied. In this study, we investigated the effect of AZO exposure on mouse oocyte maturation to elucidate the underlying mechanisms of its possible reproductive toxicity. We found that AZO exposure disturbed meiotic maturation by impairing spindle formation and chromosome alignment, which was associated with decreased microtubule organizing center (MTOC) integrity. Moreover, AZO exposure induced abnormal mitochondrial distribution and increased oxidative stress. The AZO-induced toxicity to oocytes was relieved by melatonin supplementation during meiotic maturation. Therefore, our results suggest that AZO exposure impairs oocyte maturation not only by increasing oxidative stress and mitochondrial dysfunction, but also by decreasing MTOC integrity and subsequent spindle formation and chromosome alignment.

Abstract The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor’s characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.

Chicken is one of the most widely consumed sources of protein globally. Primordial germ cells (PGCs) are the precursors for ova and sperm. One of the early embryogenesis events in most animals is the segregation of the somatic and germ lineages. PGC cultures occur in the germline, and PGCs are less studied in many species. It is relatively challenging to separate, cultivate, and genetically alter chicken without mutating the basic germline. The present study aims to gather previous research about chicken PGCs and provide a customized review of studies and developments in the field of PGCs, especially for avian species. Furthermore, we show that the propagation of chicken PGCs into embryonic germ cells that contribute to somatic tissues may be produced in vitro. Primordial germ cells offer an ideal system in developmental biology, as these cells play a vital role in the genetic modification and treatment of infertility. Cryopreservation helps to maintain genetic resources and sustainable production in the poultry industry. Keeping in mind the significance of cryopreservation for storage and gametogenesis, we discuss its role in the preservation of primordial germ cells. Transgenesis and genetic modifications in chicken lead to the development of various medicinal chicken varieties and aid in improving their production and quality for consumption purposes. Additionally, these characteristics open up new possibilities for modifying the chicken genome for agricultural and medical purposes.

BackgroundSex differentiation is a complex and precisely regulated process by multiple genes in chicken. However, it is still unclear on the key genes of sex differentiation.ObjectiveTo explore the function of Tle4z1 screened by RNA-seq sequencing on sex differentiation during the development of chicken embryos.MethodsTle4z1 was differentially expressed from the RNA-seq of ESCs and PGCs in male and female chickens. Then, we established an effective method to overexpression or knocking down the expression of Tle4z1 in ovo and in vitro, respectively. Histomorphological observation, qRT-PCR and ELISA were applied to detect the function of Tle4z1 in the process of male sex differentiation by injecting vectors into embryos at day 0.ResultsIt showed that Tle4z1 has significant male preference in embryonic day 4.5, such phenomenon persisted during the growth period of chicken embryos. Morphological observation results showed that the gonads on both sides of genetic male (ZZ) embryos with Tle4z1 knocking down developed asymmetrically, the gonadal cortex became thicker showing the typical characteristics of genetic female (ZW) gonads. Furthermore, the expression of Cyp19a1, which dominates female differentiation, was significantly increased, while the expression of male marker genes Dmrt1, Sox9, WT1 and AR was significantly downregulated. In addition, the concentration of testosterone also significantly decreased, which was positively correlated with the expression of Tle4z1 (P < 0.01). Conversely, the ZW embryo showed defeminized development when Tle4z1 was overexpressed.ConclusionWe prove that the Tle4z1 is a novel gene through the male sexual differentiation via gene regulation process and synthesis of testosterone, which construct the basis for understanding the molecular mechanism of sex differentiation in chickens.

ABSTRACT: Marketable age is an important determinant of meat quality. Cherry Valley duck (SM3 medium) is the most efficient Pekin-type duck and is the most widely farmed breed globally. However, whether marketable age determines the meat quality of Cherry Valley ducks is not well documented. The objective of this study was to investigate the effect of marketable age on the proximate composition and nutritional profile of breast meat from Cherry Valley broiler ducks. Ducks at 28, 38, 42, and 45 days old were selected and slaughtered, and their proximate composition, cholesterol and essential mineral compositions, and amino acids and fatty acid profile of breast meat lipid were determined. The results showed higher protein content and lower intramuscular fat content were observed in the 38-day-old ducks than in the 28-day-old birds (P < 0.05). Additionally, 38-day-old ducks contained higher Fe and Mg contents (P < 0.05), whereas 28-day-old birds had higher Zn and Ca contents (P < 0.05). The essential amino acid content in 38-day-old was about 95.29 g/kg, higher than that in 28-day-old birds (P < 0.05). The contents of C20:5 n-3, omega-6 polyunsaturated fatty acids, and polyunsaturated fatty acids were the highest in 38-day-old birds (P < 0.05), whereas the content of C20:4 n-6, DHA (C22:6 n-3), and saturated fatty acids in 28-day-old birds was the lowest (P < 0.05). Finally, a comprehensive evaluation model of multiple traits was developed by applying principal component analysis, and the meat nutrition of 38-day-old ducks was identified as the optimal. Taken together, the meat of 38-day-old ducks had an advantage in proximate composition, minerals content, essential amino acids, and fatty acids, and 38 d might be recommended as an appropriate marketable age to provide duck meat of high nutrition value.

Aiming at the influence of fewer feature points and dynamic obstacles on location and mapping in off-road environments, we propose a dual-constraint LiDAR-based Simultaneous Localization and Mapping (SLAM) scheme. By abstracting LiDAR registration into two constraints, namely, in-window constraints and out-of-window constraints, the in-window constraints allow our scheme to compromise between accuracy and real-time performance, and out-of-window constraints can exploit optimized variables to provide richer constraint information. The advantages of incremental SLAM map construction can be used to design a variety of map forms. Although the variables outside the window are no longer involved in the optimization, we can use the two-dimensional probability grid map to provide binary semantic information and dynamic weights for the constraints outside the window to enhance the registration accuracy. Finally, we conducted experiments in off-road environment and compared with the mainstream LiDAR SLAM scheme, which can prove that our scheme has more advantages in accuracy.

Parameter calibration is critical for self-localization based on dead reckoning in the control of intelligent vehicles such as autonomous driving. Most traditional calibration methods for robotics control based on dead reckoning rely on data collection with specially designed paths. For the calibration of parameters in the control of intelligent vehicles, the design of such paths is considered impossible due to the complexity of road conditions. To solve this problem, an optimization-based dead reckoning calibration scheme is introduced in this research using the differential global positioning system to obtain the actual positions of the intelligent vehicle. In this scheme, the difference between the positions obtained through dead reckoning and the positions obtained through the differential global positioning system is selected as the optimization objective function to be minimized. An adaptive quantum-inspired evolutionary algorithm is developed to improve the quality and efficiency of optimization. Experiments with an intelligent vehicle were also conducted to demonstrate the effectiveness of the developed calibration scheme. In addition, the newly introduced adaptive quantum-inspired evolutionary algorithm is compared with the classic genetic algorithm and the classic quantum-inspired evolutionary algorithm using eight benchmark test functions considering computation quality and efficiency.

MicroRNAs (miRNAs) are essential factors in the reproductive process of poultry. Here, we found miR-302d is a potential differentiation and negative factor of chicken embryonic stem cells (ESCs) into spermatogonia stem cells (SSCs). The competition mechanism was carried out for the preliminary exploration to determine the relationship among miR-302d, lncRNA-341(interacting with miR-302d), and target gene TLE4. The results showed that lncRNA-341 can competitively bind to miR-302d to decrease the targeted binding of miR-302d and TLE4 which promotes the differentiation of chicken SSCs. Moreover, it is suggested that miR-302d may participate in the Wnt signaling pathway through TLE4.

The production of germ cells, especially primordial germ cells (PGCs), is important for avian stem cells and reproduction biology. However, key factors involved in the regulation of PGCs remain unknown. Here, we report a PGC-related marker gene: C1EIP (Chromosome 1 Expression in PGCs), whose activation and expression are regulated by the transcription factor STAT3 (signal transducer and activator of transcription 3), histone acetylation, and promoter methylation. C1EIP regulates PGCs formation by mediating the expression of PGC-associated genes, such as CVH (Chicken Vasa Homologous) and CKIT (Chicken KIT proto-oncogene). C1EIP knockdown during embryonic development reduces PGC generation efficiency both in vitro and in ovo. Conversely, C1EIP overexpression increases the formation efficiency of PGCs. C1EIP encodes a cytoplasmic protein that interacts with ENO1 (Enolase 1) in the cytoplasm, inhibits the Notch signaling pathway, and positively regulates PGC generation. Collectively, our findings demonstrate C1EIP as a novel gene involved in PGC formation, which regulates genes involved in embryonic stem cell differentiation through interaction with ENO1 and subsequent inhibition of the Notch signaling pathway by the impression of Myc (MYC proto-oncogene).

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 μM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41±1.51% and 19.43±0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.

Abstract Background Notch signaling is mainly regulated by Notch1 during development of chicken germ stem cells; however, the molecular mechanisms that contribute to generation of these germ stem cells have not been thoroughly investigated. Results In our studies, Overexpression of the Notch1 NICD promoted development of the reproductive ridge, but inhibited the formation of seminiferous tubules. The formation efficiency of PGCs in the reproductive ridge following overexpression of NICD (7.5% ± 0.11) was significantly higher than that (4.9% ± 0.17, p

The complete mitogenome sequence of Sinogastromyzon szechuanensis was obtained using next generation sequencing and confirmed via overlap-PCR method. The genome was 16,565 bp in length and consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 23 transfer RNA genes and 1 control region. The overall nucleotide composition of heavy strand was 30.38% A (5033), 16.62% G (2753), 25.25% T (4182) and 27.75 (4597), with a slight A–T skew (55.63%), which is most obvious in the D-loop and most transfer RNA genes. Mitochondrial genome analyses based on ML analyses yielded identical phylogenetic trees.

Background Mycoplasma hyopneumoniae (Mhp) is the main pathogen causing respiratory disease in the swine industry. Mhp infection rates differ across pig breeds, with Chinese native pig breeds that exhibit high fecundity (e.g., Jiangquhai, Meishan, Erhualian) more sensitive than Duroc, Landrace, and other imported pig breeds. However, the genetic basis of the immune response to Mhp infection in different pig breeds is largely unknown. Aims The aims of this study were to determine the relative Mhp susceptibility of the Chinese native Jiangquhai breed compared to the Duroc breed, and identify molecular mechanisms of differentially expressed genes (DEGs) using an RNA-sequencing (RNA-seq) approach. Methods Jiangquhai and Duroc pigs were artificially infected with the same Mhp dose. The entire experiment lasted 28 days. Daily weight gain, Mhp-specific antibody levels, and lung lesion scores were measured to evaluate the Mhp infection susceptibility of different breeds. Experimental pigs were slaughtered on the 28th day. Lung tissues were collected for total RNA extraction. RNA-seq was performed to identify DEGs, which were enriched by gene ontology (GO) and the Kyoto Encyclopedia annotation of Genes and Genomes (KEGG) databases. DEGs were validated with real-time quantitative polymerase chain reaction (RT-qPCR). Results Infection with the same Mhp dose produced a more serious condition in Jiangquhai pigs than in Duroc pigs. Jiangquhai pigs showed poorer growth, higher Mhp antibody levels, and more serious lung lesions compared with Duroc pigs. RNA-seq identified 2,250 and 3,526 DEGs in lung tissue from Jiangquhai and Duroc pigs, respectively. The two breeds shared 1,669 DEGs, which were involved in immune-relevant pathways including cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, and chemokine signaling pathway. Compared to Jiangquhai pigs, more chemokines, interferon response factors, and interleukins were specifically activated in Duroc pigs; CXCL10, CCL4, IL6 and IFNG genes were significantly up-regulated, which may help Duroc pigs enhance immune response and reduce Mhp susceptibility. Conclusion This study demonstrated differential immune-related DEGs in lung tissue from the two breeds, and revealed an important role of genetics in the immune response to Mhp infection. The biological functions of these important DEGs should be further confirmed and maybe applied as molecular markers that improve pig health.

Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.

Primordial germ cells are the ancestors of female and male cells. Current research has shown that long non-coding RNA (lncRNA) and Histone methylation are the pivotal epigenetic factors in the PGC formation. However, there are few studies on the regulatory mechanism of lncRNA in the formation of PGC. Here, we define the lncRNA highly expressed in chicken PGC, lncCPSET1 (chicken-PGC-specifically-expressed transcript 1) This study found that compared with the interference of lncCPSET1/histone methylase Mll2 alone, the PGC formation was severely inhibited with the interference of lncCPSET1 and histone methylase Mll2 jointly in vivo and in vitro. Studies on the transcription level of lncCPSET1 found that H3K4me2 and transcription factor Jun have a positive effect on the activation of lncCPSET1; while DNA hypomethylation inhibits the expression of lncCPSET1. In terms of mechanism, compared with DNA methylation, H3K4me2 dominates lncCPSET1 activation. H3K4me2 can be enriched in the lncCPSET1 promoter, change its chromosome conformation, recruit the transcription factor Jun, and activate the expression of lncCPSET1. Taken together, we confirmed the model that H3K4me2 rather than DNA hypomethylation mediates Jun to regulate lncCPSET1 transcription, which broadens the study of lncCPSET1 pre-transcriptional mechanism.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) in vitro. Previously, a lentivirus induction strategy of introducing Oct4, Sox2, Nanog and Lin28 (OSNL) into the iPSC process has been shown as a possible way to produce chicken iPSCs from chicken embryonic fibroblasts, but the induction efficiency of this method was found to be significantly limiting. In order to help resolve this efficiency obstacle, this study seeks to clarify the associated regulation mechanisms and optimizes the reprogramming strategy of chicken iPSCs. This study showed that glycolysis and the expression of glycolysis-related genes correlate with a more efficient reprogramming process. At the same time, the transcription factors Oct4, Sox2 and Nanog were found to activate the expression of glycolysis-related genes. In addition, we introduced two small-molecule inhibitors (2i-SP) as a “glycolysis activator” together with the OSNL cocktail, and found that this significantly improved the induction efficiency of the iPSC process. As such, the study identifies direct molecular connections between core pluripotency factors and glycolysis during the chicken iPSC induction process and, with its results, provides a theoretical basis and technical support for chicken somatic reprogramming.

Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.

Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.

The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

Chicken (Gallus gallus) pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs) can be broadly applied in the research of developmental and embryonic biology, but the difference between amphoteric ESCs and PGCs is still elusive. This study determined the sex of collected samples by identifying specific sex markers via polymerase chain reaction (PCR) and fluorescence activated cell sorter (FACS). RNA-seq was utilized to investigate the transcriptomic profile of amphoteric ESCs and PGCs in chicken. The results showed no significant differentially expressed genes (DEGs) in amphoteric ESCs and 227 DEGs exhibited in amphoteric PGCs. Moreover, those 227 DEGs were mainly enriched in 17 gene ontology (GO) terms and 27 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Furthermore, qRT-PCR was performed to verify RNA-seq results, and the results demonstrated that Notch1 was highly expressed in male PGCs. In summary, our results provided a knowledge base of chicken amphoteric ESCs and PGCs, which is helpful for future research in relevant biological processes.

Germ cells have an irreplaceable role in transmitting genetic information from one generation to the next, and also play an important role in sex differentiation in poultry, while little is known about epigenetic factors that regulate germ cell differentiation. In this study, RNA-seq was used to detect the expression profiles of long non-coding RNAs (lncRNAs) during the differentiation of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The results showed that a total of 296, 280 and 357 differentially expressed lncRNAs (DELs) were screened in ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs, respectively. Gene Ontology (GO) and KEGG enrichment analysis showed that DELs in the three cell groups were mainly enriched in autophagy, Wnt/β-catenin, TGF-β, Notch and ErbB and signaling pathways. The co-expression network of 37 candidate DELs and their target genes enriched in the biological function of germ cell development showed that XLOC_612026, XLOC_612029, XLOC_240662, XLOC_362463, XLOC_023952, XLOC_674549, XLOC_160716, ALDBGALG0000001810, ALDBGALG0000002986, XLOC_657380674549, XLOC_022100 and XLOC_657380 were the key lncRNAs in the process of male germ cell formation and, moreover, the function of these DELs may be related to the interaction of their target genes. Our findings preliminarily excavated the key lncRNAs and signaling pathways in the process of male chicken germ cell formation, which could be helpful to construct the gene regulatory network of germ cell development, and also provide new ideas for further optimizing the induction efficiency of germ cells in vitro.

An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken's cells and inhibit ECSs differentiation into SSCs.

Several inducers have been used to differentiate embryonic stem cells (ESCs) into male germ cells but the induction process has been inefficient. To solve the problem of low efficiency of inducer for ESCs differentiation into male germ cells, all-trans retinoic acid (ATRA), Am80(the retinoic acid receptor agonist), and estradiol (E2) was used to induce ESCs to differentiate into male germ cells in vitro. ESCs were cultured in media containing ATRA, Am80, or E2 respectively which can differentiate ESCs into a germ cell lineage. In process of ATRA and Am80 induction Group, germ cell-like cells can be observed in 10 days; but have no in E2 induction Group. The marker genes of germ cell: Dazl, Stra8, C-kit, Cvh, integrinα6, and integrinβ1 all showed a significant up-regulation in the expression level. The ATRA-induction group showed high expression of C-kit and Cvh around 4 days, and integrinα6 and integrinβ1 were activated on day 10, respectively, while the E2-,Am80- induction group showed a high expression of C-kit as early as 4 days immunocytochemistry results shown that, integrinα6 and integrinβ1 could be detected in the ATRA-, Am80-, and E2-induction group, Positive clones in the ATRA group were greater in number than those in the other two groups. we conclued that ATRA, Am80, and E2 can promote the expression of the corresponding genes of germ cells, and had different effect on the differentiation of ESCs into male germ cells. ATRA was the most effective inducer of germ cell differentiation.

Stra8 encodes stimulated by retinoic acid gene 8, a protein that is important for initiation of meiosis in mammals and birds. This study was aimed at identifying the active control area of chicken STRA8 gene core promoter, to screen optimum inducers of the STRA8 gene, thus to enhance the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells. Fragments of chicken STRA8 gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the STRA8 gene under different inducers. Our studies showed that the promoter fragment -1055 bp to +54 bp of Suqin chicken Stra8 revealed the strongest activity. The dual-luciferase® reporter showed that Tamibarotene (Am80) and TrichostatinA (TSA) could significantly enhance STRA8 transcription. The in vitro inductive culture of chicken ESCs demonstrated that spermatogonial stem cells (SSC)-like cells appeared and Integrinβ1 protein was expressed on day 10, indicating that Am80 and TSA can promote ESCs differentiation into SSCs via regulation of Stra8.

Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.

In this study we investigated the methylome of chickens immunized with Infectious laryngotracheitis (ILT) vaccine derived from chicken embryos. Methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) method was employed in the detection of the 1,155 differentially methylated regions (DMRs) across the entire genome. After validation, we ascertained the genomic DMRs distribution and annotated them regarding genes, transcription start sites (TSS) and CpG islands. We found that global DNA methylation decreased in vaccinated birds, presenting 704 hypomethylated and 451 hypermethylated DMRs, respectively. Additionally, we performed an enrichment analysis detecting gene networks, in which cancer and RNA post-transcriptional modification appeared in the first place, followed by humoral immune response, immunological disease and inflammatory disease. The top four identified canonical pathways were EIF2 signaling, regulation of EIF4 and p70S6K signaling, axonal guidance signaling and mTOR signaling, providing new insight regarding the mechanisms of ILT etiology. Lastly, the association between DNA methylation and differentially expressed genes was examined, and detected negative correlation in seventeen of the eighteen genes.

As an attempt to increase the resistance to Newcastle Disease Virus (NDV) and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA) gene and myxo-virus resistance (Mx) and detect the gene expression in transfected mouse fibroblasts (NIH-3T3) cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF) cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3) cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA). The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P