MLL4 prepares the enhancer landscape for Foxp3 induction via chromatin looping.

MLL4 is an essential subunit of the histone H3 Lys4 (H3K4)-methylation complexes. We found that MLL4 deficiency compromised the development of regulatory T cells (Treg cells) and resulted in a substantial decrease in monomethylated H3K4 (H3K4me1) and chromatin interaction at putative gene enhancers, a considerable portion of which were not direct targets of MLL4 but were enhancers that interacted with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the enhancers not bound by MLL4 correlated with MLL4 binding at distant interacting regions. Deletion of an upstream MLL4-binding site diminished the abundance of H3K4me1 at the regulatory elements of the gene encoding the transcription factor Foxp3 that were looped to the MLL4-binding site and compromised both the thymic differentiation and the inducible differentiation of Treg cells. We found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping, which identifies a previously unknown mechanism for regulating the T cell enhancer landscape and affecting Treg cell differentiation.

Role and function of the Hintw in early sex differentiation in chicken (Gallus gallus) embryo.

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.

Integration of selection signatures and multi-trait GWAS reveals polygenic genetic architecture of carcass traits in beef cattle.

Carcass merits are widely considered as economically important traits affecting beef production in the beef cattle industry. However, the genetic basis of carcass traits remains to be well understood. Here, we applied multiple methods, including the Composite of Likelihood Ratio (CLR) and Genome-wide Association Study (GWAS), to explore the selection signatures and candidate variants affecting carcass traits. We identified 11,600 selected regions overlapping with 2214 candidate genes, and most of those were enriched in binding and gene regulation. Notably, we identified 66 and 110 potential variants significantly associated with carcass traits using single-trait and multi-traits analyses, respectively. By integrating selection signatures with single and multi-traits associations, we identified 12 and 27 putative genes, respectively. Several highly conserved missense variants were identified in OR5M13D, NCAPG, and TEX2. Our study supported polygenic genetic architecture of carcass traits and provided novel insights into the genetic basis of complex traits in beef cattle.

Narrow H3K4me2 is required for chicken PGC formation.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.

Trac-looping measures genome structure and chromatin accessibility.

Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4+ T cells revealed substantial reorganization of enhancer-promoter interactions associated with changes in gene expression after T cell receptor stimulation.

Identification of a novel differentially methylated region adjacent to ATG16L2 in lung cancer cells using methyl-CpG binding domain protein-enriched genome sequencing.

Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to ATG16L2. The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of ATG16L2 are warranted.

Pan-RNA editing analysis of the bovine genome.

RNA editing is an essential process for modifying nucleotides at specific RNA sites during post-transcription in many species. However, its genomic landscape and characters have not been systematically explored in the bovine genome. In the present study, we characterized global RNA editing profiles from 50 samples of cattle and revealed a range of RNA editing profiles in different tissues. Most editing sites were significantly enriched in specific BovB-derived SINEs, especially the dispersed Bov-tAs, which likely forms dsRNA structures similar to the primate-specific Alu elements. Interestingly, ADARB1 (ADAR2) was observed to be predominant in determining global editing in the bovine genome. Common RNA editing sites among similar tissues were associated with tissue-specific biological functions. Taken together, the wide distribution of RNA editing sites and their tissue-specific characters implied the bovine RNA editome should be further explored.

P53 and H3K4me2 activate N6-methylated LncPGCAT-1 to regulate primordial germ cell formation via MAPK signaling.

Long noncoding RNAs (lncRNAs) participate in the formation of primordial germ cells (PGCs); however, the identity of the key lncRNAs and the molecular mechanisms responsible for the formation of PGCs remain unknown. Here, we identify a key candidate lncRNA (lncRNA PGC transcript-1, LncPGCAT-1) via RNA sequencing of embryonic stem cells, PGCs, and Spermatogonial stem cells (SSCs). Functional experiments confirmed that LncPGCAT-1 positively regulated the formation of PGCs by elevating the expression of Cvh and C-kit while downregulating the pluripotency(Nanog) in vitro and in vivo; PAS staining of genital ridges in vivo also showed that interference with LncPGCAT-1 can significantly reduce the number of PGCs in genital ridges, while overexpression of LncPGCAT-1 had the opposite result. The result of luciferase reporter assay combined with CHIP-qPCR showed that the expression of LncPGCAT-1 was promoted by the transcription factor P53 and high levels of H3K4me2. Mechanistically, the luciferase reporter assay confirmed that mitogen-activated protein kinase 1 (MAPK1) was the target gene of LncPGCAT-1 and gga-mir-1591. In the ceRNA system, high levels of N6 methylation of LncPGCAT-1 enhanced the adsorption capacity of LncPGCAT-1 for gga-mir-1591. Adsorption of gga-mir-1591 activated the MAPK1/ERK signaling cascade by relieving the gga-mir-1591-dependent inhibition of MAPK1 expression. Moreover, LncPGCAT-1 interacted with interleukin enhancer binding factor 3 (ILF3) to regulate the ubiquitination of P53 and phosphorylation of JNK. Interaction with ILF3 resulted in positive self-feedback regulation of LncPGCAT-1 and activation of JNK signaling, ultimately promoting PGC formation. Altogether, the study expands our knowledge of the function and molecular mechanisms of lncRNAs in PGC development.

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing.

BACKGROUND: Marek's disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. RESULTS: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. CONCLUSIONS: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Integrated analysis of lncRNA and mRNA repertoires in Marek's disease infected spleens identifies genes relevant to resistance.

BACKGROUND: Marek's disease virus (MDV) is an oncogenic herpesvirus that can cause T-cell lymphomas in chicken. Long noncoding RNA (lncRNA) is strongly associated with various cancers and many other diseases. In chickens, lncRNAs have not been comprehensively identified. Here, we profiled mRNA and lncRNA repertoires in three groups of spleens from MDV-infected and non-infected chickens, including seven tumorous spleens (TS) from MDV-infected chickens, five spleens from the survivors (SS) without lesions after MDV infection, and five spleens from noninfected chickens (NS), to explore the underlying mechanism of host resistance in Marek's disease (MD). RESULTS: By using a precise lncRNA identification pipeline, we identified 1315 putative lncRNAs and 1166 known lncRNAs in spleen tissue. Genomic features of putative lncRNAs were characterized. Differentially expressed (DE) mRNAs, putative lncRNAs, and known lncRNAs were profiled among three groups. We found that several specific intergroup differentially expressed genes were involved in important biological processes and pathways, including B cell activation and the Wnt signaling pathway; some of these genes were also found to be the hub genes in the co-expression network analyzed by WGCNA. Network analysis depicted both intergenic correlation and correlation between genes and MD traits. Five DE lncRNAs including MSTRG.360.1, MSTRG.6725.1, MSTRG.6754.1, MSTRG.15539.1, and MSTRG.7747.5 strongly correlated with MD-resistant candidate genes, such as IGF-I, CTLA4, HDAC9, SWAP70, CD72, JCHAIN, CXCL12, and CD8B, suggesting that lncRNAs may affect MD resistance and tumorigenesis in chicken spleens through their target genes. CONCLUSIONS: Our results provide both transcriptomic and epigenetic insights on MD resistance and its pathological mechanism. The comprehensive lncRNA and mRNA transcriptomes in MDV-infected chicken spleens were profiled. Co-expression analysis identified integrated lncRNA-mRNA and gene-gene interaction networks, implying that hub genes or lncRNAs exert critical influence on MD resistance and tumorigenesis.

Functional characterization of the Sox2, c-Myc, and Oct4 promoters.

To better understand the mechanisms in transcriptional regulation, we analyzed the promoters of the reprogramming key genes Sox2, c-Myc, and Oct4. Here, we cloned different 5' deletions of the goat Sox2, c-Myc, and Oct4 promoters, and evaluated their functions by green fluorescent protein reporter system and dual-luciferase reporter system. Site-directed mugagenesis and epigenetic modifiers were used to explore the influence of transcription binding sites and epigenetic status on the promoters. The results suggested that the basal promoters were located in the - 109 to 49, - 147 to 1, and - 96 to 30 bp regions of the Sox2, c-Myc, and Oct4 promoters. The transcription factors that identified to influence the Sox2, c-Myc, and Oct4 promoter activities were Elf-1 and activating protein 2 (AP-2), C/EBP and Sp1, and Mzf1 and Sp1, respectively. The epigenetic alternation of the Sox2, c-Myc, and Oct4 promoters by 5-aza-2'-deoxycytidine or/and trichostatin A significantly increased the promoter activities. In conclusion, the result determined the core promoter areas of the Sox2, c-Myc, and Oct4 genes, and identified the transcription factors that influence their promoter activities. We also verified that the Sox2, c-Myc, and Oct4 promoters were hypermethylated and hypoacetylated.

Study on immortal conditions of chicken embryonic stem cells.

In recent years, considerable attention has been paid to chicken embryonic stem cells (ESCs) studies in relation to extensive applications in gene therapy and regenerative medicine. However, the approaches used are still immature. In this study, we showed that the chicken ESCs clones with a clear border can express alkaline phosphatase and marker proteins such as SSEA-1, SOX2, and OCT4 stably. In addition, culture medium containing 10 µmol/L of vitamin C (VC) could significantly promote the proliferation of ESCs cells. Moreover, ESCs transfected with p:enhanced green fluorescent protein (pEGFP)-hTERT could be subcultured more than tenth generations in culture medium containing exogenous factors (mLIF + bFGF + hSCF) and VC, and these ESCs clone could still be regenerated following cryopreservation. Quantitative real-time polymerase chain reaction results showed that there was no significant difference between SSEA-1, SOX2, and OCT4 expression during ESCs immortalization and that the tenth generation of ESCs was still able to express marker proteins SSEA-1, SOX2, and OCT4. Our results showed that an immobilized system for ESCs was established, and the ESCs were cultured in vitro maintaining their pluripotency.

Genetic Architecture and Selection of Chinese Cattle Revealed by Whole Genome Resequencing.

The bovine genetic resources in China are diverse, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Chinese cattle, we analyzed the whole genomes of 46 cattle from six phenotypically and geographically representative Chinese cattle breeds, together with 18 Red Angus cattle genomes, 11 Japanese black cattle genomes and taurine and indicine genomes available from previous studies. Our results showed that Chinese cattle originated from hybridization between Bos taurus and Bos indicus. Moreover, we found that the level of genetic variation in Chinese cattle depends upon the degree of indicine content. We also discovered many potential selective sweep regions associated with domestication related to breed-specific characteristics, with selective sweep regions including genes associated with coat color (ERCC2, MC1R, ZBTB17, and MAP2K1), dairy traits (NCAPG, MAPK7, FST, ITFG1, SETMAR, PAG1, CSN3, and RPL37A), and meat production/quality traits (such as BBS2, R3HDM1, IGFBP2, IGFBP5, MYH9, MYH4, and MC5R). These findings substantially expand the catalogue of genetic variants in cattle and reveal new insights into the evolutionary history and domestication traits of Chinese cattle.

Transgenerational transmission of maternal stimulatory experience in domesticated birds.

The environmental stimuli experienced by a female can influence phenotypes and gene expression in the subsequent generations. We used a specifically designed domesticated-bird model to examine the transgenerational transmission of maternal stimulus exposure, a phenomenon that has been observed but has not been understood in noninbred animals. We subjected parental generation [filial (F)0] hens to viral- or bacterial-like stimulation after artificial insemination. Subsequent filial generations F1 and F2 transmitted growth or fertility variations without further stimulation in contrast to the controls. The whole-genome bisulfite sequence and next-generation mRNA sequencing of peripheral blood lymphocytes (PBLs) from the F1 generation revealed DNA methylome and transcriptome differences in the F1 polyriboinosinic:polyribocytidylic [poly(I:C)] acid or LPS offspring, compared with the F1 controls. In the F1 offspring, DNA methylation changes induced by maternal immune stimulation may have contributed to transcriptional variation. Pathways analysis indicated that the metabolic processes of xenobiotics and drug metabolism pathways, as well as reproduction-related pathways, were involved in the transgenerational transmission of maternal stimulatory experience. Furthermore, LPS-induced transcriptional transmission may have contributed to subfertility, as indicated by the results of comparative analysis between the transcriptomes of spleen tissues across the F0 and F1 generations, as well as the correlative analysis between the transcriptome and reproductive phenotypes. Our findings provide a framework for determining the mechanisms by which maternal stimulatory factors can be inherited transgenerationally with respect to growth, fertility, DNA methylation, and transcriptional levels in outbred animals.-Liu, L., Yang, N., Xu, G., Liu, S., Wang, D., Song, J., Duan, Z., Yang, S., Yu, Y. Transgenerational transmission of maternal stimulatory experience in domesticated birds.

Marek's Disease Virus Infection Induced Mitochondria Changes in Chickens.

Mitochondria are crucial cellular organelles in eukaryotes and participate in many cell processes including immune response, growth development, and tumorigenesis. Marek's disease (MD), caused by an avian alpha-herpesvirus Marek's disease virus (MDV), is characterized with lymphomas and immunosuppression. In this research, we hypothesize that mitochondria may play roles in response to MDV infection. To test it, mitochondrial DNA (mtDNA) abundance and gene expression in immune organs were examined in two well-defined and highly inbred lines of chickens, the MD-susceptible line 72 and the MD-resistant line 63. We found that mitochondrial DNA contents decreased significantly at the transformation phase in spleen of the MD-susceptible line 72 birds in contrast to the MD-resistant line 63. The mtDNA-genes and the nucleus-genes relevant to mtDNA maintenance and transcription, however, were significantly up-regulated. Interestingly, we found that POLG2 might play a potential role that led to the imbalance of mtDNA copy number and gene expression alteration. MDV infection induced imbalance of mitochondrial contents and gene expression, demonstrating the indispensability of mitochondria in virus-induced cell transformation and subsequent lymphoma formation, such as MD development in chicken. This is the first report on relationship between virus infection and mitochondria in chicken, which provides important insights into the understanding on pathogenesis and tumorigenesis due to viral infection.

Whole-genome bisulfite sequencing of goat skins identifies signatures associated with hair cycling.

BACKGROUND: Hair follicles (HFs), upon development, undergo repetitive cycles of growth (anagen), regression (catagen), and rest (telogen). The transition between the stages is determined by multiple molecular signals, including DNA methylation, which plays important roles in mammalian cellular identity and is essential for the development of HFs. Secondary hair follicles (SHFs) in cashmere goat exhibit classic cyclic hair development, and little has been done on a genome-wide scale to examine potentially methylated genes involved in the hair cyclic transition. RESULTS: Genome-wide DNA methylation profiles between skin tissues sampled during the anagen and telogen stages in cashmere goats were investigated using whole-genome bisulfite sequencing (WGBS). The methylation status was observed to be higher in the skin samples with HFs in the telogen than those in the anagen stage. A total of 1311 differentially methylated regions (DMRs) were identified between the two groups, which contained 493 fully annotated DMR-related genes (DMGs) (269 Hyper- DMGs and 224 Hypo-DMGs). Furthermore, a significant over-representation of the functional categories for DMGs related to immune response and intercellular crosstalk during hair cycling was observed. By integrating DNA methylation and mRNA expression data, we revealed that four genes (FMN1, PCOLCE, SPTLC3, and COL5A1) are crucial factors for elucidating epigenetic mechanisms contributing to the telogen-to-anagen transition. CONCLUSION: Our study provided systematic methylome maps pertaining to the hair cycling stages (anagen vs telogen) at a single-base resolution, and revealed stage-specific methylation loci during cashmere growth or quiescence. Furthermore, we identified epigenetically regulated genes that are potentially involved in HF development and growth in cashmere goats, and likely in other mammal species.

DNA methylation profiles correlated to striped bass sperm fertility.

BACKGROUND: Striped bass (Morone saxatilis) spermatozoa are used to fertilize in vitro the eggs of white bass (M. chrysops) to produce the preferred hybrid for the striped bass aquaculture industry. Currently, only one source of domestic striped bass juveniles is available to growers that is not obtained from wild-caught parents and is thus devoid of any genetic improvement in phenotypic traits of importance to aquaculture. Sperm epigenetic modification has been predicted to be associated with fertility, which could switch genes on and off without changing the DNA sequence itself. DNA methylation is one of the most common epigenetic modification types and changes in sperm epigenetics can be correlated to sub-fertility or infertility in male striped bass. The objective of this study was to find the differentially methylated regions (DMRs) between high-fertility and sub-fertility male striped bass, which could potentially regulate the fertility performance. RESULTS: In our present study, we performed DNA methylation analysis of high-fertility and sub-fertility striped bass spermatozoa through MBD-Seq methods. A total of 171 DMRs were discovered in striped bass sperm correlated to fertility. Based on the annotation of these DMRs, we conducted a functional classification analysis and two important groups of genes including the WDR3/UTP12 and GPCR families, were discovered to be related to fertility performance of striped bass. Proteins from the WDR3/UTP12 family are involved in forming the sperm flagella apparatus in vertebrates and GPCRs are involved in hormonal signaling and regulation of tissue development, proliferation and differentiation. CONCLUSIONS: Our results contribute insights into understanding the mechanism of fertility in striped bass, which will provide powerful tools to maximize reproductive efficiencies and to identify those males with superior gametes for this important aquaculture species.

Cloning, expression pattern analysis, and subcellular localization of Capra hircus SCD1 gene with production of transgenic mice.

This study aimed to clone the Stearoyl-CoA desaturase 1 (SCD1) gene derived from Xuhuai goat (Capra hircus), and analyze the sub-cellular localization in cells and tissues. The cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR). pEGFP-SCD1 vector was constructed to detect sub-cellular localization and tissue distribution. pEGFP-SCD1 was transfected into NIH-3T3 cells using polyethylene imine (PEI) and observed under fluorescence inversion microscope system 48 h after transfection. The expression level of SCD1 was detected by RT-PCR. Testicular injection was used to produce transgenic mice with goat SCD1 gene. DNA and protein were extracted from the tail tissue of F1 mice. The expression of exogenous gene in the F1 generation was detected in both DNA and protein. The results showed that the coding sequence (CDS) fragments of C. hircus SCD1 gene was 1074 bp and encodes 360 amino acids. RT-PCR results showed that SCD1 could be expressed successfully in NIH-3T3 cells in vitro. Sub-cellular localization analysis showed that pEGFP-SCD1 fusion protein located in the cytoplasm. It can be concluded that transgenic mice with goat SCD1 expressed in sperm and tail tissue was successfully produced in the F1 mice generation.

RXRG associated in PPAR signal regulated the differentiation of primordial germ cell.

Primitive germ cells (PGCs) are the "ancestor" of sex germ cells. However, their regulation mechanism is not clear at present. The aim of this study is to investigate the effect of PPAR signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into PGCs, providing theoretical support for the further study of the mechanism of regulation of chicken PGCs. Based on the results of RNA-Seq analysis, the key signal pathway PPAR and the key signal molecule RXRG in the pathway were successfully screened. Also, we made the PPAR signal transduction pathway be activated in vivo and in vitro. The results showed that the expression of FABP3 and SCD-1 was up-regulated by high expression of RXRG, which significantly up-regulated the expression of Cvh, C-kit, and the ratio of Cvh + C-kit+ cells was higher than that of control group. The formation of PGCs was blocked by interfering with PPAR signaling pathway. We conclude that RXRG positively regulates the PPAR signaling pathway and promotes differentiation of chicken ESCs into PGCs. Thus, gene RXRG can be used to study the regulation of PGCs differentiation in chickens.

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells.

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta-hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down-regulation of the steroid hormones synthesis pathway. Furthermore, qRT-PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway.