Jiwei Hu

Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos

Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our understanding of the location-specific and breed-specific genes and pathways but also provides some candidate molecular targets for increasing muscle products and meat quality by genetic control.

Establishment of an in vitro culture model of theca cells from hierarchical follicles in ducks

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Gene expression patterns, and protein metabolic and histological analyses for muscle development in Peking duck

In this study, we aimed to use duck breast muscle and leg muscle, the 2 main productive muscle organs, as a model to elucidate the molecular mechanism controlling how the 2 muscles have different deposition capabilities, and to analyze the mechanisms facilitating duck muscle development posthatching. Peking duck breast muscle and leg muscle were collected 3, 7, and 16 wk posthatching. The morphology of the myofibers was observed by paraffin sectioning the muscles. The expression of genes involved in protein metabolism [mammalian target of rapamycin (mTOR), RPS6-p70-protein kinase (S6K), forkhead box O1 (FoxO1), muscle RING finger 1 (MuRF1), and atrogin-1 (MAFbx)] was detected using real-time quantitative PCR and Western blot assays, and the results indicated that breast muscle had a stronger capacity for both protein synthesis and protein degradation compared with leg muscle. Satellite cell frequency declined during muscle development in both tissues, and the expression of Pax3/7, satellite cell marker genes, was not significantly different between breast muscle and leg muscle. No notable apoptosis was observed in either tissue. The results of this study suggest that protein metabolism signaling is the main reason promoting duck skeletal muscle mass gain.

Comparison of growth characteristics of in vitro cultured granulosa cells from geese follicles at different developmental stages

Granulosa cells (GCs) are essential components of follicles and are involved in regulating the process of follicles development. However, comparative studies on GCs isolated from different staged follicles have not been conducted in goose. The aim of the present study was to identify the growth characteristics of goose GCs from pre-hierarchical (6–10 mm) and hierarchical (F4–F2, F1) follicles. Our results showed that the three cohorts of cells had different tolerance to collagenase and had noticeable morphological differences. The F1 granulosa layers were fully digested by 0.1% collagenase, while higher concentration (0.3%) was used for both F4–F2 and pre-hierarchical granulosa layers. In the state of suspension, the diameter of F1 individual cell was larger than the other two cohorts. However, after adhering to the culture plate, cells of F1 just had changes in the diameter accompanied by small bright spots, while both pre-hierarchical and F4–F2 GCs proliferated rapidly with spreading and irregularly shaped voids. Furthermore, all attached cells could be stained by the follicle-stimulating hormone receptor antibody. Analyses of both growth curve and the mRNA expression profiles of genes related to cellular proliferation, apoptosis, and steroidogenesis suggested that three cohorts of in vitro cultured GCs had different physiological viability and functions. Taken together, the present study not only revealed differences of the growth characteristics among three cohorts of goose GCs from pre-hierarchical, F4–F2 and F1 follicles, but also optimized the in vitro culture system of geese different staged GCs.

CYP17 gene plays a key role in goose genital growth by influencing the testosterone level at puberty

&NA; All birds reproduce by internal fertilization, but only 3% of birds have external genitalia. Hormone secretions and body size influence genital growth, but the actual regulatory mechanism is rarely reported. Thus, using 35 geese as experimental material, the regulatory mechanism of goose external genitalia growth was explored by measuring body size parameters, serum hormone concentrations, and related gene expression. In this study, genital growth was different among tested geese, but histological and morphological results showed that all geese external genitalia contained complete tissues. Measurements of hormone levels showed that at puberty, as the genital length increased, irregular decreases were observed in the levels of follicle‐stimulating hormone (FSH) and luteinizing hormone (LH), whereas an irregular increase was observed in the levels of testosterone (T); furthermore, the levels of testosterone (T) gradually increased to a peak at 34 weeks. Based on RT‐PCR results, as the genital length increased, only the expression of 17&agr;‐hydroxylase/17, 20‐lyase (CYP17) mRNA slightly decreased at first, and then significantly increased to a peak, whereas the expression patterns of other genes were irregular. Furthermore, the CYP17 immunohistochemistry results also showed a pattern that was highly consistent with the patterns of mRNA expression and T secretion. In addition, based on body measurements, as body weight increased, the genital length increased. Thus, these results suggested that the CYP17 gene plays a key role in goose genital growth.

Characterization of the Goose CAPN3 Gene and its Expression Pattern in Muscle Tissues of Sichuan White Geese at Different Growth Stages

Calpain 3 (CAPN3), also known as p94, is associated with multiple production traits in domestic animals. However, the molecular characteristics of the CAPN3 gene and its expression profile in goose tissues have not been reported. In this study, CAPN3 cDNA of the Sichuan white goose was cloned, sequenced, and characterized. The CAPN3 full-length cDNA sequence consists of a 2,316-bp coding sequence (CDS) that encodes 771 amino acids with a molecular mass of 89,019 kDa. The protein was predicted to have no signal peptide, but several N-glycosylation, O-glycosylation, and phosphorylation sites. The secondary structure of CAPN3 was predicted to be 38.65% α-helical. Sequence alignment showed that CAPN3 of Sichuan white goose shared more than 90% amino acid sequence similarity with those of Japanese quail, turkey, helmeted guineafowl, duck, pigeon, and chicken. Phylogenetic tree analysis showed that goose CAPN3 has a close genetic relationship and small evolutionary distance with those of the birds. qRT-PCR analysis showed that in 15-day-old animals, the expression level of CAPN3 was significantly higher in breast muscle than in thigh tissues. These results serve as a foundation for further investigations of the function of the goose CAPN3 gene.

Molecular characterization, expression and cellular localization of CYP17 gene during geese ( Anser cygnoides ) follicular development

As a key member of the cytochrome P450 gene superfamily, CYP17 gene encodes 17α-hydroxylase/C17,20-lyase that is critical for directing androgen synthesis. The CYP17 gene has been identified in several species, yet little is known about its distribution and expression profile during goose follicular development. In the present study, we obtained the full-length coding sequence of goose CYP17 (gCYP17) gene for the first time using RACE method. Its sequence alignment and phylogenetic analysis suggested that gCYP17 was highly conserved with those of other birds and consisted of four main functional domains like other species. Results from immunohistochemistry, quantitative real-time PCR and Western blotting suggested that gCYP17 was predominantly located in theca interna throughout follicular development. Furthermore, levels of gCYP17 reached the maximum in theca layer of the 6-8 mm follicles which were significantly higher than in those of other follicles (P < 0.05). In addition, gCYP17 was expressed at much higher levels in the F4 theca layer than the F1 follicle (P < 0.05). Therefore, these results indicated that the fluctuating expression pattern and specific cellular localization of gCYP17 during follicular development might be closely related to androgen secretion, and thereby follicular maturation.

Molecular cloning, characterization and expression analysis of C/EBP α, β and δ in adipose-related tissues and adipocyte of duck (Anas platyrhynchos)

CCAAT/enhancer binding protein α, β, δ (C/EBP α, β, δ) are essential transcriptional factors in regulating adipose development. However, information about their sequence characteristics and functions during adipocyte development still remains scarce in birds. In present study, we found that duck C/EBP α, β, δ differed in their phosphorylation sites and low complexity regions (LCRs) among their orthologs and paralogs. Phylogenetic analysis showed that C/EBP α, β, δ had different evolutionary patterns, and each of duck C/EBP α, β, δ was strikingly diverged from orthologs of other Aves. Results of quantitative real-time PCR exhibited that C/EBP α, β, δ were all highly expressed in duck adipose tissues. Indeed, investigations of changes in both their mRNA levels and lipid droplet content during duck adipocytes differentiation showed that their expression profiles were closely related to cellular lipid accumulation. Furthermore, hierarchical clustering analysis of the C/EBPs and lipid metabolism-related genes expression profiles showed that C/EBP α was clustered with genes related to lipolysis, lipogenesis and fatty acid desaturation, whereas C/EBP β, δ were clustered with genes related to de novo lipogenesis and fatty acid elongation, which were different from mammals. In summary, C/EBP α, β, δ of duck differ from other species in their structures and have different effects on lipid metabolism during adipocytes differentiation. This research serve as a foundation for further investigations about avian C/EBP α, β, δ in adipocytes differentiation and adipose development.

Long-term thermal manipulation in the late incubation period can inhibit breast muscle development by activating endoplasmic reticulum stress in duck ( Anasplatyrhynchos domestica )

Poultry embryos are easily affected by environmental changings during incubation, thereinto, the temperature modification is the most important one, but the mechanism of temperature effects on bird eggs is not clear. By using RNA-seq, we have previously found that endoplasmic reticulum stress (ERS) may involve in regulating embryonic muscle development of duck under the influence of temperature alteration. To further clarify the role of ERS in the effect, in the present study, we detected the impact of increasing the incubation temperature by 1℃ during embryonic days 10-27 (E10-27) on the development of duck embryos, and investigated the changes in mRNA and protein expression of ERS marker genes and muscle-related genes under the thermal manipulation (TM). The results of relative weight comparison showed that only the relative weight of breast muscle was steadily decreased by TM from E10 to the first day after hatching (W0). Meanwhile, the real-time PCR and western-blot analysis revealed that raising the incubation temperature stimulated the expression of ERS marker genes in breast muscle at E20. The mRNA expressions of muscle hypertrophy and atrophy-related genes were also detected, and were not changed regularly, however, the protein expressions of hypertrophy-related genes were all decreased at both E20 and W0, and the protein expression of atrophy-related genes were up-regulated at E20. The protein expression of muscle proliferation-related genes were also decreased at E20. Additionally, these results were the same as that in the ERS positive control groups. Taken together, these results indicated that long-term TM during late embryonic period could block the development of duck breast muscle by inhibiting muscle hypertrophy and proliferation, and promoting muscle atrophy at a post-transcriptional level via the activation of ERS.