Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds.

BACKGROUND: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. METHODS: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. RESULTS: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. CONCLUSION: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

Investigating the genetic determination of duration-of-fertility trait in breeding hens.

The duration-of-fertility (DF), which was defined as the number of days when breeding hens lay fertile eggs following copulation or artificial insemination (AI), is an important economic trait in chick production when it has strong effects on fertile egg output and production costs. Little is known about the underlying genes and molecular markers related to DF trait to date. Here, we measured the DF of 701 Chinese Jinghong hens and 408 Jingfen hens. The DF showed high individual variability and potential for genetic improvement. Then, 192 Jinghong breeding hens were provided for a genome-wide association study, 27 SNPs respectively located in three genomic linkage regions (GGA1:41Kb; GGA3:39Kb and GGA8:39Kb) were suggested to be significantly associated with DF. Particularly, 6 of these 27 SNPs were further verified to be associated with DF in the 701 Jinghong and 408 Jingfen hens using PCR-RFLP genotyping method. These 27 SNPs were also mapped to 7 genes according to their genomic position. Furtherly, 5 of these 7 genes were tested using qPCR. Results show that the CYP2D6, WBP2NL, ESR1 and TGFBR3 mRNA expression levels of hens with long DF were significantly higher than the hens with short DF (P < 0.05). Overall, findings in our research provide new insight into the genetic basis of duration-of-fertility in breeding hens while providing new clues for further functional validation on the DF-related genetic regulation mechanism and improvement of DF through chicken breeding.

Transcriptome sequencing revealed that knocking down FOXL2 affected cell proliferation, the cell cycle, and DNA replication in chicken pre-ovulatory follicle cells.

Forkhead box L2 (FOXL2) is a single-exon gene encoding a forkhead transcription factor, which is mainly expressed in the ovary, eyelids and the pituitary gland. FOXL2 plays an essential role in ovarian development. To reveal the effects of FOXL2 on the biological process and gene expression of ovarian granulosa cells (GCs), we established stable FOXL2-knockdown GCs and then analysed them using transcriptome sequencing. It was observed that knocking down FOXL2 affected the biological processes of cell proliferation, DNA replication, and apoptosis and affected cell cycle progression. FOXL2 knockdown promoted cell proliferation and DNA replication, decreased cell apoptosis, and promoted mitosis. In addition, by comparing the transcriptome after FOXL2 knockdown, we found a series of DEGs (differentially expressed genes) and related pathways. These results indicated that, through mediating these genes and pathways, the FOXL2 might induce the cell proliferation, cycle, and DNA replication, and play a key role during ovarian development and maintenance.

Gene co-expression network analysis reveals key potential gene modules in utero-vaginal junction associated with duration of fertility trait of breeder hens.

The number of days (DN) when hens lay fertile eggs as well as the number of fertile eggs (FN) were produced after a single artificial insemination (AI), including the two duration of fertility (DF) traits. Indeed, they are the key production performance that associates with the production cost of hatching egg when its determination the interval between successive artificial inseminations. However, the relevant genes response for regulating the DF has not been uncovered yet. Therefore, we performed a weighted gene co-expression network analysis (WGCNA) to investigate the insight into co-expression gene modules on DF process in hens. The total mRNA was extracted from the utero-vaginal junction (UVJ, with the sperm storage function in hen's oviduct which is the biological basis for DF) of 20 hens with several levels of DF traits, and performed transcriptome sequences of mRNA. As a result, three co-expression gene modules were identified to be highly correlated with DF traits. Moreover, the expression changes of top 5 hub genes in each module with DF traits were further confirmed in other 20 hens by RT-PCR. These findings highlighted the co-expression modules and their affiliated genes as playing important roles in the regulation of DF traits.

Transcriptome sequencing reveals key potential long non-coding RNAs related to duration of fertility trait in the uterovaginal junction of egg-laying hens.

Duration of fertility, (DF) is an important functional trait in poultry production and lncRNAs have emerged as important regulators of various process including fertility. In this study we applied a genome-guided strategy to reconstruct the uterovaginal junction (UVJ) transcriptome of 14 egg-laying birds with long- and short-DF (n = 7); and sought to uncover key lncRNAs related to duration of fertility traits by RNA-sequencing technology. Examination of RNA-seq data revealed a total of 9977 lncRNAs including 2576 novel lncRNAs. Differential expression (DE) analysis of lncRNA identified 223 lncRNAs differentially expressed between the two groups. DE-lncRNA target genes prediction uncovered over 200 lncRNA target genes and functional enrichment tests predict a potential function of DE-lncRNAs. Gene ontology classification and pathway analysis revealed 8 DE-lncRNAs, with the majority of their target genes enriched in biological functions such as reproductive structure development, developmental process involved in reproduction, response to cytokine, carbohydrate binding, chromatin organization, and immune pathways. Differential expression of lncRNAs and target genes were confirmed by qPCR. Together, these results significantly expand the utility of the UVJ transcriptome and our analysis identification of key lncRNAs and their target genes regulating DF will form the baseline for understanding the molecular functions of lncRNAs regulating DF.

A transcriptomic comparison of theca and granulosa cells in chicken and cattle follicles reveals ESR2 as a potential regulator of CYP19A1 expression in the theca cells of chicken follicles.

Previous studies have shown that theca and granulosa cell layers in follicles do not play the same roles in mammals and birds, especially regarding the synthesis of estrogen. The functions of these two cell types have been well characterized in cattle, but they remain unclear in chickens. To clarify this issue, a comparison of small yellow follicles (SYFs) in chickens and cattle at different follicular development stages was done by weighted gene co-expression network analysis (WGCNA). The modules obtained from WGCNA were used for further identification of the key genes associated with CYP19A1 expression. Module preservation analysis showed high similarity between cow_D (the follicular phase before the LH surge) and chicken_SYF (small yellow follicle between 6 and 8 mm in diameter) datasets, and 10 top hub genes highly associated with CYP19A1 expression in chicken SYFs were identified in each module. A comparison of the transcriptomes of theca and granulosa cells (TCs and GCs) between chicken SYFs and cattle follicles at the differentiation stage, as well as the aforementioned hub genes, revealed that ESR2 is a potential regulator of CYP19A1 expression in the theca cells of chicken SYFs. Furthermore, 197 cell-specific (179 in theca and 18 in granulosa) and 235 cell-biased expressed genes (196 in theca and 39 in granulosa) in chicken small yellow follicles were also identified by transcriptomic comparison of theca and granulosa cells.

Identifying artificial selection signals in the chicken genome.

Identifying the signals of artificial selection can contribute to further shaping economically important traits. Here, a chicken 600k SNP-array was employed to detect the signals of artificial selection using 331 individuals from 9 breeds, including Jingfen (JF), Jinghong (JH), Araucanas (AR), White Leghorn (WL), Pekin-Bantam (PB), Shamo (SH), Gallus-Gallus-Spadiceus (GA), Rheinlander (RH) and Vorwerkhuhn (VO). Per the population genetic structure, 9 breeds were combined into 5 breed-pools, and a 'two-step' strategy was used to reveal the signals of artificial selection. GA, which has little artificial selection, was defined as the reference population, and a total of 204, 155, 305 and 323 potential artificial selection signals were identified in AR_VO, PB, RH_WL and JH_JF, respectively. We also found signals derived from standing and de-novo genetic variations have contributed to adaptive evolution during artificial selection. Further enrichment analysis suggests that the genomic regions of artificial selection signals harbour genes, including THSR, PTHLH and PMCH, responsible for economic traits, such as fertility, growth and immunization. Overall, this study found a series of genes that contribute to the improvement of chicken breeds and revealed the genetic mechanisms of adaptive evolution, which can be used as fundamental information in future chicken functional genomics study.

Novel copy number variation of the TGFß3 gene is associated with TGFß3 gene expression and duration of fertility traits in hens.

Improvements in the duration of fertility (DF) could increase the interval between successive artificial inseminations, thereby decreasing the cost associated with production of hatching eggs. The molecular mechanisms involved in DF in hens remains under-explored. In this study, expression levels of the transforming growth factor-ß genes (TGFßs: TGFß1, TGFß2, TGFß3) were investigated in utero-vaginal junctions (UVJs) of hens with long DF (Group L, n = 10) and short DF (Group S, n = 10). TGFß1 and 2 tended to exhibit higher expression levels in UVJs from Group L hens. The expression levels of TGFß3 mRNA and protein were significantly increased in UVJs of hens from Group L compared to hens in Group S. Consistently, six TGFßs downstream genes (DAXX, MEKK1, T-BET, GATA-3, TAK1, and FOXP3) associated with the immune response were found to be significantly differentially expressed in UVJs of Group L than Group S hens. In addition, four SNPs were identified in intron 1 of TGFß3, and these SNPs were significantly associated with DF traits (P < 0.05). Furthermore, we identified multi-copy and copy number variants (CNVs) in chicken TGFß3 and later determined significant associations between TGFß3 CNVs and DF traits in hens. Specifically, TGFß3 copy number exhibited a significant positive correlation with its expression (P < 0.05). Collectively, our results suggest that chicken DF traits may be regulated by the expression of TGFß3 in UVJ. Meanwhile, the copy number variation in the TGFß3 gene identified in this study seems to be one marker for DF traits.