Circular RNA (circRNA) is a type of noncoding RNA involved in a variety of biological processes, especially in post-transcriptional regulation. The granulosa cells of follicles play a determining role in ovarian development. However, the function of circRNA in chicken follicles is unclear. To better understand the molecular mechanism underlying follicular development and granulosa cell function, we performed a strategy of second-generation sequencing and linear RNA depletion for granulosa cells from small yellow follicles (SYF, 5–8 mm), the smallest hierarchal follicles (F6, 9–12 mm), and the largest hierarchal follicles (F1, ~ 40 mm). We predicted a total of 11,642 circRNAs that distributed on almost all chromosomes. The majority of the splice lengths of circRNAs were 200–500 nt and mainly produced from intron and CDS regions. During follicle growth, differentially expressed (DE) circRNAs showed dynamic changes which were tissue- and stage-specific. The host genes of DE circRNAs were functionally enriched in GTPase activity and several pathways involved in reproduction. Moreover, bioinformatic prediction analysis for circRalGPS2 demonstrated that circRNAs from the same genes may share common miRNA to act as a sponge. The predicted target genes were enriched in various biological processes including cognition, cell communication, and regulation of signaling, and several pathways related to reproduction such as tight junction, oocyte meiosis, progesterone-mediated oocyte maturation, and GnRH signaling. This study provides a starting point for further experimental investigations into chicken circRNAs and casts a light on the understanding of follicle development.

MicroRNAs play an important role in the growth and development of chicken embryos, including the regulation of skeletal muscle genesis, myoblast proliferation, differentiation, and apoptosis. Our previous RNA-seq studies showed that microRNA-27b-3p (miR-27b-3p) might play an important role in regulating the proliferation and differentiation of chicken primary myoblasts (CPMs). However, the mechanism of miR-27b-3p regulating the proliferation and differentiation of CPMs is still unclear. In this study, the results showed that miR-27b-3p significantly promoted the proliferation of CPMs and inhibited the differentiation of CPMs. Then, myostatin (MSTN) was confirmed to be the target gene of miR-27b-3p by double luciferase reporter assay, RT-qPCR, and Western blot. By overexpressing and interfering with MSTN expression in CPMs, the results showed that overexpression of MSTN significantly inhibited the proliferation and differentiation of CPMs. In contrast, interference of MSTN expression had the opposite effect. This study showed that miR-27b-3p could promote the proliferation of CPMs by targeting MSTN. Interestingly, both miR-27b-3p and MSTN can inhibit the differentiation of CPMs. These results provide a theoretical basis for further understanding the function of miR-27b-3p in chicken and revealing its regulation mechanism on chicken muscle growth.

Development of ovarian follicles requires interactions between granulosa cells, theca cells, and oocytes. Multiple transcription levels are involved but information about the role of noncoding RNAs, especially circular RNAs (circRNAs), is lacking. Here, we used RNA sequencing to profile circRNAs and mRNAs in theca cells from three types of follicle: small yellow follicles (SYF), the smallest hierarchical follicles (F6), and the largest hierarchical follicles (F1). Using bioinformatics analysis, we identified a total of 14,502 circRNAs in all theca cells, with 5622 widely distributed in all stages of development. Differential expression analysis suggested that some genes display differential isoforms during follicular development. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed enrichment of both differentially expressed circRNAs and mRNAs in pathways associated with reproduction, including the TGF-β signaling pathway, oocyte meiosis, and vascular smooth muscle contraction. Our study provides the first visual information about circRNAs and mRNAs in theca cells during follicle development in chickens and adds to the growing body of knowledge about theca cells.

Background: Phosphorus is essential for bone mineralization in broilers, however, the underlying mechanisms remain unclear. We aimed to investigate whether bone phosphorus retention and bone development might be regulated by related hormones and local bone-derived regulators in broilers. Methods: Broilers were fed diets containing different levels of non-phytate phosphorus (NPP) 0.15%, 0.25%, 0.35%, 0.45% and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% and 0.43% from 1 to 21 or 22 to 42 days of age. Serum and tibia samples were collected for determinations of bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators of broiler chickens on d 14, 28 and 42, respectively. Results: Tibia ash phosphorus, total phosphorus accumulation in tibia ash (TP_TA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS), and ash on d 14, 28 or 42, serum 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on d 28 and 42, mRNA expressions of tibia fibroblast growth factor 23 (FGF23) and dentin matrix protein 1 (DMP1) on d 14 and 28 increased linearly or quadratically (P < 0.05), while serum parathyroid hormone (PTH) on d 28, tibia alkaline phosphatase (ALP) on d 14, 28 and 42, bone gal protein (BGP) on d 14, and mRNA expression of tibia phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) on d 14 and 28 decreased linearly or quadratically (P < 0.04) as dietary NPP level increased. TP_TA, BMC, BMD, and ash on d 28 and 42, BBS on d 28, and ash phosphorus on d 42 were positively correlated (r = 0.389 to 0.486, P < 0.03) with serum 1,25(OH)₂D₃. All of the above parameters were positively correlated (r = 0.380 to 0.689, P < 0.05) with tibia DMP1 mRNA expression on d 14, 28 and 42, but negatively correlated (r = − 0.609 to − 0.538, P < 0.02) with serum PTH on d 28, tibia ALP on d 14, 28 and 42, and BGP on d 14. TP_TA, BMC and ash on d 14 and BMD on d 28 were negatively correlated (r = − 0.397 to − 0.362, P < 0.03) with tibia PHEX mRNA expression, and BMD on d 28 was positively correlated (r = 0.384, P = 0.04) with tibia FGF23 mRNA expression. Conclusions: These results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH)₂D₃ and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators.

A series of elaborately regulated and orchestrated changes in gene expression profiles leads to muscle growth and development. In this study, RNA sequencing was used to profile embryonic chicken myoblasts and fused myotube transcriptomes, long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) at four stages of myoblast differentiation. Of a total of 2484 lncRNA transcripts, 2288 were long intergenic non-coding RNAs (lincRNAs) and 198 were antisense lncRNAs. Additionally, 1530 lncRNAs were neighboring 2041 protein-coding genes (<10 kb upstream and downstream) and functionally enriched in several pathways related to skeletal muscle development that have been extensively studied, indicating that these genes may be in cis-regulatory relationships. In addition, Pearson’s correlation coefficients demonstrated that 990 lncRNAs and 7436 mRNAs were possibly in trans-regulatory relationships. These co-expressed mRNAs were enriched in various developmentally-related biological processes, such as myocyte proliferation and differentiation, myoblast differentiation, and myoblast fusion. The number of transcripts (906 lncRNAs and 4422 mRNAs) differentially expressed across various stages declined with the progression of differentiation. Then, 4422 differentially expressed genes were assigned to four clusters according to K-means analysis. Genes in the K1 cluster likely play important roles in myoblast proliferation and those in the K4 cluster were likely associated with the initiation of myoblast differentiation, while genes in the K2 and K3 clusters were likely related to myoblast fusion. This study provides a catalog of chicken lncRNAs and mRNAs for further experimental investigations and facilitates a better understanding of skeletal muscle development.

The proliferation and differentiation of myoblasts is an important process of skeletal muscle development. In this process, microRNAs (miRNAs) play an important role in the proliferation and differentiation of chicken primary myoblasts (CPMs). Our previous study found that miR-214 and the tRNA methyltransferase 61A (TRMT61A) gene were differentially expressed in different stages of proliferation and differentiation. Therefore, this study aimed to explore the effect of miR-214 on the proliferation and differentiation of CPMs and the functional relationship between miR-214 and TRMT61A. In this study, we detected the effect of miR-214 on the proliferation of CPMs by qPCR, flow cytometry, CCK-8, and EdU after the overexpression and interference of miR-214. qPCR, Western blotting, and indirect immunofluorescence were used to detect the effect of miR-214 on the differentiation of the CPMs. The expression patterns of miR-214 and TRMT61A were observed at different time points of differentiation induced by the CPMs. The results show that miR-214 inhibited the proliferation of the CPMs and promoted the differentiation of the CPMs. The Dual-Luciferase Reporter assay and the expression pattern of miR-214 and TRMT61A suggested that they had a negative regulatory target relationship. This study revealed the function and regulatory mechanism of miR-214 in the proliferation and differentiation of CPMs.