Background: MicroRNAs have been suggested to play important roles in the regulation of gene expression in various biological processes. To investigate the function of miRNAs in chicken ovarian development and folliculogenesis, two small RNA libraries constructed from sexually mature (162-day old) and immature (42-day old) ovary tissues of Single Comb White Leghorn chicken were sequenced using Illumina small RNA deep sequencing. Results: In the present study, 14,545,100 and 14,774,864 clean reads were obtained from sexually mature (162-d) and sexually immature (42-d) ovaries, respectively. In total, 202 known miRNAs were identified, and 93 of them were found to be significantly differentially expressed: 42 miRNAs were up-regulated and 51 miRNAs were down-regulated in the mature ovary compared to the immature ovary. Among the up-regulated miRNAs, gga-miR-1a has the largest fold-change (6.405-fold), while gga-miR-375 has the largest fold-change (11.345-fold) among the down-regulated miRNAs. The three most abundant miRNAs in the chicken ovary are gga-miR-10a, gga-let-7 and gga-miR-21. Five differentially expressed miRNAs (gga-miR-1a, 21, 26a, 137 and 375) were validated by real-time quantitative RT-PCR (qRT-PCR). Furthermore, the expression patterns of the five miRNAs were analyzed in different developmental stages of chicken ovary and follicles of various sizes. Conclusion: The present study provides the first miRNA profile in sexually immature and mature chicken ovaries. Some miRNAs such as gga-miR-1a and gga-miR-21are expressed differentially in immature and mature chicken ovaries as well as among different sized follicles, suggesting an important role in the follicular growth or ovulation mechanism in the chicken.

Ovarian follicle selection is an important process impacting the laying performance and fecundity of hens, and is regulated by follicle-stimulating hormone (FSH) through binding to its receptor (FSHR). In laying hens, the small yellow follicle (6–8 mm in diameter) with the highest expression of FSHR will be recruited into the preovulatory hierarchy during ovarian follicle development.The study of molecular mechanism of chicken follicle selection is helpful for the identification of genes underlying egg laying traits in chicken and other poultry species. Herein, the transcriptomes of chicken small yellow follicles differing in the mRNA expression of FSHR were compared, and a total of 17,993 genes were identified in three pairs of small yellow follicles. The Wnt4 signaling pathway was significantly enriched in the follicles with the greatest fold change in FSHR expression. In this pathway, the expression level of Wnt4 mRNA was significantly upregulated with a log2(fold change) of 2.12. We further investigated the expression, function, and regulation of Wnt4 during chicken follicle selection and found that Wnt4 mRNA reached its peak in small yellow follicles; Wnt4 stimulated the proliferation of follicular granulosa cells, increased the expression of StAR and CYP11A1 mRNA in prehierarchical and hierarchical follicles, increased the expression of FSHR mRNA, and decreased the expression of AMH and OCLN mRNA. Treatment with FSH significantly increased Wnt4 expression in granulosa cells. Moreover, Wnt4 facilitated the effects of FSH on the production of progesterone (P4) and the mRNA expression of steroidogenic enzyme genes in the granulosa cells of hierarchical follicles, but inhibited the effects of FSH in the granulosa cells of prehierarchical follicles. Collectively, these data suggest that Wnt4 plays an important role in chicken follicle selection by stimulating granulosa cell proliferation and steroidogenesis. This study provides a theoretical basis for improving the egg laying performance of chicken, and a reference for the elucidation of the molecular mechanism of follicular selection in mammals.

Background: Follicle selection in chickens refers to the process of selecting one follicle from a group of small yellow follicles (SY, 6–8 mm in diameter) for development into 12–15 mm hierarchical follicles (usually F6 follicles), which is an important process affecting laying performance in the poultry industry. Although transcriptomic analysis of chicken ovarian follicles has been reported, integrated analysis of chicken follicles for selection by using both transcriptomic and proteomic approaches is still rarely performed. In this study, we compared the proteomes and transcriptomes of SY and F6 follicles in laying hens and identified several genes involved in chicken follicle selection. Results: Transcriptomic analysis revealed 855 differentially expressed genes (DEGs) between SY follicles and F6 follicles in laying hens, among which 202 were upregulated and 653 were downregulated. Proteomic analysis revealed 259 differentially expressed proteins (DEPs), including 175 upregulated and 84 downregulated proteins. Among the identified DEGs and DEPs, changes in the expression of seven genes, including VLDLR1, WIF1, NGFR, AMH, BMP15, GDF6 and MMP13, and nine proteins, including VLDLR, VTG1, VTG3, PSCA, APOB, APOV1, F10, ZP2 and ZP3L2, were validated. Further analysis indicated that the mRNA level of chicken VLDLR was higher in F6 follicles than in SY follicles and was also higher in granulosa cells (GCs) than in thecal cells (TCs), and it was stimulated by FSH in GCs. Conclusions: By comparing the proteomes and transcriptomes of SY and F6 follicles in laying hens, we identified several differentially expressed proteins/genes that might play certain roles in chicken follicle selection. These data may contribute to the identification of functional genes and proteins involved in chicken follicle selection.

The intramuscular fat content (IMF) refers to the amount of fat within muscles, including the sum of phospholipids mainly found in cell membranes, triglycerides and cholesterol, and is determined both by hyperplasia and hypertrophy of adipocyte during the development of pigs. The IMF content is an important economic trait that is genetically controlled by multiple genes. The Laiwu pig is an indigenous fatty pig breed distributed in North China, characterized by excessively higher level of IMF content (9%~12%), therefore, is suitable for the identification of genes controlling IMF variations. To identify genes underlying IMF deposition, we performed genome-wide transcriptome and methylome analyses on longissimus dorsi (LD) muscle in Laiwu pigs across four developmental stages. A total of 22,524 expressed genes were detected and 1158 differentially expressed genes (DEGs) were hierarchically clustered in the LD muscle over four developmental stages from 60 d to 400 d. These genes were significantly clustered into four temporal expression profiles, and genes participating in fat cell differentiation and lipid biosynthesis processes were identified. From 120 d to 240 d, the period with the maximum IMF deposition rate, the lipid biosynthesis related genes (FOSL1, FAM213B and G0S2), transcription factors (TFs) (EGR1, KLF5, SREBF2, TP53 and TWIST1) and enriched pathways (steroid biosynthesis and fatty acid biosynthesis) were revealed; and fat biosynthesis relevant genes showing differences in DNA methylation in gene body or intergenic region were detected, such as FASN, PVALB, ID2, SH3PXD2B and EGR1. This study provides a comprehensive landscape of transcriptome of the LD muscle in Laiwu pigs ranging from 60 to 400 days old, and methylome of the LD muscle in 120 d and 240 d Laiwu pigs. A set of candidate genes and TFs involved in fat biosynthesis process were identified, which were probably responsible for IMF deposition. The results from this study would provide a reference for the identification of genes controlling IMF variation, and for exploring molecular mechanisms underlying IMF deposition in pigs.

The Jining Gray goat is famous for its sexual precocity; however, the exact regulatory mechanism is still unknown. The hypothalamus is the key centrum in the process of animal reproduction, especially in signal transduction, and the initiation of puberty. The identification of potential genes and pathways in the hypothalamus of Jining Gray goat is critical to understanding the regulatory mechanism of sexual precocity in these goats. In this study, mRNA transcriptome analysis of the hypothalamus of juvenile and pubertal goats revealed eight genes (NTS, ADORA1, CRH, UCN3, E2F2, PDGFRB, GNRH1, and CACNA1C) and three pathways [neuroactive ligand-receptor interaction; gonadotropin-releasing hormone (GnRH) signal; melanoma] that are involved in this regulation. Subsequent methylation analysis on differentially methylated region (DMR) genes revealed the potential regulation network that influences pubertal onset. Correlation analysis verified the methylation level of some DMR genes correlates negatively with expression level. Integrated analysis between transcriptomes and methylomes identified 80 candidate genes involved in GnRH and neuroactive ligand signal pathways, of which CACNA1C and CRH were differentially expressed genes (DEGs) influenced by methylation level. The GnRH gene was the only DEG not affected by its methylation level. In summary, in this study, we identified eight genes and three pathways that are related to pubertal onset in Jining Gray goats, and the expression of CACNA1C and CRH genes of the GnRH and neuroactive ligand signal pathways were influenced by DNA methylation, while that of the GnRH gene was not affected.