CircEML1 facilitates the steroid synthesis in follicular granulosa cells of chicken through sponging gga-miR-449a to release IGF2BP3 expression.

Non-coding RNAs (ncRNAs) induced competing endogenous RNAs (ceRNA) play crucial roles in various biological process by regulating target gene expression. However, the studies of ceRNA networks in the regulation of ovarian ovulation processing of chicken remains deficient compared to that in mammals. Our present study revealed that circEML1 was differential expressed in hen's ovarian tissues at different ages (15 W/20 W/30 W/68 W) and identified as a loop structure from EML1 pre-mRNA, which promoted the expressions of CYP19A1/StAR and E2/P4 secretion in follicular granulosa cells (GCs). Furthermore, circEML1 could serve as a sponge of gga-miR-449a and also found that IGF2BP3 was targeted by gga-miR-449a to co-participate in the steroidogenesis, which possibly act the regulatory role via mTOR/p38MAPK pathways. Meanwhile, in the rescue experiment, gga-miR-449a could reverse the promoting role of circEML1 to IGF2BP3 and steroidogenesis. Eventually, this study suggested that circEML1/gga-miR-449a/IGF2BP3 axis exerted an important role in the steroidogenesis in GCs of chicken.

Adiponectin modulates steroid hormone secretion, granulosa cell proliferation and apoptosis via binding its receptors during hens' high laying period.

Adiponectin is an important adipocytokine and plays the roles in multiple metabolic processes via binding its receptors - AdipoR1 and AdipoR2, which has also been found to participate in the regulation of the reproductive system of animals, in particular by influencing the secretion of ovarian steroid hormones. To further investigate the expression of adiponectin and its receptors in follicles after in vitro incubation, and their role in the steroid synthesis of laying hens' ovaries, we performed qRT-PCR and ELISA to detect the expressions of AdipoQ, AdipoR1, and AidpoR2, and determined the key genes involved in steroidogenesis and the secretion of estradiol (E2) and progesterone (P4) through the in vitro activation of adiponectin (AipoRon) and overexpression or knockdown of AdipoR1 and AdipoR2. Our results revealed that adiponectin and its receptors wildly exist in follicles and granulosa cells, and AdipoRon (5 and 10 µg/mL) had no effect on granulosa cell proliferation and apoptosis but significantly stimulated the secretion of adiponectin and its receptors in granulosa cells after incubation for 24 h. Furthermore, AdipoRon could significantly stimulate the secretion of P4 and inhibit E2 level compared to those of the control group through modulating the key genes expression of steroidogenesis (CYP19A1, StAR, CYP11A1, FSHR, and LHR). The secretion of E2 was also decreased in granulosa cells by the treatments of overexpression and knockdown of AdipoR1/2, however, there was no difference in terms of the level of P4 and StAR expression between them if there was overexpression or knockdown of AdipoR1/2. In addition, it was shown that the secretion of E2 only exhibits a marked drop if co-processing 10 µg/mL AdipoRon and pGMLV AdipoR2 compared to single treatments. Taken together, the study highlighted the role of adiponectin and its receptors in the regulation of steroid synthesis and secretion in ovarian granulosa cells in laying hens.

Identification of the Key microRNAs and miRNA-mRNA Interaction Networks during the Ovarian Development of Hens.

It is well-known that multiple functional miRNAs are found in mammals' ovaries, which are linked not only to ovarian development, but also to maturation and apoptosis. However, there is still a lack of knowledge regarding the role of miRNAs in the hen ovary. In the present study, we analyzed the miRNA sequencing libraries of ovaries at the four different developmental stages of hens (15, 20, 30, and 68 W) and a total of 677 known miRNAs and 61 novel miRNAs were identified. In total, 209 of them were differently expressed miRNAs (DE miRNAs) obtained from comparisons of the four stages, including 84 upregulated and 125 downregulated DE miRNAs. Furthermore, the five key DE miRNAs gga-miR-2954, gga-miR-6634-5p, gga-miR-449b-5p, gga-miR-449c-3p, and gga-miR449c-5p were screened using an analysis of the miRNA-mRNA interaction network and functional enrichment annotated in seven significantly enriched pathways, such as endocytosis, lysine degradation, the biosynthesis of amino acids, and the MAPK signaling pathway, which may primarily participate in cell differentiation and proliferation, steroid hormone biosynthesis, and angiogenesis by targeting the related genes. For instance, gga-miR-449 family members were predicted to target 15 genes, including TGFB1, TPM1, TPM3, and CAMKB2, which were reported to regulate follicular growth, selection, and the ovulatory cycle. Taken together, our results illustrate the ovarian miRNA profiles of the four classic developmental stages of hens and highlight the significant role of miRNAs in ovarian development and functions. However, in-depth research needs to be carried out to validate the potential functional miRNAs found in this study.

Estrogen Abolishes the Repression Role of gga-miR-221-5p Targeting ELOVL6 and SQLE to Promote Lipid Synthesis in Chicken Liver.

Few studies have been conducted regarding the biological function and regulation role of gga-miR-221-5p in the liver. We compared the conservation of miR-221-5p among species and investigated the expression pattern of gga-miR-221-5p, validating the direct target genes of gga-miR-221-5p by dual luciferase reporter assay, the biological function of gga-miR-221-5p in the liver was studied by gga-miR-221-5p overexpression and inhibition. Furthermore, we explored the regulation of gga-miR-221-5p and its target genes by treatment with estrogen and estrogen antagonists in vivo and in vitro. The results showed that miR-221-5p was highly conserved among species, expressed in all tested tissues and significantly downregulated in peak-laying hen liver compared to pre-laying hen liver. Gga-miR-221-5p could directly target the expression of elongase of very long chain fatty acids 6 (ELOVL6) and squalene epoxidase (SQLE) genes to affect triglyceride and total cholesterol content in the liver. 17ß-estradiol could significantly inhibit the expression of gga-miR-221-5p but promote the expression of ELOVL6 and SQLE genes. In conclusion, the highly conservative gga-miR-221-5p could directly target ELOVL6 and SQLE mRNAs to affect the level of intracellular triglyceride and total cholesterol. Meanwhile, 17ß-estradiol could repress the expression of gga-miR-221-5p but increase the expression of ELOVL6 and SQLE, therefore promoting the synthesis of intracellular triglyceride and cholesterol levels in the liver of egg-laying chicken.

Genome-Wide Analysis of the FABP Gene Family in Liver of Chicken (Gallus gallus): Identification,Dynamic Expression Profile, and RegulatoryMechanism.

The fatty acid-binding protein (FABP) gene family, which encodes a group of fatty acid-trafficking molecules that affect cellular functions, has been studied extensively in mammals. However, little is known about the gene structure, expression profile, and regulatory mechanism of the gene family in chickens. In the present study, bioinformatics-based methods were used to identify the family members and investigate their evolutionary history and features of gene structure. Real-time PCR combined with in vivo and in vitro experiments were used to examine the spatiotemporal expression pattern, and explore the regulatory mechanism of FABP genes. The results show that nine members of the FABP gene family, which branched into two clusters and shared a conserved FATTYACIDBP domain, exist in the genome of chickens. Of these, seven FABP genes, including FABP1, FABP3-7, and FABP10 were abundantly expressed in the liver of hens. The expression levels of FABP1, FABP3, and FABP10 were significantly increased, FABP5 and FABP7 were significantly decreased, and FABP4 and FABP6 remained unchanged in hens at the peak laying stage in comparison to those at the pre-laying stage. Transcription of FABP1 and FABP3 were activated by estrogen via estrogen receptor (ER) α, whilst FABP10 was activated by estrogen via ERß. Meanwhile, the expression of FABP1 was regulated by peroxisome proliferator activated receptor (PPAR) isoforms, of which tested PPARα and PPARß agonists significantly inhibited the expression of FABP1, while tested PPARγ agonists significantly increased the expression of FABP1, but downregulated it when the concentration of the PPARγ agonist reached 100 nM. The expression of FABP3 was upregulated via tested PPARß and PPARγ agonists, and the expression of FABP7 was selectively promoted via PPARγ. The expression of FABP10 was activated by all of the three tested PPAR agonists, but the expression of FABP4-6 was not affected by any of the PPAR agonists. In conclusion, members of the FABP gene family in chickens shared similar functional domains, gene structures, and evolutionary histories with mammalian species, but exhibited varying expression profiles and regulatory mechanisms. The results provide a valuable resource for better understanding the biological functions of individual FABP genes in chickens.

Identification of differentially expressed genes and pathways between intramuscular and abdominal fat-derived preadipocyte differentiation of chickens in vitro.

BACKGROUND: The distribution and deposition of fat tissue in different parts of the body are the key factors affecting the carcass quality and meat flavour of chickens. Intramuscular fat (IMF) content is an important factor associated with meat quality, while abdominal fat (AbF) is regarded as one of the main factors affecting poultry slaughter efficiency. To investigate the differentially expressed genes (DEGs) and molecular regulatory mechanisms related to adipogenic differentiation between IMF- and AbF-derived preadipocytes, we analysed the mRNA expression profiles in preadipocytes (0d, Pre-) and adipocytes (10d, Ad-) from IMF and AbF of Gushi chickens. RESULTS: AbF-derived preadipocytes exhibited a higher adipogenic differentiation ability (96.4% + 0.6) than IMF-derived preadipocytes (86.0% + 0.4) (p < 0.01). By Ribo-Zero RNA sequencing, we obtained 4403 (2055 upregulated and 2348 downregulated) and 4693 (2797 upregulated and 1896 downregulated) DEGs between preadipocytes and adipocytes in the IMF and Ad groups, respectively. For IMF-derived preadipocyte differentiation, pathways related to the PPAR signalling pathway, ECM-receptor interaction and focal adhesion pathway were significantly enriched. For AbF-derived preadipocyte differentiation, the steroid biosynthesis pathways, calcium signaling pathway and ECM-receptor interaction pathway were significantly enriched. A large number of DEGs related to lipid metabolism, fatty acid metabolism and preadipocyte differentiation, such as PPARG, ACSBG2, FABP4, FASN, APOA1 and INSIG1, were identified in our study. CONCLUSION: This study revealed large transcriptomic differences between IMF- and AbF-derived preadipocyte differentiation. A large number of DEGs and transcription factors that were closely related to fatty acid metabolism, lipid metabolism and preadipocyte differentiation were identified in the present study. Additionally, the microenvironment of IMF- and AbF-derived preadipocyte may play a significant role in adipogenic differentiation. This study provides valuable evidence to understand the molecular mechanisms underlying adipogenesis and fat deposition in chickens.

miR-34a-5p Increases Hepatic Triglycerides and Total Cholesterol Levels by Regulating ACSL1 Protein Expression in Laying Hens.

Accumulating evidence has shown that miR-34a serves as a posttranscriptional regulatory molecule of lipid metabolism in mammals. However, little studies about miR-34a on lipid metabolism in poultry have been reported until now. To gain insight into the biological functions and action mechanisms of miR-34a on hepatic lipid metabolism in poultry, we firstly investigated the expression pattern of miR-34a-5p, a member of miR-34a family, in liver of chicken, and determined its function in hepatocyte lipid metabolism by miR-34a-5p overexpression and inhibition, respectively. We then validated the interaction between miR-34a-5p and its target using dual-luciferase reporter assay, and explored the action mechanism of miR-34a-5p on its target by qPCR and Western blotting. Additionally, we looked into the function of the target gene on hepatocyte lipid metabolism by gain- and loss-of-function experiments. Our results indicated that miR-34a-5p showed a significantly higher expression level in livers in peak-laying hens than that in pre-laying hens. miR-34a-5p could increase the intracellular levels of triglycerides and total cholesterol in hepatocyte. Furthermore, miR-34a-5p functioned by inhibiting the translation of its target gene, long-chain acyl-CoA synthetase 1 (ACSL1), which negatively regulates hepatocyte lipid content. In conclusion, miR-34a-5p could increase intracellular lipid content by reducing the protein level, without influencing mRNA stability of the ACSL1 gene in chickens.

LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p.

Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects the tenderness, juiciness, and flavor of chicken. An increasing number of studies are focusing on the functions of lncRNAs in adipocyte differentiation. However, little is known about lncRNAs associated with intramuscular adipocyte differentiation. In the present study, we focused on an up-regulated lncRNA during intramuscular adipogenetic differentiation, which we named intramuscular fat-associated long non-coding RNA (IMFNCR). IMFNCR promotes intramuscular adipocyte differentiation. In-depth analyses showed that IMFNCR acts as a molecular sponge for miR-128-3p and miR-27b-3p and that PPARG is a direct target of miR-128-3p and miR-27b-3p in chicken. High-fat and high-protein diet inhibited chicken IMFNCR level in vivo. Moreover, IMFNCR level was positively correlated with PPARG mRNA level in chicken breast muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that IMFNCR acts as a ceRNA to sequester miR-128-3p and miR-27b-3p, leading to heightened PPARG expression, and thus promotes intramuscular adipocyte differentiation. Taken together, our findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Characterisation of the genetic effects of the ADFP gene and its association with production traits in dairy goats.

Adipose differentiation-related protein (ADFP) is important for regulation of lipid metabolism and insulin secretion in beta-cells. In this study, we investigated polymorphisms within the caprine ADFP gene and determined its relationship with production traits. As there was no sequence information available for the caprine ADFP gene, we generated DNA sequence data and examined the genomic organisation. The caprine ADFP gene is organised into 7 exons and 6 introns that span approximately 8.7 kbp and is transcribed into mRNA containing 1,353 bp of sequence coding for a protein of 450 amino acids. The protein sequences showed substantial similarity (71-99%) to orthologues from cattle, human and mouse. We identified polymorphisms in the sequences using DNA sequencing, PCR-RFLP and forced PCR-RFLP methods. Seven single nucleotide polymorphisms (SNPs) were identified using samples from 4 different goat populations consisting of 1408 healthy and unrelated individuals. Six haplotypes involving the 7 SNPs from the caprine ADFP gene were identified and their effects on production traits were analysed. Haplotype 6 had the highest haplotype frequency and was highly significantly associated with chest circumference and milk yield in the analysed populations. The results of this study suggest that the ADFP gene is a strong candidate gene affecting production traits and may be used for marker-assisted selection and management in Chinese dairy goat breeding programmes.

MicroRNAs-1614-3p gene seed region polymorphisms and association analysis with chicken production traits.

In the present study, a total of 860 chickens from a Gushi-Anka F2 resource population were used to evaluate the genetic effect of the gga-miR-1614-3p gene. A novel, silent, single nucleotide polymorphism (SNP, +5 C>T) was detected in the gga-miR-1614-3p gene seed region through AvaII polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR products sequencing methods. Associations between the SNP and chicken growth, meat quality and carcass traits were performed by association analysis. The results showed that the SNP was significantly associated with breast muscle shear force and leg muscle water loss rate, wing weight, liver weight and heart weight (p<0.05), and highly significantly associated with the weight of the abdominal fat (p<0.01). The secondary structure of gga-miR-1614 and the free energy were altered due to the variation predicted by the M-fold program.