Epigenetic Regulation of miR-25 and Lnc107153 on Expression of Seasonal Estrus Key Gene CHGA in Sheep.

Pituitary pars tuberalis (PT) plays an important role as the transmission center in the seasonal reproduction of animals. It helps convert external photoperiod signals into intrinsic seasonal reproduction signals. In sheep PT, specific expression patterns of several genes (including short photoperiod-induced gene CHGA and long photoperiod genes EYA3 and TSHß) under different photoperiods are crucial characteristics during this signal transduction. Recent studies have revealed the role of epigenetics in regulating the expression of seasonal reproductive key genes. Therefore, we explored whether microRNAs and LncRNAs regulated the expressions of the above key genes. Firstly, the expression of miR-25 and CHGA showed a significant negative correlation in sheep PT. Results of the dual luciferase reporter assay and miR-25 overexpression indicated that miR-25 could inhibit the expression of CHGA by specifically binding to its 3'UTR region in pituitary cells. Then, expression negative correlation and dual luciferase reporter analyses were used to screen and identify the candidate LncRNA (Lnc107153) targeted by miR-25. Finally, the results of fluorescence in situ hybridization and Lnc107153 overexpression suggested that Lnc107153 and miR-25 were involved in the epigenetic regulation of CHGA expression. However, the expressions of EYA3 and TSHß were not regulated by miRNAs. These results will provide new insights into the epigenetic regulatory network of key genes in sheep seasonal reproduction.

Associations of CALM1 and DRD1 polymorphisms, and their expression levels, with Taihang chicken egg-production traits.

Egg production by hens is an important reproductive performance index in the poultry industry. To investigate the effects of the CALM1 and DRD1 genes on egg production in chicken, their mRNA expression and single nucleotide polymorphisms (SNP) levels were investigated, and bioinformatics and egg-production association analyses were performed. Three SNPs (g.44069941G > A and g.44069889A > G in CALM1 and g.10742639C > T in DRD1) were detected in the exons and introns of CALM1 and DRD1 in 400 Taihang chickens. Among them, g.44069941G > A was significantly associated with Taihang chicken egg production on the 500th day (p < 0.05), whereas g.10742639C > T was significantly associated with the 300th day (p < 0.05). The expression levels of CALM1 and DRD1 in ovarian tissues of a high-yielding Taihang group were greater than in a low-yielding group (p < 0.05). The bioinformatics analysis revealed that the mutations influenced the mRNA secondary structures of CALM1 and DRD1. This study provides new insights into the potential effects of CALM1 and DRD1 polymorphisms on chicken egg production. The two SNPs g.44069941G > A and g.10742639C > T are potential molecular markers for improving the reproductive traits of Taihang chicken.

CALM1 and DRD1 were two important genes for reproduction. In this study, the entire coding regions of both genes were sequenced and mutations were detected in Taihang chickens. The results showed that two single nucleotide polymorphisms (SNPs), g.44069941G > A in the CALM1 gene and g.10742639C > T in the DRD1 gene, were associated with egg-laying traits. g.10742639C > T is a synonymous mutation predicted to affect the secondary structure of mRNA. Therefore, these two mutations might be potential molecular markers for improving reproductive traits in Taihang chickens.

Effect of polymorphisms in the 5'-flanking sequence of MC1R on feather color in Taihang chickens.

MC1R plays an important role in the regulation of the formation, transfer, and deposition of melanin in animals and is important for determining coat color. Many studies have reported on single nucleotide polymorphisms (SNPs) in the coding sequence of MC1R. However, few studies have investigated the polymorphisms in the 5'-flanking sequence of MC1R. In this study, we sequenced 2000 bp of the 5'-flanking sequence of MC1R in 300 Taihang chickens with brown feathers (MTH) and 300 Taihang chickens with black feathers (HTH). The sequencing results showed that 4 SNPs (MC1R g.18838722 G > C, g.18838624 T > C, g.18838694 G > A, and g.18838624 C > T) were located in the 5'-flanking sequence of MC1R between the MTH and HTH groups. Association analysis showed that there was a significant correlation between the 4 SNPs and feather color in Taihang chickens. The correlation between MC1R g.18838624 T >C and feather color of Taihang chicken was 100%, of which the CC (E1) genotype is MTH and the TT (E2) genotype is HTH. Furthermore, there was a significant correlation between MC1R g.18838624 T > C and egg production at 302 d. E1 (184.14 ± 0.674) was significantly higher than that in E2 (181.75 ± 0.577) (P < 0.05). Luciferase reporter assays were used to detect the transcriptional activity of MC1R with different SNP genotypes. The results showed that the luciferase activity of E2 was significantly higher than that of E1 (P < 0.05). In addition, transcription factor-binding site predictions showed that E2 creates a new binding site for ZEB1. RT‒qPCR results revealed that the expression of MC1R in E2 was significantly lower than that in E1 (P < 0.05), and the expression of ZEB1 in E2 was significantly higher than that in E1 (P < 0.05). Overexpression and shRNA experiments demonstrated that ZEB1 regulates the expression of MC1R in DF1 cells. ZEB1 has a negative regulatory effect on the transcriptional activity of MC1R; it inhibits the expression of MC1R and affects the feather color of Taihang chickens. This study provides new insight into the molecular mechanism of feather color formation and the transcriptional regulation of MC1R in Taihang chickens.

Identification and Characterization of lncRNAs Expression Profile Related to Goat Skeletal Muscle at Different Development Stages.

LncRNAs are essential for regulating skeletal muscle. However, the expression profile and function of lncRNAs in goat muscle remains unclear. Here, an average of ~14.58 Gb high-quality reads were obtained from longissimus dorsi tissues of 1-month-old (n = 3) and 9-month-old (n = 3) Wu'an black goats using RNA sequencing. Of a total of 3441 lncRNAs, 1281 were lincRNAs, 805 were antisense lncRNAs, and 1355 were sense_overlapping lncRNAs. These lncRNAs shared some properties with goats, such as fewer exons, shorter transcript, and open reading frames (ORFs) length. Among them, 36 differentially expressed lncRNAs (DE lncRNA) were identified, and then 10 random lncRNAs were validated by RT-qPCR. Furthermore, 30 DE lncRNAs were neighboring 71 mRNAs and several genes were functionally enriched in muscle development-related pathways, such as APC, IFRD1, NKX2-5, and others. Additionally, 36 DE lncRNAs and 2684 mRNAs were included in co-expression interactions. A lncRNA-miRNA-mRNA network containing 4 lncRNAs, 3 miRNAs, and 8 mRNAs was finally constructed, of which XR_001296113.2 might regulate PDLIM7 expression by interaction with chi-miR-1296 to affect skeletal muscle development. This study revealed the expression profile of goat lncRNAs for further investigative studies and provides a fuller understanding of skeletal muscle development.

Association analysis and expression level of ACE polymorphisms with egg-laying trait in Taihang chicken.

The number of egg-laying is an important indicator of reproduction performance in poultry breeding. To investigate the relationship between the function of Angiotensin-converting enzyme (ACE) and egg-laying performance of Taihang chicken, the mRNA and protein expression and single nucleotide polymorphism (SNP) of ACE were detected. Analysis of ACE bioinformatics and association analysis of polymorphisms were then performed. The polymorphisms analysis of ACE showed that three SNP loci (g.5066812A>C, g.5080076G>A, and g.5072728A>G) were detected in 800 Taihang chickens with egg-laying records. Association analysis of egg-laying found that ACE g.5066812A>C mutation was significantly associated with the egg-laying performance of Taihang chickens (P < 0.05), and the individuals with the g.5066812A>C mutation showed significantly increasing egg-laying. The mRNA expression was significantly higher in individuals with the AA genotype mutation than those with the AC and CC genotypes (P < 0.01), and the expression of ACE protein levels was consistent with the mRNA expression. Bioinformatics analysis indicated that these mutations affected the secondary and tertiary structure of ACE. This study provides new insights into ACE affecting chicken egg production and some basis for improving the egg production rate of Taihang chickens.

Microbial Diversity and Community Composition of Duodenum Microbiota of High and Low Egg-Yielding Taihang Chickens Identified Using 16S rRNA Amplicon Sequencing.

The duodenum is an important digestive organ for poultry and houses a variety of microbes that help chickens to enhance nutrient absorption and improve production. To evaluate the characteristic of gut microbiome, duodenum content samples from 42-week-old native Taihang chickens with high (H) and low (L) egg-yielding were collected for 16S rRNA amplicon sequencing analysis. Consequently, 1,361,341 sequences were clustered into 2055 OTUs, with percentages of affiliation of 96.50 and 57.30% at phylum and genus levels. Firmicutes, Proteobacteria, Cyanobacteria and Bacteroidetes were the dominant phylum, with a lower ratio of Firmicutes/Bacteroidetes in H group than in L group (p < 0.05). At genus level, overrepresentation of Bacteroides, Faecalibacterim, and Enterococcus and underrepresentation of Romboutsia were found in H group. No significant difference in overall diversity of microbiota was observed between two groups. LEFSe analysis revealed Enterococcus was significantly enriched in H group. Importantly, Enterococcus and Lactobacillus were negatively correlated. Functional prediction analysis showed the proportion of microbiota involved in the metabolism process was the highest and enriched in H group. Differences in microbiota composition between the two groups, which may be related to intestinal function difference, also provide promising biomarkers for improving laying hen production.

Transcriptome Analysis Reveals Key miRNA-mRNA Pathways in Ovarian Tissues of Yunshang Black Goats With Different Kidding Numbers.

The granulosa cell (GC) is the basic functional unit of follicles, and it is important for promoting follicle growth and sex hormones, as well as growth factor secretion in the process of reproduction. A variety of factors influence granulocyte proliferation, yet there are still many gaps to be filled in target and non-coding RNA regulation. In our study, the differentially expressed (DE) mRNAs and miRNAs were detected by using RNA-seq, and we constructed a mRNA-miRNA network related to goat prolificacy. Then, the goat primary GCs were isolated from the follicle for the function validation of candidate genes and their regulator miRNAs. A total of 2,968 DE mRNAs and 99 DE miRNAs were identified in the high- and low-prolificacy goat by RNA-seq, of which there were 1,553 upregulated and 1,415 downregulated mRNAs, and 80 upregulated and 19 downregulated miRNAs, respectively. JAK3 was identified as highly expressed in the low-prolificacy goats (3 times higher than high-prolificacy goats), and the integrated analysis showed that chi-miR-493-3p was a potential regulator of JAK3. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that JAK3 was involved in the PI3K-Akt signaling pathway, the Jak-STAT signaling pathway, and signaling pathways regulating pluripotency of stem cells. In particular, the PI3K-Akt signaling pathway was a typical pathway for cell proliferation, differentiation, apoptosis, and migration. We found that the chi-miR-493-3p targets JAK3 directly via RT-qPCR, dual fluorescence assays, and Western blot. Furthermore, the expression of JAK3 was significantly decreased by the chi-miR-493-3p mimic and increased by the chi-miR-493-3p inhibitor. The CCK-8 assay showed that overexpression of JAK3 promoted cell proliferation, while inhibiting JAK3 had the opposite effect. The expression of cell proliferation markers CDK4 and cyclin D2 also showed the same results. Moreover, the enzyme-linked immunosorbent assay showed that steroid hormones E2 and PROG were increased by overexpressing JAK3 and decreased by inhibiting JAK3. Therefore, our results identified a chi-miR-439-3p-JAK3 regulatory pathway, which provided a new insight into the GC proliferation and prolificacy of goat.

Expression Profile Analysis to Identify Circular RNA Expression Signatures in Muscle Development of Wu'an Goat Longissimus Dorsi Tissues.

The growth and development of skeletal muscle is a physiological process regulated by a variety of genes and signaling pathways. As a posttranscriptional regulatory factor, circRNA plays a certain regulatory role in the development of animal skeletal muscle in the form of a miRNA sponge. However, the role of circRNAs in muscle development and growth in goats is still unclear. In our study, apparent differences in muscle fibers in Wu'an goats of different ages was firstly detected by hematoxylin-eosin (HE) staining, the circRNA expression profiles of longissimus dorsi muscles from 1-month-old (mon1) and 9-month-old (mon9) goats were screened by RNA-seq and verified by RT-qPCR. The host genes of differentially expressed (DE) circRNAs were predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) of host genes with DE circRNAs were performed to explore the functions of circRNAs. The circRNA-miRNA-mRNA networks were then constructed using Cytoscape software. Ten significantly differentially expressed circRNAs were also verified in the mon1 and mon9 groups by RT-qPCR. Luciferase Reporter Assay was used to verify the binding site between circRNA and its targeted miRNA. The results showed that a total of 686 DE circRNAs were identified between the mon9 and mon1 groups, of which 357 were upregulated and 329 were downregulated. Subsequently, the 467 host genes of DE circRNAs were predicted using Find_circ and CIRI software. The circRNA-miRNA-mRNA network contained 201 circRNAs, 85 miRNAs, and 581 mRNAs; the host mRNAs were associated with "muscle fiber development" and "AMPK signaling pathway" and were enriched in the FoxO signaling pathway. Competing endogenous RNA (ceRNA) network analysis showed that novel_circ_0005314, novel_circ_0005319, novel_circ_0009256, novel_circ_0009845, novel_circ_0005934 and novel_circ_0000134 may play important roles in skeletal muscle growth and development between the mon9 and mon1 groups. Luciferase Reporter Assay confirmed the combination between novel_circ_0005319 and chi-miR-199a-5p, novel_circ_0005934 and chi-miR-450-3p and novel_circ_0000134 and chi-miR-655. Our results provide specific information related to goat muscle development and a reference for the goat circRNA profile.