Cross-talk between competitive endogenous RNAs (ceRNAs) may play a critical role in revealing potential mechanism of bovine follicular cysts. Ovarian cyst has always been an intractable scientific problem and has led to considerable economic losses to bovine breeding industry. However, its pathogenesis and molecular mechanisms are still not well understood. Here, this study aimed to investigate the role of non-coding RNAs (ncRNAs) and the ceRNA networks in bovine follicular cyst. Whole transcriptome sequencing of bovine follicular granulosa cells (GCs) was conducted to obtain the expression profiles of mRNAs, lncRNAs and miRNAs. The results for the identified expressions of 8,003 mRNAs, 579 lncRNAs and 205 miRNAs were often altered between cystic and normal follicular GCs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on these differentially expressed mRNAs. Furthermore, the ceRNA network combining mRNAs, miRNAs, and lncRNAs using several bioinformatics methods based on co-expression analysis between the differentially expressed RNAs was conducted. Finally, the lncRNA NONBTAT027373.1-miR-664b-HSD17B7 pathway was verified by dual-luciferase reporting assay and RNA binding protein immunoprecipitation (RIP) assay. LncRNA NONBTAT027373.1 sponged miR-664b in GCs and prevented miR-664b from binding to the HSD17B7 3′-UTR. These results indicated that genes and lncRNAs related to steroid hormone synthesis and energy metabolism could play important roles in the formation of bovine cystic follicles through the ceRNA mechanism and represent candidate targets for further research. This can be used as a practical guideline for promoting healthy and highly efficient development in the bovine industry.

Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. Small white follicles (1–2 mm in diameter) were treated for RNA isolation; Small white follicles (1–2 mm in diameter) and large white follicles (4–6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4–6 mm in diameter), small yellow follicles (6–8 mm in diameter), large yellow follicles (9–12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6–8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4–6 mm follicles relative to follicles of other sizes (P < 0.05). Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

Ovarian follicular GCs are strongly implicated in the growth, development, and atresia of ovarian follicles. The Wnt/β-catenin and Notch signaling pathways participate in GC proliferation, differentiation, apoptosis, and steroid hormone production during follicular development. However, the crosstalk between Wnt and Notch signaling in GCs remains unclear. This study investigated this crosstalk and the roles of these pathways in apoptosis, cell cycle progression, cell proliferation, and steroid hormone secretion in bovine follicular GCs. The interaction between β-catenin and Notch2 in GCs was assessed by overexpressing CTNNB1, which encodes β-catenin. The results showed that inhibiting the Notch pathway by Notch2 silencing in GCs arrested the cell cycle, promoted apoptosis, reduced progesterone (P4) production, and inhibited the Wnt2-mediated Wnt/β-catenin pathway in GCs. IWR-1 inhibited Wnt2/β-catenin and Notch signaling, reduced GC proliferation, stimulated apoptosis, induced G1 cell cycle arrest, and reduced P4 production. CTNNB1 overexpression had the opposite effect and increased 17β-estradiol (E2) production and Notch2 protein expression. Co-immunoprecipitation assays revealed that Notch2 interacted with β-catenin. These results elucidate the crosstalk between the Wnt/β-catenin and Notch pathways and the role of these pathways in bovine follicular GC development.