The potential biological functions of circular RNAs during the initiation of atresia in pig follicles

https://doi.org/10.1016/j.domaniend.2019.106401Get rights and content

Highlights

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    Genome-wide deep circular RNA (circRNA) sequencing was applied to screen circRNAs in healthy and early atretic porcine medium antral follicles.

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    One hundred and eight upregulated and 89 downregulated circRNAs were significantly shifted during early atresia process.

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    Comparison analysis revealed the cotranscription of circRNAs and their linear messenger RNA and highlighted a series of key pathways during early follicular atresia.

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    Our study offered a valuable reference for circRNA transcriptomic profiles in initiation of follicular atresia.

Abstract

The specific expression profile and function of circular RNAs (circRNAs) in mammalian ovarian follicles, especially during the atresia process, are unclear. In this study, genome-wide deep circRNA sequencing was applied to screen circRNAs in healthy and early atretic antral follicles in pig ovaries. A total of 40,567 distinct circRNAs were identified in follicles, among which 197 circRNAs (108 upregulated and 89 downregulated) were significantly shifted during the early atresia process. Most differentially expressed circRNAs (DECs) lacked protein-coding potential. Annotation analysis of the DECs revealed 162 known host genes, or noncoding RNAs, and 10 intergenic regions. The key pathways in which these host genes are involved include the focal adhesion-PI3K-Akt-mTOR signaling pathway, vascular endothelial growth factor A (VEGFA)–vascular endothelial growth factor receptor 2 signaling pathway and transforming growth factor-beta signaling pathway. Further comparison analysis between host genes of DECs and the differentially expressed linear messenger RNA transcripts revealed the cotranscription of circRNAs and their linear mRNAs in inhibin beta units (INHBA and INHBB), glutathione S-transferase (GSTA1), and VEGFA. In addition, we predicted 196 pairs of potential circRNA-micro RNA (miRNA) interactions among 77 DECs and 101 porcine miRNAs. We have identified 16 functional miRNAs by comparing the 101 miRNAs to the functional miRNAs reported in mammal ovarian follicle atresia and granulosa cell apoptosis studies. Our study adds new knowledge to circRNA distribution profiles in pig ovarian follicles, offers a valuable reference for transcriptomic profiles in the initiation of follicular atresia, highlights warranted circRNAs for further functional investigation, and provides possible biomarkers for ovarian dysfunctions.

Introduction

Circular ribonucleic acids (circRNAs) belong to a unique class of endogenous noncoding RNA families in eukaryotes and have attracted major attention because of their functions in posttranscriptional regulation of gene expression [1,2]. Their circular structure allows circRNAs to avoid digestion by RNA exonuclease and thus maintains their stability [3]. Most circRNAs are conserved among species [[4], [5], [6]]. Currently, 4 basic types of canonical alternative splicing have been found in circRNAs, including cassette exons, intron retention, alternative 5′ splicing, and alternative 3′ splicing [7]. Accordingly, circRNAs are classified into 3 types: circular exonic RNAs, which are the most common, circular intronic RNAs and exon–intron circRNAs [8,9]. Recent studies have suggested that circRNAs play important roles in physiological and pathologic conditions, such as neurogenesis [10], cancer development [11] and innate immune responses [12]. The known regulatory mechanisms of circRNAs include but are not limited to (1) affecting the splicing of their linear transcripts, (2) acting as sponges for micro RNAs (miRNAs), and (3) interacting with associated proteins [13].

In mammals, ovarian follicular development is a continuous process throughout the reproductive life span. Pig primordial follicle reserve is formed in the fetal ovary, and approximately 5 million primordial follicles are available at puberty [14]. A number of primordial follicles start to grow and may either be eventually selected for ovulation or undergo atresia at any stage of development [15,16] during each estrous cycle, thus limiting the final number of ovulations [17,18]. Quite a few studies have indicated that follicular atresia can be triggered by granulosa cell (GC) apoptosis, which is regulated by a delicate balance between prosurvival and proapoptotic factors [14,19,20]. Our earlier transcriptome profiling [21] and miRNA profiling [22] studies revealed molecular mechanisms involved in atresia initiation of medium (3–5 mm in size) pig follicles from different aspects. However, the expression and function of circRNAs during atresia requires further investigation.

In the present study, we performed genome-wide deep circRNA sequencing to reveal the shifted expression profiles of circRNAs between healthy (H) and early atretic (EA) ovarian follicles in pigs. Combined with previous research, an in-depth analysis was carried out to explore and discuss the potential biological functions of the specific circRNAs for follicular atresia in pigs. This study adds new comprehensive knowledge to circRNA expression in ovarian tissue, supplements the critical pathways in the initiation of follicular atresia, and provides candidate circRNAs for further functional studies and biomarker evaluation.

Section snippets

Animal and follicle collection

All experimental procedures were undertaken according to the guidelines of the Administration of Animal Care and Use and were approved by the Animal Ethics Committee of Nanjing Agricultural University, Nanjing, Jiangsu, China (SYXK2011–0036; December 6, 2011). Commercial large white pig populations were involved in the present study. The pigs were aged 7 mo, healthy, unstimulated replacement gilts. Pig ovaries were obtained from a local slaughterhouse and transferred to the laboratory within

circRNA expression profiles

The circRNA expression profiles were determined in porcine ovarian follicles by deep sequencing and bioinformatic analysis. A total of 40,567 circRNAs were identified from the circRNA-seq data with 34,270 (84.48% of all circRNAs) in the H follicles, 33,929 (83.64%) in the EA follicles, and 27,632 (68.11%) in both. Genome-wide distribution analysis showed that although the number of circRNAs was divergent across the chromosomes, the distribution density appeared to be rather even (Fig. 1A). The

Discussion

It has been revealed that circRNAs are abundant in animal transcriptomes and have been identified in various cell types of different species ranging from Drosophila [37,38] to human [6,39,40]. The pig is not only an important farm animal but also an ideal nonrodent animal model for biomedical research. Porcine circRNA profiles were detected first in brain tissue during development by Veno et al [41], and 4,634 circRNAs generated from 2,195 host genes were identified. Later, Liang et al [42]

Conclusion

In conclusion, our research represents the first description of circRNA profiles in ovarian follicles of pigs. The predicted miRNA–circRNA interaction provides an important basis for miRNA–circRNA relationships during the follicle atresia process. The circRNAs generated from INHA, INHB, GSTA1, and VEGFA and critical pathways such as TGF-beta and FoxO signaling were highlighted for further investigations. Because whole follicles were used in this study, further investigation will be required to

Acknowledgments

This work was supported by the National Natural Science Foundation of China, China (grant number 31672421), the Natural Science Foundation of Jiangsu Province, China (grant numbers BK20161453 and BK20160721), and the Fundamental Research Funds for the Central Universities, China (grant number KYZ201644).

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    Declarations of interest: none.

    Authors’ Contributions: T.Y.G., L.H., and W.Y. performed the experiments and analyzed the data; X.D., Q.Q.L., M.L.M., and Q.F.L. provided assistance in designing the study and revised the article; J.B.Z. and Z.X.P. designed and supervised the study, analyzed data, and wrote and finalized the article. All authors read and approved the final article.

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