Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus

Abstract

Background: The expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied.

Results: Seven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c.

Conclusion: Our results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Keywords: Expression pattern; Foxl2; Gonadal differentiation; Monopterus albus; Reproductive; circRNA.

Fig. 1

Characterization and classification of circRNAs. A CircRNA length and the number of circRNAs in the corresponding length range. B The relationship between the paternal genes and the circRNAs. “1” means that one paternal gene corresponds to one circRNA, “2” means that one paternal gene corresponds to two circRNAs, and so on. C CircRNA category and the number and proportion of circRNAs in the corresponding category. One circRNA contains one or more exons (D), introns (E), and intergene regions (F)

Fig. 2

Differentially expressed circRNAs during gonadal development. A OV vs. TE. B IE vs. TE. C IM vs. TE. D IL vs. TE. E Venn diagram of differentially expressed circRNAs. OV: ovary, IE: early intersexual gonad, IM: middle intersexual gonad, IL: late intersexual gonad, TE: testis

Fig. 3

GO and KEGG enrichment of the parent genes of the DE circRNAs. A GO enrichment of the parent genes of the DE circRNAs in the IE vs. TE groups. IE: early intersexual gonad, TE: testis, BP: biological process, CC: cellular component, MF: molecular function. B KEGG enrichment of the parent genes of the DE circRNAs

Fig. 4

Schematic diagram of interactions between DE circRNAs, miRNAs and mRNAs. For presentation purposes, “novel_circ_000” and  "mal-miR-" were shortened to “circ” and "miR-", respectively in the diagram

Fig. 5

Characterization of the predicted circRNAs. A Amplification of the circRNAs using divergent primers with cDNA. Lanes 1, 3 and 4 refer to the cDNA samples transcribed by random hexamer primers, and lane 2 refers to the cDNA samples transcribed by oligo (dT)₁₈. The cDNA used in lane 3 was synthesized by total RNA digested with RNase R, and lane 4 was a control. : represents back-splicing sites. B Verification of circRNA expression patterns. The circRNA expression level of RNA-seq was calculated as Log₅₀(TPM), and the RT–qPCR data were computed as the mean ± S.E.M. TPM: transcripts per million

Fig. 6

circSnd1 interacts with mal-miR-135b/c. A The genomic loci of circSnd1 and divergent primers were designed (F: forward primer, R: reverse primer), and the expected size of the product was amplified and verified by Sanger sequencing; the cDNA samples in lanes 1-4 were the same as those in Fig. 5A. B Quantitative results of circSnd1 in the nucleus and cytoplasm. C Schematic representation of the mal-miR-135b/c target sequence within circSnd1. D, E Dual-luciferase assays for validating the interaction of mal-miR-135b/c with circSnd1. Values are the mean ± S.E.M., and the experiment was repeated at least three times. The letters above the bar chart indicated P<0.05

Fig. 7

CircSnd1 as a potential modulator of foxl2. A-C Expression patterns of circSnd1, mal-miR-135b/c and foxl2 during the sex change. D CircSnd1, mal-miR-135b/c and foxl2 were enriched in the RNA-induced silencing complex (RISC). E Schematic representation of the mal-miR-135b/c target sequence within the foxl2 3' UTR. F Dual-luciferase assays validating the -135b/c with foxl2. Values are the mean ± SEM, and the experiment was repeated at least three time. The letters above the bar chart indicated P<0.05

Fig. 8

Results of identification of the gonad development stages of ricefield eel. A Ovary (OV), B early intersexual gonad (IE), C middle intersexual gonad (IM), D late intersexual gonad (IL), E testis (TE). CAO: cortical alveolar oocyte, PGO: primary growth stage oocyte, GR: gonadal ridge, SC: spermatocyte, ST: spermatid, BV: blood vessel, EVO: early vitellogenesis oocyte