Dynamic expression and functional analysis of circRNA in granulosa cells during follicular development in chicken

- Dynamic expression and functional analysis of circRNA in granulosa cells during.
- follicular development in chicken.
- The granulosa cells of follicles play a determining role in ovarian development..
- The majority of the splice lengths of circRNAs were 200 – 500 nt and mainly produced from intron and CDS regions.
- During follicle growth, differentially expressed (DE) circRNAs showed dynamic changes which were tissue- and stage-specific..
- The host genes of DE circRNAs were functionally enriched in GTPase activity and several pathways involved in reproduction.
- One of the striking characteristics of the bird follicle is the presence of 5–7 hierarchal follicles before ovulation.
- The basic structure of the follicular wall consists of the theca and granulosa.
- Full list of author information is available at the end of the article.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Elucidating the features of the molecular mechanism during GC development could pro- vide a reference to understanding molecular development and improving assisted reproduction in humans..
- In par- ticular, the expression patterns of circRNAs are often tis- sue- and stage-specific [17], and highly conserved across the human brain and other species [18].
- With the fast de- velopment of bioinformatics techniques and deep sequen- cing, a number of circRNAs has been detected in animal follicle development.
- reports that the host genes of the circRNAs in goat pre-ovulatory ovarian follicles are involved in the ovarian steroidogenesis pathway and the p53 signaling pathway [19], chi-circ_0008219 modulates follicle growth by sponging three miRNAs to achieve.
- However, data associated with the function of circRNAs in chicken is still scarce, es- pecially with respect to follicle development.
- This led us to systematically study the expression of circRNAs in chicken follicle GCs..
- In the present study, we performed RNA-seq for cir- cRNAs during chicken follicle development to explore the structure and expression profile of GCs..
- Granulosa cells obtained for circular RNA-seq.
- Only hens with a soft eggshell egg in the oviduct were considered in our study.
- After ade- nylation of the 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structures were ligated to pre- pare for hybridization.
- Validation of circRNAs by sanger sequencing and qRT-PCR The circRNAs were validated with convergent and diver- gent primers according to a previous study [18].
- The expression of circRNAs at different ovary tissues was calculated via the 2 - ΔΔ Ct method..
- Overview of circRNAs.
- 1, which showed that the numbers of circRNAs in GCs were distributed almost on all chromosome, with the largest on chromosome 1 (chr1) and the smallest on the chr16..
- Multiple circRNAs were produced by one gene and the majority of circRNAs have two exons, followed by three exons and one exon (Fig.
- Table 1 Summary of sequencing results in chicken follicle granulosa cells at three stages.
- 9165 and 9149 flank introns (42.17%) of circRNAs were above nt, respectively (Fig.
- Differentially expressed analysis.
- Volcano plots of circRNAs expressed differentially in GCs are displayed in Fig.
- In the hierarchal fol- licle GC libraries, a total of 412 circRNAs with 191 up- regulated and 221 downregulated in F6 GCs were significantly differentially expressed (Fig.
- A heatmap of all differentially expressed circRNAs based on transcript per million (TPM) values is shown.
- Functional analyses of host genes of differentially expressed circRNAs.
- These three types of circRNAs were all detected in all reproduction tissues.
- Interest- ingly, the expression level in the ovary stroma was the highest.
- The expression level was lower in the hierarchal.
- 2 Profiling of circular RNAs in chicken follicle granulosa cells.
- Note: (a), splice length of circRNAs.
- (c), source of circRNAs.
- (f), flank intron length distributed in granulosa cells.
- It was observed that five miRNAs (miR-1625-3p, miR-1552-3p, miR-16-2-3p, miR-18b-3p, and miR-200a-3p) were com- mon among the three types of circRNAs sourced from the same gene RalGPS2 (Fig.
- Note: (a), Venn analysis of differentially expressed circRNAs in three stages.
- b, volcano map of differentially expressed circRNAs (DEcircs) between SYF and F6 granulosa cells.
- c, volcano map of DEcircs between SYF and F1 granulosa cells.
- d, volcano map of DEcircs between F6 and F1 granulosa cells.
- Folliculogenesis in birds is a very complex process, which includes recruitment of the primitive follicle, devel- opment or atresia of small follicles and 6–8 mm follicle se- lection, hierarchal follicle rapid growth, and ovulation..
- Though many studies have reported that circRNAs were widespread in mammals [32] and plants [14], little is known about circRNAs in chickens, especially in the folliculogenesis process..
- In the current study, a strategy of second generation sequencing for circRNAs in chicken follicle GCs was.
- 4 Heat map representing the relative expression levels of all differentially expressed circRNAs in the three stages (Fold change >.
- 6 Differentially expressed circRNAs in three different stages of granulosa cells.
- Note: a, Venn diagram of differentially expressed circRNAs ( n = 891, fold change >.
- G: granulosa cells..
- 5 GO and KEGG analysis for the host genes of all differentially expressed circRNAs.
- Note: (a), The top 10 GO enrichment term in BP, CC, and MF for the host genes of all differentially expressed circRNAs.
- (b), the top 20 KEGG enrichment term for the host genes of all differentially expressed circRNAs.
- GGA1 is the longest chromosome in the entire genome of the chicken and chr16 is the shortest [33].
- We predicted a total of 11,642 circRNAs in three types of follicle GC, a number which is less than that detected in the muscle of embryonic chicken and more than that found in the liver of ALVJ-resistant and susceptible chicken which suggested that the expression of circRNAs were tissue-specific and assisted in the hy- pothesis that the circularization is a tightly regulated event in different tissues [32].
- In our study, the spliced length of the majority of circRNAs was 200~300 nt, which was longer than those obtained in a previous study from pre-implantation embryos (124–227 nt) [36]..
- The difference of length in different animals may be due to a species dif- ference in the function performed by circRNAs.
- With re- spect to the source of circRNAs in GCs, it was shown that circRNAs mainly came from introns, which was un- like in previous research where most circRNAs came from coding exons [34].
- production rate or exon number in circRNAs were similar with previous studies, for instance, the production rate of circRNAs is determined by intronic sequence [38] and a single gene can produce multiple circRNAs [39].
- It was concluded that circRNAs may exert different functions on different cell morphology of the same tissue..
- Moreover, GO and KEGG pathway analyses for these differentially expressed circRNAs were performed.
- 7 Validation of circRNAs by experimental and sequencing.
- b, Sanger sequencing confirmed the back-splicing junction of circRNAs.
- (c), RT-qPCR validation of nine differentially expressed circRNAs in three types of granulosa cell.
- GTPases of the Ras super- family are omnipresent signaling proteins that play im- portant roles in a wide range of vital cellular processes [40], including ovarian follicle development [41, 42].
- 6, a total of 36 differentially expressed circRNAs showed pervasive expression pat- terns in three types of GCs.
- Previous work has re- ported that the abundance of circRNAs were negatively Table 2 Thirty-six pervasive DE circRNAs with miRNA binding sites.
- chr exon ORC miR-1620, miR-1620, miR-1596-5p, miR-1596-5p, miR-20b-3p chr exon DGAT miR-301a-3p, miR-9-5p, miR-17-5p, miR-106-5p, miR-1641 chr exon GDPD miR-17-5p, miR-20a-5p, miR-106-5p,miR-454-3p,miR-215-5p chr intergenic miR-1658-3p, miR-1655-5p,miR-1c,miR-15b-5p,miR-15a chr intergenic miR-1631, miR-125b-5p,miR-1611,miR-489-3p,miR-1647 chr exon NCKIPSD miR-22-5p, miR-1611,miR-183,miR-30c-1-3p,miR-1553-3p chr exon CLEC19A miR-1550-5p, miR-1594,miR-1635,miR-365-3p,miR-1564-3p chr intergenic miR-1650, miR-1628, miR-20b-3p,miR-219a,miR-1617 chr exon ST3GAL miR-20a-5p,miR-20b-5p,miR-1649-5p,miR-1553-3p,miR-17-5p chr intergenic miR-217-5p,miR-20a-5p,miR-20b-5p,miR-9-5p,miR-1612 chr intergenic miR-217-5p,miR-142-5p,miR-1646,miR-1612,miR-18a-3p chr exon TOX miR-1464,miR-1556,miR-1648-3p,miR-140-3p,miR-1614-3p chr exon PTP4A miR-1b-5p,miR-1a-2-5p,miR-1a-1-5p,miR-1565,miR-1649-5p chr exon FOXP miR-1595-3p,miR-29b-2-5p,miR-1653,miR-460b-5p,miR-29b-1-5p chr exon R3HDM miR-30c-1-3p,miR-1589,miR-181b-5p,miR-181a-5p,miR-1647 chr exon UTRN miR-1651-3p,miR-1453,miR-29b-1-5p,miR-1615,miR-138-1-3p chr exon SLC16A miR-1562-5p,miR-215-3p,miR-1625-3p,miR-365-3p,miR-146b-3p chr exon SLC16A miR-1585,miR-128-3p,miR-1574-3p,miR-455-3p,miR-124b chr exon FAM13A miR-1632-5p,miR-29a-5p,miR-1b-3p,miR-1464,miR-100-3p chr intergenic miR-1615,miR-1556,miR-456-3p,miR-142-5p,miR-1609 chr exon miR-148a-3p,miR-30a-3p,miR-30e-5p,miR-27b-3p,miR-33-5p chr exon miR-205a,miR-205b,miR-1650,miR-456-3p,miR-19b-5p chr intergenic miR-302a,miR-146a-3p,miR-34a-5p,miR-34c-5p,miR-449a chr intergenic miR-1655-5p,miR-1616,miR-30e-3p,miR-1610,miR-1632-5p chr intergenic miR-130a-5p,miR-1570,miR-204,miR-211,miR-1588 chr exon FAF miR-183,miR-1552-3p,miR-26a-5p,miR-1649-5p,miR-302b-5p chr exon miR-757,miR-15b-5p,miR-15a,miR-16-5p,miR-1630 chr exon RALGPS miR-16-2-3p,miR-137-3p,miR-1645,miR-1640,miR-217-5p chr exon RALGPS miR-16-2-3p,miR-1645,miR-1625-3p,miR-18b-3p,miR-1560-3p chr exon RALGPS miR-16-2-3p,miR-137-3p,miR-1640,miR-217-5p,miR-1658-5p chr intergenic miR-130b-5p,miR-130b-5p,miR-106-3p,miR-106-3p,miR-17-3p chr exon MED12L miR-15b-5p,miR-15a,miR-16-5p,miR-20b-5p,miR-7.
- It is tempting to speculate that these upregulated circRNAs may inhibit GC proliferation and downregulated circRNAs may affect follicle number by participating in the regulation of follicle recruitment..
- The results showed that the expression of all circRalGPS2s were predomin- ant in the ovary stroma and a decreased level was found in follicle growth, suggesting that circularization of cir- cRNAs may not accompany GCs development.
- With the.
- combined analysis of the abovementioned reports and current results, it is plausible to hypothesize that the lower the level of circRNAs, the more abundant are the display of biological activities, and circRNA from differ- ent host genes may exert different functions in different tissues, organs, or stages..
- The main mechanism of circRNAs may act as a miRNA sponge [16] to modulate post-transcriptional regulation [23].
- The higher level of circRNAs, the stron- ger is the efficiency of the sponge [16].
- GO and KEGG analysis for the predicted target genes of circRalGPS2 showed that the target genes were enriched in the regulation of signaling, secretion by the cell, and cell motility (Fig.
- PIK3R1 is involved in the PI3K/AKT pathway that is responsible for chicken GC pro- liferation [53].
- Furthermore, on the basis of the ceRNA hypothesis [55], circRalGPS2 can compete with miRNA to modulate mRNA expression that regulates downstream target genes.
- Thus, an assessment of the function of circRNAs as a miRNA sponge to modulate gene expression during fol- licle development needs tentative overexpression and knockdown experimental designs..
- In particular, we found a hot spot gene RalGPS2 that produced three isoforms of circRNAs which exhibited a similar expression pattern during.
- follicle development.
- All novel circRNAs detected in granulosa cells.
- Differentially expressed circRNAs (Fold change >.
- 0.05) in different granulosa cells.
- KEGG pathway for host genes of all differentially expressed circRNAs in three granulosa cells stage.
- The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript..
- The datasets generated and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) repository with the accessions SRP153771, BioProject accession number PRJNA481176 (https://.
- All experimental procedures were conducted in compliance with Experimental Animals Regulations and all efforts were made to alleviate the suffering of the birds.
- Follicle selection in the avian ovary.
- Characterization of a chicken luteinizing hormone receptor (cLH-R) complementary deoxyribonucleic acid, and expression of cLH-R messenger ribonucleic acid in the ovary.
- Dynamic changes in the follicular transcriptome and promoter DNA methylation pattern of steroidogenic genes in chicken follicles throughout the ovulation cycle.
- Role of FSH and epidermal growth factor (EGF) in the initiation of steroidogenesis in granulosa cells associated with follicular selection in chicken ovaries.
- Transcriptome analysis of the potential roles of FOXL2 in chicken pre-hierarchical and pre-ovulatory granulosa cells..
- Circular RNA expression profiling of human granulosa cells during maternal aging reveals novel transcripts associated with assisted reproductive technology outcomes.
- A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus).
- Functional genomics of the chicken--a model organism.
- Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed.
- Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.
- Inhibitory effect of SLIT2 on granulosa cell proliferation mediated by the CDC42-PAKs-ERK1/2 MAPK pathway in the prehierarchical follicles of the chicken ovary.
- Identification of novel candidate genes for follicle selection in the broiler breeder ovary

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