CircEpha5 regulates the synthesis and secretion of androgen in mouse preantral follicles by targeting miR-758-5p

Abstract Circular RNAs are involved in the pathogenesis of various diseases, although its expression pattern and role in polycystic ovary syndrome (PCOS), characterised by hyperandrogenism, are not very clear. This article assessed the circRNAs expression profile in the ovaries of PCOS mice by circRNAs high-throughput sequencing and explored the role of circEpha5 in hyperandrogenism. The results showed that the overexpression of circEpha5 in mouse preantral follicles could increase the expression of Cyp17a1, an androgen synthesis-related gene, which resulted in a higher serum level of testosterone. Dual-luciferase reporter gene studies identified miR-758-5p as a direct target of circEpha5. Consequently, miR-758-5p expression was downregulated upon circEpha5 overexpression. Ectopically expressed miR-758-5p reversed the stimulation effects of circEpha5 on steroidogenesis-related gene expression and testosterone release. Therefore, circEpha5 could sponge miR-758-5p to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in the preantral follicles. This work is contributed to the understanding of the pathogenesis of hyperandrogenemia and lays the foundation for the development of therapeutic targets of PCOS hyperandrogenism. IMPACT STATEMENT What is already known on this subject? PCOS is a complex endocrine and metabolic disorders with hyperandrogenism as the main clinical manifestation. There are a variety of abnormal expression circRNAs in PCOS, however, the relationship between circEpha5 and hyperandrogenism has yet to be fully elucidated. What do the results of this study add? We first found that expression levels of serum circEpha5 were significantly higher in PCOS than in a normal group. Using mouse preantral follicle culture model and the letrozole-induced PCOS mouse model, the mechanism of CircEpha5 regulating androgen secretion was studied. What the implications are of these findings for clinical practice and/or further research? It was revealed that CircEpha5 can absorb miR-758-5p in the sponge to regulate the expression of Cyp17a1, thereby promoting the synthesis and secretion of androgen in preantral follicles, which may become a key target for the screening and treatment of PCOS hyperandrogenism.


Introduction
Polycystic ovary syndrome (PCOS) is the most known endocrine disorder among reproductive-age women, with at least two of the following three features: hyperandrogenism, oligomenorrhea or amenorrhoea, and polycystic ovaries (Patel 2018).
It has been reported that most PCOS patients with hyperandrogenism exhibit steroid secretion defects, which can lead to abnormal folliculogenesis and a failure in dominant follicle selection (Zeng et al. 2020).For example, overexpression of steroidogenesis acute regulator (StAR) in PCOS exhibited a state of hyperandrogenism (Kalyani et al. 2018).The elevation of luteinizing hormone receptor (LHR) promotes the conversion of cholesterol into testosterone and androsterone, thus stimulating ovarian theca cells to produce excessive androgen (Ritu et al. 2019).Studies have shown that CAG repeat polymorphisms in androgen receptor (AR) gene may cause hyperandrogenemia in PCOS (Ryan et al. 2018).Theca cells from polycystic ovaries of classic PCOS patients overexpress most steroidogenic enzymes.Cytochrome P450 cholesterol side chain lyase (P450c11, encoded by CYP11A1 gene), catalyses the conversion of cholesterol to progesterenolone during steroid hormone synthesis.Cytochrome P450 17 A-hydroxylase and 17 and 20 carbon chain lyase (P450c17, CYP17A1 gene code) are rate-limiting enzymes for androgen production in the ovary and adrenal cortex (Belani et al. 2018).The expression of enzymes related to androgen biosynthesis increases in follicular membrane cells of PCOS mice, and the level of testosterone also rises in the culture medium of PCOS follicular membrane cells (Jakimiuk et al. 2001, Madeleine et al. 2018).Studying the synthesis and secretion of androgen in follicles may provide new insight into the pathogenesis of PCOS hyperandrogenemia.
Circular RNAs (circRNAs) are a class of tissue-specific, highly conserved, and biologically stable non-coding RNAs (ncRNAs) (Yu et al 2019).CircRNAs can regulate transcription or post-transcription of multiple genes by sponging microRNAs in the development of tumours including ovarian cancer (Zhang et al. 2019), breast cancer (Liu et al. 2022), or other diseases, such as preeclampsia (Wu et al. 2023).It has been found that circRNAs are differentially expressed in the follicular exosomes (Wang et al. 2019) and the cumulus cells of PCOS patients (Che et al. 2019).CircRHBG is significantly increased in granulosa cells of PCOS patients, while knockout of circRHBG can promote iron ptosis in PCOS (Zhang et al. 2021).Knockdown of circ-FURIN suppresses the proliferation and induces apoptosis of granular cells in PCOS (Chen et al. 2021).CircLDLR in the follicular fluid of patients with polycystic ovary syndrome is related to production of oestradiol through CYP19A1 (Huang et al. 2020).However, the pathogenic mechanism of circRNAs in hyperandrogenism of PCOS is still unclear.
In this study, we described the circRNA expression profile in the ovaries of PCOS mice using RNA sequencing technology and investigated the role of circEpha5, which is 664 bp and derived from exon 3 of the host gene Epha5, in androgen synthesis and secretion.

Patients
The serum samples were collected from women of reproductive age (18 to 35 years), who did not receive any hormonotherapy within at least 3 months before serum sampling.A total of 37 participants (22 PCOS and 15 controls) were included.PCOS patients were diagnosed based on the revised Rotterdam criteria (American College of Obstetricians and Gynecologists 2018).Women with regular menstrual cycles and normal ovarian morphology were included in the control group.Additionally, the follicular fluids were collected from six infertility patients (3 PCOS, and 3 controls assisted reproductive technology due to husband factor) during treatment with standard ovarian stimulation.The present study has received ethical approval from the Second Affiliated Hospital of Nanjing Medical University.The clinical characteristics of the PCOS and control patients are shown in Table 1.

Mouse model
Thirty C57BL/6 mice (3-week-old C57BL/6 mice with bodyweight of 9-11 g) were purchased from and reared in the Animal Management Centre of Nanjing Medical University, then randomly divided into 2 groups.The model group was given letrozole gavage solution at 1 ml/(100g/day) for 21 consecutive days, while the control group was given normal saline according to the same standard (Kauffman et al. 2015).From the 14th day, insert a wet cotton swab into the vagina of the mouse for 1 cm and rotate it for several circles, smear it on a clean slide with physiological saline, and then conduct toluidine blue staining after natural drying to distinguish the oestrus cycle of the mouse under the light microscope.Sex hormone levels and biochemical indices in the serum of treated mice were obtained by radioimmunoassay on day 21.After embedding, sectioning, HE staining, and photographing, the ovarian morphology in the PCOS mouse model was demonstrated.The animal experiment was approved by the Ethics Committee of Nanjing Medical University (IACUC-2007007).

High-throughput sequencing
High-purity RNA was extracted from the ovaries of PCOS and control mice to establish a library.The differentially expressed circRNAs (DEcircRNAs) were screened with Illumina sequencing, and their functions with bioinformatics analysis.The top DEcircRNAs were introduced to Enrichr for bioinformatics analysis.In addition, Gene ontology (GO) and KEGG enrichment analyses were performed to reveal the functions and pathways of DEcircRNAs.

Quantitative reverse transcription polymerase chain reaction (qRT -PCR) analysis
The expression levels of circEpha5, miR-758-5p and Cyp17a1 in the experimental groups were determined by qRT-PCR.Total RNA was extracted from human serum, mouse ovaries and follicles using Trizol reagent (Thermo Fisher Scientific, USA) or thermophenol method.HiFairV R reverse transcription kit (Yesen, China) and Hieff UniconV R qPCR kit (Yesen, China) were used for qRT-PCR of mRNAs, circRNAs, and miRNAs, separately.Before calculation, we respectively normalised the circRNA and miRNA expression levels by GAPDH or U6.The key primers are listed in Table 2.

Fluorescent in situ hybridisation (FISH)
FISH was performed according to a standard protocol for circRNA FISH from Exiqon (Jemma, China).Normal ovarian tissue of 3-week-old mice (used for the location of circRNA in the normal ovary) was fixed in 4% paraformaldehyde, dehydrated in gradient alcohol, and prepared into paraffin sections 3 -5um thick.circRNA detection probes for circEpha5 were purchased from GenePharma (China).

Culture of mouse preantral follicles
Mouse preantral follicles were extracted from the ovaries of 3-week-old mice and placed in pre-warmed Leibovitz's L-15 medium (Invitrogen, USA) containing 10% FBS.Individual follicles with a diameter of 180-200 lm and with intact basement membranes and theca layers were isolated under an anatomical microscope, using 28.5-gauge needles attached to 1 ml syringe barrels.These follicles were then incubated in a-MEM medium (Invitrogen, USA) with 5% FBS, 18.35 nmol/L recombinant follicle-stimulating hormone (r-FSH, GONAL-fV R , Meck KgaA, Darmstadt, Germany), and 1% Insulin-Transferrin-Selenium (ITS, Invitrogen, USA) at 37 � C.

Luciferase reporter assay
The pGLO-basic vectors (GenePharma, China) with either circEpha5-WT or circEpha5-MUT were transfected into 293 T cells together with the miRNA (miR-758-5p) mimics or negative control mimics (100 nM) using Lipofectamine Reagent (Yesen, China).After 24h post-transfection, cells were lysed and the Dual-Luciferase Reporter Assay System was used to measure luciferase activity.Firefly luciferase activities were normalised to Renilla luciferase activities.

Statistical method
SPSS 22.0 statistical software was used for data processing.Measurement data were expressed as mean ± SD, and compared with T-test.p < 0.05 indicated that the difference was statistically significant.RT-qPCR data charts were processed by GraphPad Prism 6.0.We used the ARRIVE checklist when writing our report (Percie du Sert N et al. 2020).

Successful establishment of PCOS mouse model
We first established letrozole-induced HA-PCOS mouse model.No differences were found in baseline body weight between control mice (NC group) and the letrozole-induced PCOS mice (PCOS group).The body weights of mice in the two groups increased with time, which demonstrated a significant difference after two weeks of treatment (17.64 ± 1.24g in NC group and 18.70 ± 1.17g in PCOS group, p ¼ 0.021).After 21 days of intragastric administration of letrozole, the average body weight in PCOS group was significantly higher than that in the control group (19.31 ± 1.15g vs. 17.92 ± 0.86g, p ¼ 0.0008) (Figure 1(A)).In addition, the sex hormone levels and biochemical indices showed remarkable differences between the two groups.The serum levels of testosterone (NC group, 0.01 ± 0.00 ng/ml; PCOS group, 0.8 ± 0.18 ng/ml; p ¼ 0.001) and oestradiol (NC group, 13.75 ± 2.33 pg/ml; PCOS group, 18.90 ± 3.16 pg/ml; p ¼ 0.039) increased markedly in the PCOS group.In addition, the ratio of LH/FSH in the PCOS group was distinctly higher than that in the control group (0.98 ± 0.12 in NC group, 1.60 ± 0.34 in PCOS group, p ¼ 0.043) (Figure 1(C)).The profile of sex hormones in the PCOS group was basically consistent with those observed in PCOS patients (Table 1).
We then determined the mRNA levels of sex hormonerelated genes Lhr and Cyp17a1 in the ovarian tissue through RT-qPCR.The results indicated that the expression levels of Cyp17a1 and Lhr were higher in the PCOS group compared with the control group (Figure 1(D)).
We also found that, during the treatment period, the mice in the control group underwent two regular oestrous cycles, with follicles at different developmental stages and multiple luteal bodies, while those in the PCOS group underwent oestrous interphase (Figure 1(e1�e4), (f1/f2)), with only haemorrhagic or cystic dilated follicles (Figure 1(f3/f4)).

CircRNA expression profiles in PCOS mouse ovarian tissue
By the high-throughput RNA-sequencing, 6586 circRNAs were significantly up-regulated, and 6117 circRNAs were significantly down-regulated from the ovaries in letrozole-induced PCOS mice compared with the control group (fold change �2 and adjusted p < 0.001) (Figure S1(A)).The products of parent genes were involved in various signalling pathways, including intracellular phagocytosis, MAPK signalling pathway, and liposome metabolism (Figure S1(B)).We set the standard (high expression reading, fold change > 2 and adjusted p < 0.0001, recorded in the circBase) to screen 16 differentially expressed circRNAs for verification, including 8 up-regulated circRNAs and 8 down-regulated circRNAs (Figure S1(C)).The RT-qPCR results confirmed 7 out of 8 upregulated circRNAs and 5 out of 8 down-regulated circRNAs (Figure S1(c1,c2)).

Highly expressed circEpha5 in the cytoplasm of follicular membrane cells/mesenchymal cells
Among the DEcircRNAs, the expression of mmu_circ_0001362 was significantly higher in the ovaries from PCOS mice, which is 664 bp and derived from exon 3 of the host gene Epha5 (Figure S2(A)).The sequence of mmu_circ_0001362 is highly conserved among different species (Figure S2(B)).In human, HSA_CIRC_0126824 (CHR4:66467358-66468022, exon3) shares a similar sequence with mmu_circ_0001362, and is also derived from the EPHA5.We named the mmu_circ_0001362 and HSA_CIRC_0126824 as circEpha5.
The distribution of circEpha5 in the cells of primary follicles was detected by FISH.Gene mapping was carried out in paraffin sections of mouse ovarian tissue (Figure S2(C)).The result showed that the circEpha5 was mainly expressed in the cytoplasm of ovarian Sertoli-Leydig cells, as well as follicular membrane cells, ovarian stromal cells and peripheral granulosa cells of primary follicles.

CircEpha5 binds and targets miR-758-5p
We next detected the expression level of circEpha5 in the serum and ovarian of mice by RT-PCR, and found that it was much higher in PCOS group than that in the NC group (Figure 2(a1)).And this increasing pattern was also observed in the serum (Figure 2(a2)) and follicle fluid (Figure 2(a3)) of PCOS patients.To predict the potential targets for circEpha5, we searched them through TargetScan (http://www.targetscan.org/),and found that miR-758-5p contained the target seed binding sequence of circEpha5 (Figure 2(B)).To validate this prediction, we constructed luciferase reporter containing putative binding sequence of circEpha5 and co-transfected it into 293 T cells with miR-758-5p mimic or miR-758-5p inhibitor.Luciferase reporter results demonstrated that miR-758-5p mimic could significantly suppress the luciferase reporter signal.However, the disruption of the putative binding sequence within circEpha5 did not cause any significant changes in the luciferase reporter intensity (Figure 2(D)).These data suggested that circEpha5 may sponge miR-758-5p by directly binding it.Therefore, we expected to observe the reversed expression of miR-758-5p relative to circEpha5.The RT-PCR results of both the ovarian tissue and the serum of PCOS mice, and the serum of PCOS patients verified our hypothesis (Figure 2(C)).

miR-758-5p overexpression rescued the negative effect of circEpha5 on testosterone secretion in preantral follicles
Functional experiments were conducted in mouse preantral follicles to investigate the possible role of circEpha5 in regulating testosterone hormone secretion in follicles.We first transfected the circEpha5 mimics into the cultured preantral follicles, and found that the miR-758-5p was significantly decreased by RT-PCR (Figure 3(a1)).Meanwhile, the gene expression levels of androgen-related enzyme Star and Cyp17a1 were increased (Figure 3(a2)).Correspondingly, the level of testosterone in follicular culture medium was also elevated (Figure 3(a3)).
Then, we overexpressed miR-758-5p in the cultured follicles with the miR-758-5p mimics.Interestingly, the expression of circEpha5 was declined (Figure 3(b1)), indicating the presence of negative feedback regulation between miR-758-5p and circEpha5.Meanwhile, the gene expression of androgen-related enzymes Cyp17a1 was decreased (Figure 3(b2)), and the level of testosterone in follicular culture medium was reduced (Figure 3(b3)).However, when we co-transfected circEpha5 and miR-758-5p into the follicles, both the expression levels of Cyp17a1 and the androgen secretion returned to the level of control group (Figure 3(C)).Together, these results demonstrated that there may exist the circEpha5/miR-758-5p/Cyp17a1 regulation axis to regulate androgen production in the follicles.
psychological stress to women.However, the possible mechanisms of androgen production and its wide-ranging effects on PCOS have not been clarified yet.This study firstly explored the expression profile of circRNA in the ovaries of PCOS mice, and it was found that circEpha5 played an important role in the synthesis and secretion of androgens in preantral follicles.
Emphasising the molecular biology of diseases is crucial for understanding physiology and pathology (Cuccu et al. 2023, andGolia D'Aug� e et al. 2023).Studies have indicated that circRNAs could participate in biological processes through various mechanisms, including binding to RBPs, acting as miRNA sponges, and even encoding peptides (Holdt et al. 2016, Zheng et al. 2016, Wang et al. 2017).The bio function of binding to miRNAs has been widely studied, which can promote miRNA degradation (Wang et al. 2017).In our study, miR-758-5p was predicted as a potential target of circEpha5 by bioinformatics analysis.We further confirmed the direct binding between circEpha5 and miR-758-5p in 293 T cells though luciferase reporter assays.Previous Studies have shown that miR-758-5p can regulate cholesterol uptake by targeting CD36 3'UTR (Li et al. 2017).Studies (Yao et al. 2018) have demonstrated that down-regulation of miR-758 disrupts cholesterol metabolism by increasing ATP-binding cassette transporter A1(ABCA1) expression.The profile of miR-758 in cholesterol metabolism is a hot research topic (Zaiou et al. 2018).Given that cholesterol is a raw material for ovarian sex hormone production, we found that PCOS mice had significantly elevated cholesterol levels and decreased expression of miR-758-5p.Thus, we believe that the abnormal cholesterol modulation in PCOS condition may be associated with miR-758-5p.
In addition, the synthesis reaction of steroids depends on a cytochrome P450 enzyme system.Disturbance of follicular development and maturation resulting in premature arrest of follicular growth in PCOS is largely attributed to changes in the expression of steroidogenic enzymes and hormonal secretion in granulosa cells (Salilew-Wondim et al. 2015, Heidarzadehpilehrood et al. 2022).Among those, CYP17A1 is a key steroidogenic enzyme, which has both 17?-hydroxylase and C17, 20 lyase activities (Auchus 2017).Studies have shown that infertility risk, insulin resistance, blood pressure, Figure 2. miR-758-5p sponged by circEpha5.Compared with the control group, mouse ovarian tissue and PCOS patient serum showed higher level of circEpha5 and lower expression of miR-758-5p (A, C).In addition, higher circEpha5 levels were also reflected in mouse serum and human follicular fluid (a2-a3).Bioinformatics analysis showed that circEpha5 and miR-758-5p had binding sites (B).circEpha5 and miR-758-5p had a direct binding site by double luciferase reporter assay(D).� p < 0.05, �� p < 0.01, and ��� p < 0.001 indicate statistical significance.and androgen production in PCOS patients are significantly associated with the occurrence of CYP17A1 polymorphisms (Wang et al. 2018, Lone et al. 2021).Ashraf et al measured hormonal and biochemical tests and anthropometer measurements between the 394 patients with PCOS against 306 control samples.In addition, PCR-RFLP was applied to analyse the genotype.The results of their study showed that there is a significant relationship between the T/C polymorphism in 5'UTR of CYP17 gene with PCOS and hyperandrogenism in the Kashmiri population (Ashraf et al. 2021).These studies indicated that altering the expression and activity of Cyp17a1 may lead to the perturbed homeostasis of steroid hormones commonly seen in PCOS.However, the molecular mechanisms underlying the abnormal expression of these enzymes in PCOS are still unclear.Here, our study showed that, in letrozole-induced PCOS mice, Cyp17a1 was also increased in PCOS than that in the control group.Besides we demonstrated that the overexpression of CircEpha5 in preantral follicles could result in an increased level of Cyp17a1 expression, while forced expression of miR-758-5p decreased Cyp17a1 expression, raising the possibility that Cyp17a1 may be a potential target of CircEpha5/miR-758-5 pathway.Cyp17a1 has a clear physiological role in increasing the expression level of testosterone (Yang et al. 2021, Chen et al. 2022).Higher levels of Cyp17a1 and androgens were respectively found in follicles and their culture media overexpressing circEpha5.In addition, we have found lower levels of Cyp17a1 in follicles overexpressing miR-758-5p.This indicates that circEpha5 may regulate the synthesis and secretion of androgens in follicles by targeting miR-758-5p.It is undeniable that this study has certain limitations.In the future, more experiments, such as circEpha5 knockdown experiments, need to be conducted to demonstrate the role of circEpha5/miR-758-5p/Cyp17a1 cascade regulation in steroid production in polycystic ovary syndrome; More clinical samples need to be collected to clarify the reliability of molecular markers in disease diagnosis and treatment; meanwhile, the role of circEpha5 in improving PCOS symptoms such as reducing androgens and improving fertility (Lagan� a et al. 2022, Miku� s et al. 2022) needs to be focused.
In summary, our study demonstrates that circEpha5 acts as a sponge to depress the function of miR-758-5p, furtherly promotes the expression of the key steroidogenesis-related enzyme Cyp17a1, and finally affects the ovarian androgen release.

Figure 3 .
Figure 3. circEpha5 targets miR-758-5p to regulate the synthesis and secretion of androgens in follicles through Cyp17a1 pathway.In preantral follicles, circEpha5 increased the expression of Star and Cyp17a1, and the level of testosterone in follicular culture medium (A).miR-758-5p decreased the expression of Star and Cyp17a1, and reduced the level of testosterone in follicular culture medium (B).Overexpression of miR-758-5p reversed some changes brought about by circEpha5(C).� p < 0.05, �� p < 0.01, and ��� p < 0.001 indicate statistical significance.

Table 1 .
The clinical characteristics of patients with PCOS.The Bold values with asterisk indicate there are significant statistical differences between the Control group and PCOS group.

Table 2 .
Primers sequences used in this study.