miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

ZHU Lu,JlNG Jing,QlN Shuai-qi,LU Jia-ni,ZHU Cui-yun,ZHENG Qi,LlU Ya,FANG Fu-gui,Ll Yun-sheng,ZHANG Yun-hai,LlNG Ying-hui

1 College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,P.R.China

2 Local Animal Genetic Resources Conservation and Biobreeding Laboratory of Anhui Province,Hefei 230036,P.R.China

Abstract MicroRNA (miRNA) has vital regulatory effects on the proliferation,differentiation and secretion of ovarian granulosa cells,but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear.Both miR-99a-5p and Frizzled-5 (FZD5)were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry,respectively,and FZD5 was verified (P＜0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments.Furthermore,FZD5 mRNA and protein expression were both found to be regulated (P＜0.05) by miR-99a-5p in GCs.Moreover,the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P＜0.05) estradiol and progesterone secretion from the GCs,as determined by ELISA.In summary,miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs.Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

Keywords:miRNA,Frizzled-5,follicular development,estradiol,progesterone

1.lntroduction

Follicular growth and development in the ovaries play an important role in the reproductive processes in female mammals,including the alternation of the estrous cycle.The follicular structure includes oocytes,granulosa cells(GCs),and follicular membrane cells (Barnettet al.2006).Among these cells,ovarian GCs affect the development of follicles and reproductive processes (Jin and Liu 2003).The secretion of steroid hormones by GCs is the most critical regulator in this regard and has thus been a principal focus of much of the research in this field.

miRNAs are involved in a variety of physiological processes as a key gene expression regulator,including metabolism,tumorigenesis,cell proliferation,differentiation,and apoptosis (Bartel 2004;Zhouet al.2017;Linget al.2018,2020;Paceet al.2019;Ranet al.2019;Tanget al.2019;Honget al.2020).miRNA also plays the specific roles in the reproductive functions of female mammals,including ovarian development,follicle maturation,ovarian granulosa cell proliferation,apoptosis,estrogen and progesterone secretion,among others (Tanet al.2014;Godboleet al.2017;Rashmiet al.2019;Zhouet al.2019;Donadeuet al.2020;Zhuet al.2021).Yanget al.(2013) reported previously that miR-143,miR-145,and miR-376a participate in the regulation of primary follicle recruitment and the maintenance of primary follicle numbers in mice from fetal development to birth.In recent years,research into the regulation of steroid hormone secretion by animal ovarian GCsviathe miRNA targeting of specific genes has been increasing.However,few studies have been conducted on this system in goat ovarian GCs.miR-143 has been shown to have a negative effect on the regulation of GC proliferation and cell cycle-related gene expression,and also GC estrogen production (Zhanget al.2017).Zhuet al.(2016) reported that miR-99a-5p may be involved in the regulation of the estrus cycle in goats,but the specific functional effects of miR-99a-5p on goat ovarian GCs have not yet been explored.

The Wnt signal transduction pathway is involved in regulating various physiological activities of cells and thereby participates in organogenesis and body system development (Stolz and Bastians 2015).The Frizzled protein family is divided into 10 categories (FZD1-10),all of which are transmembrane receptors.When the Wnt ligand binds to an FZD receptor,the classic Wnt signaling pathway is activated and subsequently regulates cell proliferation,differentiation and secretion functions (Jiet al.2018).It was further shown that Wnt/β-catenin signaling is activated byFZD5(Carmon and Loose 2008)and that this pathway is involved in the regulation of follicular development (Fanet al.2010).However,the effects ofFZD5on the development of goat follicles and the functions of ovarian GCs in the goat have not yet been investigated.

The hypothesis of this study is that miR-99a-5p andFZD5could be involved in the regulation of hormone secretion from goat ovarian granulosa cells.Here we investigated the role of miR-99a-5p andFZD5in the secretion of steroid hormones from goat GCs culturedin vitro.Our results provide initial reference data for future research into the molecular mechanisms of GC steroid hormone secretion.

2.Materials and methods

2.1.Tissue collection and cell culture

Fresh goat ovaries were collected from the slaughterhouse in Boda Co.(Hefei,Anhui,China).The ovaries were flushed with saline,and then immersed with 37°C saline and transported to the laboratory in a thermos flask within 4 h.After further rinsing of the ovaries 3 times with 37°C saline and wiping with sterile gauze,follicular fluid containing GCs was obtained from the translucent follicles (Guoet al.2018),with a diameter of between 3 and 5 mm,viaa 1-mL sterile syringe.The collected follicular fluid was filtered through 400 mesh sterile screens,centrifuged,and washed with phosphatebuffered saline (PBS;Biosharp,Hefei,China).Finally,cells were seeded in a 60-mm cell culture dish using MEMF-12 (HyClone,USA) containing 10% fetal bovine serum (FBS;Gibco,USA),and then cultured at 37°C in 5%CO2incubator.When the culture density reached 80%,the cells were seeded into 6-or 96-well plates for further experimental processing.293T cells were obtained from our laboratory and cultured in a high-sugar medium(HyClone,USA) containing 10% FBS at 37°C in a 5%CO2cell incubator.

2.2.Fluorescence in situ hybridization (FlSH) of miR-99a-5p

The fresh ovaries were fixed in 30 mL of formaldehyde for 24 h and tissue blocks were then made using conventional paraffin embedding methods.Paraffin sections were subsequently cut to generate tissue sections.For FISH analysis,these sections were deparaffinized and subjected to protein digestion,and a miR-99a-5p probe hybridization solution at a concentration of 8 ng μL-1was added at 37°C for a 12 h incubation.The sections were then washed,and the nuclei were counterstained with DAPI to observe nuclear fluorescence.

2.3.lmmunohistochemical staining of FZD5

For immunohistochemical staining ofFZD5,goat ovarian tissue sections were deparaffinized,hydrated,and then pretreated with citrate buffer for 25 min.The sections were then immersed in PBS containing 3% H2O2for 25 min and incubated with rabbit polyclonal anti-FZD5(dilution 1:100) at 4°C overnight.Thereafter,the sections were incubated with a HRP-conjugated secondary antibody for 50 min at room temperature and then stained using a DAB color substrate solution followed by hematoxylin.The slides were then observed under a microscope.

2.4.Cell transfection

GCs were transfected when the cell density reached 70-80% confluence with a miR-99a-5p mimic,inhibitor and negative control (NC) obtained from GenePharma(Shanghai,China),using Lipofectamine? 2000 (Thermo Fisher Scientific,USA).The cells were harvested 48 h later.

2.5.RNA isolation,reverse transcription and qPCR

Total RNA was extracted fromin vitrocultured GCs using the Total RNA Kit I (OMEGA,USA) in accordance with the manufacturer’s instructions.Total RNA (500 ng) was reverse-transcribed using Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech,China).For miR-99a-5p samples,however,we used special stem-loop primers for reverse transcription.These primer sequences are listed in Table 1.Real-time quantitative PCR (qPCR) was then conducted using a Real-Time PCR System from Applied Biosystems (ABI,USA) with SYBR Green Mix (Tolobi,China).The reaction mixture was amplified using a hot-start at 95°C for 5 min followed by 40 cycles of 95°C for 15 s,55°C for 30 s,and 72°C for 15 s.All primers were synthesized by Sangon Biotech Company (Shanghai,China).The expression levels of the amplified products were calculated using the 2-ΔΔCtmethod.

Table 1 Sequences of the primers used for reverse transcription and qPCR

2.6.Luciferase reporter assay

The goat miR-99a-5p sequence was obtained from the miRBase website (http://www.mirbase.org) and submitted to GenePharma (Shanghai,China) for synthesis.The targeted binding sites of miR-99a-5p andFZD5were determined using TargetScan (http://www.targetscan.org).TheFZD53′UTR and mutation site sequences were obtained using a PCR Kit (TaKaRa,Japan).The obtained target fragment was inserted into a pGL3-control vector (digested with the restriction enzymeXbaI) using a cloning kit (Vazyme,China) and then verified by sequencing.Recombinant plasmids and mimics or inhibitors were transfected into 293T cells(70% density in a 96-well cell plate at transfection) using lipo2000 (Thermo Fisher Scientific,USA).After the cells were lysed,double luciferase activity was detected using a Dual-Luciferase Assay System (Promega,Madison,WI,USA).

2.7.Goat FZD5 lentiviral vector infection

When the goat GC density in the 6-well cell plates reached 30%,10 μL of interferenceFZD5lentiviral vectors was added (constructedviaa three-plasmid method in our laboratory).At the same time,polybrene was added at a concentration of 5 ng mL-1.The complete medium was changed after 6 h,and the cells and supernatant were collected after 48 h.

2.8.Western blotting

Protein lysates were generated from the goat GCs and the protein concentrations were determined using a BCA Protein Assay Kit (Beyotime,China).For Western blotting(WB) analysis,the samples were resolved using 12%SDS-PAGE and transferred onto a PVDF membrane(Beyotime).The membrane was then incubated with the primary antibodies against FZD5 and β-actin (1:1 000,Abcam,UK) overnight and with a secondary antibody at room temperature for 30 min on the following day.Protein bands were visualized using an imaging system after the addition of developer.Bands were quantified using ImageJ Software.

2.9.E2 and P4 detection

The supernatants collected from the GC cultures were tested for estrogen (E2) and progesterone (P4)concentrations using goat E2 and P4 ELISA kits (Shanghai Keshun,China),respectively,in accordance with the manufacturer’s instructions (goat P4 ELISA Kit:tolerance within batch,CV＜10%;tolerance between batches,CV＜15%;sensitivity,0.625-20 ng mL-1;goat E2 ELISA Kit:tolerance within batch,CV＜10%;tolerance between batches,CV＜15%;sensitivity,5-160 pg mL-1).

2.10.Statistical analysis

Data were analyzed using SPSS 19.0 Software.Each replicate of GCs or 293T cells served as an experimental unit.The Student’st-test (two-tailed) was used to measure the significance of differences between two treatments in the data for qPCR and WB cells that were treated with a mimic or inhibitor of miR-99a-5p,as well as ELISA of the E2 and P4 assays.The data in qPCR and WB from cells infected withFZD5lentivirus and results of dual-luciferase assays were analyzed using a one-way analysis of variance (ANOVA),and the least significant difference (LSD) method was used to test the significance of differences among treatments.P-values of less than 0.05 were considered to indicate a significant difference.

3.Results

3.1.miR-99a-5p and FZD5 expression in goat ovarian granulosa cells

To explore the expression profiles of miR-99a-5p andFZD5in goat ovaries,we collected goat ovarian tissue from which we made paraffin sections.A red fluorescent probe for goat miR-99a-5p was designed and synthesized,and used for FISH analyses of the tissue sections.The positive expression of miR-99a-5p in goat ovarian tissues could be observed under low magnification (Fig.1).At the same time,DAPI staining revealed the composition of the follicles.It was found that miR-99a-5p was expressed in GCs and mainly present in the cytoplasm.In addition,the results of immunohistochemistry showed thatFZD5was also expressed in the granulosa cells in goat follicles(Fig.2).These results suggested the possibility that miR-99a-5p andFZD5have functional roles in GCs.

Fig.1 miR-99a-5p expression in goat ovarian granulosa cells.Fluorescent images of goat granulosa cells in ovarian tissue.Red fluorescence indicates positive miR-99a-5p expression,and blue fluorescence demarcates the nucleus.The merged fluorescence image illustrates that miR-99a-5p is expressed in the granulosa cells in goat ovarian tissue.Three biological replicates represent ovaries from different individual goats.

Fig.2 FZD5 expression in goat ovarian granulosa cells.A,micrograph showing secondary follicle tissues in goat ovaries.B,micrograph showing antral follicle tissues in goat ovaries.C,negative control (PBS substituted FZD5 antibody).Cells indicated by the red arrows are ovarian granulosa cells.Positive FZD5 expression is evident in the granulosa cells of both the primary follicle and the antral follicle.Three biological replicates represent ovaries from different individual goats.

3.2.miR-99a-5p targets FZD5 expression in GCs

The miR-99a-5p mimics,inhibitors or controls were transfected into GCs and then subjected to qPCR analysis.The expression of miR-99a-5p was found to be significantly increased (P＜0.001) in the cells of the mimic group,while the expression of the inhibitor group was significantly decreased (P＜0.05) (Fig.3-A).To explore the targeted regulatory relationship between miR-99a-5p andFZD5,dual luciferase reporter experiments were designed.Recombinant dual luciferase reporter vectors containing the original or mutant sequences of the target binding site were constructed.The target binding site of miR-99a-5p on the FDZ5 3′UTR was predicted to be 5′-ACCCGUA-3′ using the Targetscan website.Vectors containing wild-type sites or mutant sites and miR-99a-5p mimics or their controls were transfected into 293T cells to detect the luciferase expression.It was found that the fluorescence ratio of the WT and miR-99a-5p mimic group was significantly lower (P＜0.001) than those of either the WT and NC (mimic NC),or the MUT and miR-99a-5p group.Moreover,there were no significant differences(P＞0.05) between the WT and NC and the MUT and miR-99a-5p groups (Fig.3-B).These data suggested thatFZD5is a target gene for miR-99a-5p.

Fig.3 miR-99a-5p targets and represses FZD5 mRNA and protein expression in goat granular cells.A,miR-99a-5p expression(normalized to snoRNA U6) in granulosa cells (GCs) cultured in vitro and treated with a miR-99a-5p mimic or inhibitor for 48 h.B,predicted miR-99a-5p target site within goat FZD5 (wild type,WT) and the mutated sequence (MUT) at this target site.Data are expressed as relative luciferase activities in 293T cells that were cotransfected with WT or MUT reporter constructs and miR-99a-5p mimic or mimic NC after 48 h.C and D,FZD5 mRNA (C) and protein (D) expression levels in GCs cultured in vitro and treated with a miR-99a-5p mimic or inhibitor for 48 h.m-NC,negative control of miR-99a-5p mimic;i-NC,negative control of miR-99a-5p inhibitor;NC,negative control of miR-99a-5p mimic;mimic,miR-99a-5p mimic;inhibitor,miR-99a-5p inhibitor.Data represent the mean value±SE from three biological replicates (n=3).Different letters indicate significant differences (P＜0.05).

To further determine the targeted regulatory relationship between miR-99a-5p andFZD5in GCs,miR-99a-5p mimics and inhibitors were transfected into GCs culturedin vitro.Fig.3-C shows that the level ofFZD5mRNA was significantly decreased (P＜0.01) after transfection of the mimics into GCs,whereas the expression increased(P＜0.01) significantly after transfection of the inhibitors.The expression of FZD5 protein was significantly reduced(P＜0.05) after the transfection of miR-99a-5p mimics and significantly increased after transfection of miR-99a-5p inhibitors (Fig.3-D).These findings indicated that miR-99a-5p inhibitsFZD5expression in GCs culturedin vitro.

3.3.Effects of miR-99a-5p and FZD5 on steroid secretion from GCs

After confirming thatFZD5is a target gene of miR-99a-5p,we further explored the effects ofFZD5and miR-99a-5p on steroid hormone secretion from goat GCs.First,GCs were infected with either anFZD5-interfering lentivirus(FZD5shRNA) or a control lentivirus (NC).The results of qPCR and Western blotting revealed significantly reduced (P＜0.01)FZD5mRNA and protein contents in GCs expressingFZD5shRNA (Fig.4-A and B).After knocking down the expression ofFZD5in GCs,it was found by ELISA that the estradiol (E2) and progesterone(P4) contents were also significantly reduced (P＜0.05)(Fig.4-C).In addition,the E2 and P4 concentrations in the supernatant of cultured GCs were also decreased significantly (P＜0.05) after increasing the expression of miR-99a-5p in these cells (Fig.4-D and E).

Fig.4 Estradiol and progesterone secretion by granulosa cells in vitro were decreased by overexpression of miR-99a-5p and knockdown of FZD5.A and B,FZD5 mRNA (A) and protein (B) expression in granulosa cells (GCs) cultured in vitro and infected with an interfering lentivirus (FZD5 shRNA) for 48 h.C and E,relative contents of estradiol and progesterone in GCs infected with FZD5 shRNA or transfected with miR-99a-5p for 48 h.The expression levels were determined by enzyme linked immunosorbent assay (ELISA).Blank,blank contrast;m-NC,negative control of miR-99a-5p mimic;i-NC,negative control of miR-99a-5p inhibitor;NC,negative control of FZD5 shRNA lentivirus;mimic,miR-99a-5p mimic;inhibitor,miR-99a-5p inhibitor.Data represent the mean value±SE of three biological replicates (n=3).Different letters indicate significant differences (P＜0.05).

4.Discussion

In this study,we first analyzed the expression of miR-99a-5p andFZD5in goat ovaries by FISH and immunohistochemistry.We found that miR-99a-5p is significantly expressed in goat ovarian GCs.Software predictions and luciferase double-reporter assay results indicate thatFZD5is a target gene of miR-99a-5p,and that the expression ofFZD5in GCs is regulated by this miRNA.ELISA results further indicated that miR-99a-5p andFZD5inhibited steroid hormone secretion by goat GCs,and that miR-99a-5p exerted this functional role by down-regulating the expression of theFZD5gene.These results have supported our hypothesis that miR-99a-5p andFZD5could be involved in the regulation of hormone secretion from goat ovarian granulosa cells.This provides initial reference data for future research into the molecular mechanisms of GC steroid hormone secretion.

A large body of data has revealed that miRNAs are involved in the regulation of the hypothalamicpituitary-ovary reproductive axis in mammals.Hohjoh and Fukushima (2007) reported that more than 50%of miRNAs are expressed in the central nervous system of mice,suggesting that they may regulate animal reproductionviathe hypothalamus (Hohjoh and Fukushima 2007).Huanget al.(2016) found differences in the expression of miR-99a,miR-224 and let-7c in the ovaries of pigs with high and low litter sizes (Huanget al.2016),further indicating that miR-99a may be involved in regulating the reproductive processes of mammals.It has also been shown that miR-21-3p and miR-433 directly targetFSHb3′UTR and downregulateFSHbexpression in rat pituitary cells,which affectsFSHsecretion (Hanet al.2017).Additional studies have reported an involvement of miRNAs in the growth and development of follicles.For example,Ziet al.(2017) observed differences in the expression of miR-99a,miR-21 and miRNA-143 in the follicular stage ovaries between prolific and non-prolific goats.Together with our data,it is hypothesized that miR-99a-5p may affect follicular development and alter the estrous cycle by regulating steroid hormone secretion from the ovarian granulosa cells of goats.

In this study,the results of FISH analyses demonstrated that miR-99a-5p is expressed in the goat ovary.The use of DAPI nuclear staining further indicated that miR-99a-5p is expressed in the GCs in goat follicles.We thus came to a conclusion that miR-99a-5p is likely involved in the development of goat follicles and in the regulation of goat GC function.To test this hypothesis,mimics and inhibitors of miR-99a-5p were transfected into goat ovarian GC cultures.After verifying the expected effects of the mimics and inhibitors,the estradiol and progesterone contents in the culture supernatants were detected by ELISA.The levels of these secreted hormones were decreased by the miR-99a-5p mimic and increased by the inhibition of this miRNA.Hence,miR-99a-5p inhibits the secretion of estradiol and progesterone by goat ovarian GCs.

To further explore the mechanisms underlying the effects of miR-99a-5p on the secretion of steroid hormones from ovarian GCs,the target genes of miR-99a-5p were predicted using TargetScan.Interestingly,FZD5was identified as one such target.Studies by Petersonet al.(2017) have shown that the combination of theFZD5receptor andSFRP2(secreted frizzledrelated protein 2) activates the Wnt/Ca2+pathway,leading to intracellular Ca2+release and activation of the calcineurin/NFATC3 pathway to induce endothelial cell angiogenesis.Luet al.(2013) found thatFZD5knockout mice had reduced placental angiogenesis and that embryonic death occurred after 10.5 days of pregnancy.Hence,FZD5was selected for further analysis in our present study,i.e.,the next step of verification.We first found by immunohistochemistry thatFZD5is expressed in the primary follicles,and in the GCs in stimulated follicles,in the goat.This result was consistent with the results and conclusions from the aforementioned studies of Luet al.(2013) and Petersonet al.(2017).Second,double luciferase gene reporter experiments revealed that miR-99a-5p binds to the binding site of theFZD53′UTR and thereby reduces the fluorescence ratio,which was blocked by a binding site mutation.Consistently,the expression ofFZD5,at both the mRNA and protein levels,was reduced upon miR-99a-5p transfection into GCs.In addition,the expression ofFZD5was increased by miR-99a-5p inhibition.These results indicated that miR-99a-5p targets and downregulates theFZD5gene in GCs.

We further constructed anFZD5shRNA in a lentiviral vector,verified the interference effect though qPCR and Western blotting,and then found using ELISA that knocking down the expression ofFZD5in GCs significantly reduced the secreted levels of E2 and P4.However,whether miR-99a-5p regulates steroid hormone synthesis by targetingFZD5remains to be further explored.In addition,the specific molecular mechanism by whichFZD5regulates steroid hormone synthesis is not clear.

5.Conclusion

miR-99a-5p inhibits target geneFZD5expression and estradiol and progesterone synthesis in GCs.Among them,FZD5promotes E2 and P4 synthesis but miR-99a-5p inhibits their synthesis.These functional insights into the roles of miR-99a-5p andFZD5in ovarian GCs provide important basic data for future studies on the regulation of follicular development by miRNAs.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31772566 and 31972629)and the Central Guidance on Local Science and Technology Development Fund of Anhui Province,China(202007d06020005).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal tissues conformed to the standards of the ethics committee of Anhui Agricultural University,Anhui,China (permit no.AHAU20101025).