Molecular and Cellular Endocrinology 382 (2014) 244–253

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MicroRNA-224 is involved in the regulation of mouse cumulus expansion by targeting Ptx3 Guidong Yao a,b,c,1, Meng Liang a,b,1, Ning Liang a,b, Mianmian Yin a,b, Mingrong Lü a,b, Jie Lian a,b, Yong Wang a,b, Fei Sun a,b,⇑ a b c

Department of Cell and Developmental Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui 230027, People’s Republic of China Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, People’s Republic of China

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Article history: Received 11 June 2013 Received in revised form 7 October 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: MicroRNA-224 Mouse cumulus expansion Ptx3 EGF

a b s t r a c t MicroRNAs (miRNAs) are indicated to regulate ovarian development in a cell- or stage-specific manner. Our previous study showed that miR-224 is involved in TGF-b1-mediated follicular granulosa cell (GC) growth and estradiol (E2) production during the transition from the preantral to early antral stage by targeting Smad4. In this study, miR-224 was found to target pentraxin 3 (Ptx3), a gene critical for cumulus expansion during ovulation. In addition, PTX3 was up-regulated in mouse mural GCs and cumulus-oocyte complexes (COCs) by TGF-b1 treatment, which was partially mediated by miR-224. The effect of miR-224 during ovulation was further examined in vitro and in vivo by construction of an adenovirus-mediated expression vector for miR-224 (Ad-miR-224). In vitro studies demonstrated that miR-224 could perturb cumulus expansion in EGF-stimulated COCs by decreasing PTX3 secretion. In vivo studies also showed that injection of Ad-miR-224 into ovarian bursa decreased PTX3 expression and disrupted ovulation, which led to a decreased number of implantation sites and offspring being born. These results indicate that miR-224 may affect ovulation and subsequent embryo development by targeting Ptx3, suggesting potential roles for miRNAs in offering new treatments for ovulation disorder-associated infertility, or, conversely, designing new contraceptives. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction MicroRNAs (miRNAs) are endogenous small noncoding RNAs (-22 nt) that serve as post-transcriptional regulators of gene expression (Ambros, 2004; Bartel, 2004; Zamore and Haley, 2005). The vital role of miRNAs in the development and functioning of reproductive tissues is now becoming increasingly understood. In the ovary, targeted deletion of Dicer1, a key enzyme in miRNA biogenesis, provided the first evidence that miRNAs were critically involved in multiple aspects of ovarian function. Female

Abbreviations: miRNAs, microRNAs; Ptx3, pentraxin 3; UTRs, untranslated regions; CL, corpus luteum; DMSO, dimethylsulfoxide; PND, postnatal day; eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin; ELISA, enzyme-linked immunosorbent assay; GCs, granulosa cells; mpGCs, mouse preantral granulosa cells; muGCs, mouse mural granulosa cells; COCs, cumulus-oocyte complexes. ⇑ Corresponding author at: Department of Cell and Developmental Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China. Tel.: +86 551 6360 0847; fax: +86 551 6360 2703. E-mail address: [email protected] (F. Sun). 1 These authors contributed equally to this work. 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.10.014

mice with global miRNA deficiency as a consequence of conditional Dicer1 knock-out are sterile; the causes are: meiotic defects secondary to disorganized spindle formation (Murchison et al., 2007; Tang et al., 2007), multiple reproductive defects including decreased ovulation rates, compromised oocyte and embryo integrity, prominent bilateral tubal cysts and shorter uterine horns (Gonzalez and Behringer, 2009; Hong et al., 2008; Nagaraja et al., 2008), as well as defects in corpus luteum (CL) angiogenesis accompanied by decreased progesterone concentrations (Otsuka et al., 2008). Yang et al. demonstrated that injection of miR17-5p and let-7b into the ovarian bursa of Dicer knock-out mice could restore the vasculature within the CL and increase progesterone concentrations but failed to maintain pregnancy, indicating that other miRNA(s) might still be involved (Gonzalez and Behringer, 2009). These studies demonstrate important roles of miRNAs during ovarian development and female reproduction, although knowledge about miRNAs in the context of control of ovarian physiology is still limited. Several studies have resulted in miRNAs being recognized as important regulators of female reproduction in a cell- or stagespecific manner. For example, miR-21, which is induced by the LH surge, blocks apoptosis in mouse periovulatory granulosa cells

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(GCs) and plays an important role in maintaining CL function (Carletti et al., 2010). By regulating PTGS2 and RECK and fine tuning the uterine functions of estrogen and progesterone during implantation in mammals, miR-21, up-regulated in implantation sites, ensures precise control of implantation events (Hu et al., 2008). And miR-21 represses TGF-b1 mRNA translation (Lu et al., 2009), which is down-regulated in endometriosis and could account for the enhanced TGF-b activity with endometriosis (Teague et al., 2010). However, detailed studies of miRNAs in the control of ovarian physiology at different developmental stages are still limited. In our previous studies, during the transition from the preantral to early antral stage, miR-224 was shown to be involved in TGF-b1-mediated follicular GC growth and estradiol production by targeting Smad4 (Yao et al., 2010) and to be transcriptionally regulated by p53 and NF-jB p65 in mouse preantral GCs (Liang et al., 2013); miR-383 functions to promote steroidogenesis by targeting Rbms1 and through inactivation of c-Myc (Yin et al., 2012). In addition, Ptx3, a gene critical for cumulus expansion (Salustri et al., 2004), is predicted to be another target for miR-224 by PicTar (Krek et al., 2005) and TargetScan (Lewis et al., 2005). Cumulus expansion is a critical process during ovulation; furthermore, Ptx3-deficient mice show a severe defect in female fertility (Garlanda et al., 2002; Salustri et al., 2004; Varani et al., 2002). These results indicate that miR-224 may also function in later follicular development, such as ovulation. In this study, the targeting effect of miR-224 on Ptx3 and corresponding function during ovulation were validated, in vitro and in vivo, by construction of an adenovirus-mediated expression vector for miR-224. 2. Materials and methods 2.1. Animals ICR mice were obtained from the Animal Center, University of Science and Technology of China (USTC), and housed in temperature (22 C) and light cycle (14 h light, 10 h dark) controlled quarters. Mice were provided food and water ad libitum. This study received ethical approval from the institutional review boards of USTC. 2.2. Reagents, follicle isolation, cell and cell culture Human TGF-b1 (R&D Systems, Minneapolis, MN) was reconstituted in acidified buffer [4 mM HCl (pH 5.7), 1 mg/ml BSA] to prepare a 10 lg/ml stock solution. Human EGF (R&D Systems, Minneapolis, MN) was dissolved in PBS to prepare a 5 lg/ml stock solution. SB431542 (an inhibitor of Smad2/3 activation) (Sigma) was reconstituted in dimethylsulfoxide (DMSO) to a concentration of 10 mM. These reagents were stored at 20 C. The different developmental stage follicles with different sizes (measured with an ocular micrometer) were isolated mechanically from postnatal mouse ovaries (Liang et al., 2011; Shen et al., 2007). Ovarian follicles with 60–80 lm in diameter (number of follicles isolated, n = 150), 125–155 lm in diameter (n = 100), and 280– 320 lm in diameter (n = 40) were isolated from postnatal day (PND) 8 (8 mice), PND14 (6 mice), and PND21 (4 mice), respectively, and three independent pools of each size of ovarian follicles were obtained from different batches of mice. Mouse preantral granulosa cells (mpGCs) were obtained and cultured as described previously (Yao et al., 2010). Briefly, the preantral follicles of PND 10-12 mouse ovaries were isolated with No. 5 fine needles (Sigma). The preantral follicles were then treated with type IV collagenase (Sigma) and Tryple Express (Life Technologies, Inc.), and the GCs were washed and collected by brief

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centrifugation. Mouse mural granulosa cells (muGCs) were collected from 21–23 day postpartum mice primed with 5 IU equine chorionic gonadotropin (eCG) for 44–48 h. The antral follicles were punctured with needles to release muGCs and cumulus cell-oocyte complexes (COCs) that were collected with micropipettes. MuGCs were separated from COCs using a 50 lm Nitex size exclusion filter (Wildlife Supply Company, Buffalo, NY). These cells were then pelleted by centrifugation (200g for 5 min) and cultured at 37 C and 5% CO2 in DMEM/F12 (1:1, Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 1% antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin; Life Technologies, Inc.). In each experiment of mpGCs, muGCs or COCs, three independent pools of mpGCs, muGCs or COCs were obtained from different batches of mice. The HEK293T, HEK293A and NIH3T3 cell lines were kindly donated by Professors Mian Wu, Guanghui Wang and Tao Zhu, respectively (School of Life Sciences, USTC). Each cell type was grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 1% antibiotics (100 U/ ml penicillin and 100 lg/ml streptomycin; Life Technologies), and cultured at 37 C under 5% CO2 atmosphere. 2.3. mRNA microarray analysis The mpGCs were serum starved for 24 h and then treated with or without 5 ng/ml TGF-b1 for 6 h. MiRNA expression profiles of mpGCs either treated or untreated with TGF-b1 were described previously (Yao et al., 2010). For mRNA microarray, the samples were amplified and labeled using the Agilent Quick Amp labeling kit and hybridized with Agilent whole genome oligo microarray in Agilent’s SureHyb Hybridization Chambers. Differentially expressed mRNAs were defined as genes whose expression in the study group (TGF-b1-treated mpGCs) were consistently altered 2-fold (either higher or lower) compared with the control group (untreated mpGCs). 2.4. Plasmid construction To experimentally verify whether the Ptx3 gene is a direct target of miR-224, the 30 UTR of the Ptx3 was amplified from the genomic DNA of the mouse ovary using primers as shown in Supplemental Table S1A. Mutant Ptx3 30 UTRs were also generated for each of the two miR-224 target recognition sites (seed sequences), as shown in Supplemental Fig. S1. Both the wild-type and mutated 30 UTRs of the Ptx3 gene were cloned into the psiCHECK-2 dual luciferase reporter vector using Xho I and Not I restriction sites. The constructs generated were confirmed by sequencing. 2.5. Transient transfection and luciferase activity assay Transient transfections of mpGCs and HEK293T cells were described previously (Yao et al., 2010). MuGCs were transfected with oligonucleotides using HiPerFect Transfection Reagent (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. For reporter assays, HEK293T cells were transiently transfected with Ptx3 30 UTR or its mutant reporter construct together with miR-224 mimic/mimic negative control (mimic NC) or miR-224 inhibitor/inhibitor negative control (inhibitor NC). MiR-224 mimic and mimic NC, as well as miR-224 inhibitor and inhibitor NC were chemically synthesized and purified by Shanghai Gene-Pharma Co. (Shanghai, China). MiR-224 mimic is chemically synthesized, double-stranded RNAs that mimic mature endogenous miR-224, whereas miR-224 inhibitor is a single-stranded 2-O-methylmodified oligoribonucleotide fragment exactly antisense to

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miR-224. Mimic NC is double-stranded RNAs with a scrambled nucleotide sequence and inhibitor NC is single-stranded RNAs with a scrambled nucleotide sequence. Cell extracts were prepared 30 h after transfection and the ratio of Renilla to firefly luciferase was measured with the Dual-Luciferase Reporter Assay System (Promega Corp.).

2.6. RNA extraction and real-time PCR Total RNA from mouse ovarian follicles or cultured cells was extracted with Trizol (Invitrogen, Carlsbad, CA). Real-time PCR assays of mRNA or miRNA have been described previously (Bustin et al., 2009; Liang et al., 2011; Yao et al., 2010). Briefly, for mRNA assay, 500 ng of RNA was reverse-transcribed into cDNA using PrimeScript RT Enzyme Mix I (PrimeScript RT reagent kit; TaKaRa). The SYBR Green I dye chemistry (SYBR premix Ex Taq kit; TaKaRa) was used to detect mRNA expression levels according to the manufacturer’s protocols. For miRNA assay, 20 ng of RNA was reversetranscribed into cDNA using MultiScribe Reverse Transcriptase (TaqMan MicroRNA RT Kit; Applied Biosystems). The TaqMan chemistry (TaqMan microRNA assays; Applied Biosystems) was used to detect expression level of miR-224 according to the manufacturer’s instructions. The relative expression levels of mRNAs and miRNA were calculated using the comparative 2-DDCt method (Livak and Schmittgen, 2001) and were normalized to endogenous GAPDH mRNA and U6 small nuclear RNA, respectively. Each PCR reaction for individual pool of cells or follicles was repeated in triplicate and all real-time PCR experiments were performed on three independent pools of cells or follicles. The calibrator was one of the negative control sample or the first sample of Fig. 2 and Supplemental Fig. S4/S7 in three independent pools of cells or follicles. The primer sequences for mouse TGF-b1 were previously described (Liang et al., 2011). Primers for Ptx3, Has2, Ptgs2, Tnfaip6, and GAPDH are listed in Supplemental Table S1B. The probes specific for miR-224 and U6 small nuclear RNA were purchased from Applied Biosystems.

2.7. Western blotting Western blotting analysis has been described previously (Yao et al., 2010). The following antibodies were used for immunoblotting: anti-Smad4 (Santa Cruz, CA), anti-PTX3 (Abcam, Cambridge, MA) and anti-GAPDH (Cell Signaling Technology, MA).

2.8. Enzyme-linked immunosorbent assay (ELISA) The concentrations of PTX3 in the supernatants were assayed using the Quantikine Mouse Pentraxin 3/TSG-14 Immunoassay Kit (R&D Systems, Minneapolis, MN); the procedures were performed according to the manufacturer’s instructions. In brief, 50 ll of Assay Diluent RD1-41™ were added to each well before adding 50 ll of the supernatants (some required further dilution), then incubated for 2 h at room temperature on a horizontal shaker. After five washes, 100 ll of mouse PTX3 Conjugate was added to each well and incubation was continued for 2 h at room temperature. The previous washing steps were repeated, then 100 ll of Substrate Solution was added into each well and the samples were incubated for an additional 30 min without shaking. After adding 100 ll of Stop Solution into each well, the concentrations of PTX3 were measured in a 96-well format plate reader (ELX 800 Universal Microplate Reader; BioTek, Inc., Highland Park, IL) by measuring the absorbance at a wavelength of 450 nm. All measurements were performed in triplicate.

2.9. Adenovirus preparation Replication incompetent recombinant adenoviruses expressing miRNAs were constructed using the BLOCK-It™ Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and Sugiura et al. (2009) with slight modifications. Two single-stranded DNA oligonucleotides, one encoding precursor miR-224 and an additional CACC sequence at its 50 end (50 -CACCGGGGCTTTTAAGTCACTAGTGGTTCCGTTTAG TAGATGGTTTGTGCATTGTTTCAAAATGGTGCCCTAGTGACTACAAAG CCC-30 ) and the other encoding complementary sequences and an additional AAAA sequence at its 50 end (50 -AAAAGGGCTTTGTAGTCACTAGGGCACCATTTTGAAACAATGCACAAACCATCTACTAAACGGA ACCACTAGTGACTTAAAAGCCCC-30 ) were synthesized. The CACC and AAAA sequences are complementary to the overhang sequences in the pENTR™/U6 vector. These two single-stranded DNA oligonucleotides were annealed and then the generated double-stranded DNA oligonucleotide was cloned into the pENTR™/U6 vector to produce the pENTR™/U6-miR-224 vector using the BLOCK-iT™ U6 RNAi Entry Vector Kit (Invitrogen). After introducing the ligation reaction product into competent E. coli, individual clones were isolated and sequenced. The U6 RNAi cassette in the pENTR™/U6- miR-224 vector was transferred to the adenoviral expression plasmid by LR recombination reaction using the GatewayÒ LR Clonase™ II Enzyme Mix and pAd/BLOCK-iT™-DEST GatewayÒ Vector Kit (Invitrogen). The reaction product was introduced into competent E. coli and individual clones were isolated and sequenced. Then the isolated adenoviral expression and control (pAd-GW/U6-laminshRNA) plasmids were digested with the restriction enzyme Pac I (Fermentas life sciences). The digested plasmids were transfected into 293A cells using Lipofectamine™ 2000 (Invitrogen) to produce crude adenoviral stocks. The crude adenoviral stocks were then used to infect 293A cells to amplify the adenoviral stocks. The amplified stocks were purified using the Adeno-X™ Virus Purification Kit (Clontech Laboratories, Inc., Palo Alto, CA) and stored at 80 C. The titers of the stocks were determined using the Adeno-X™ Rapid Titer Kit (Clontech Laboratories) on 293A cells. The purified adenoviruses expressing miR-224 and lamin A/CshRNA were defined as Ad-miR224 and Ad- laminshRNA, respectively.

2.10. COC isolation and viral infection ICR female mice, 21–23 days old were sacrificed by cervical dislocation 44–48 h after i.p. injection of 5 IU eCG, and their ovaries were dissected. Ovaries were cleaned free of adherent adipose and connective tissues and placed in MEM-a (Life Technologies) supplemented with 3 mg/ml BSA (Sigma). COCs were mechanically isolated by puncturing antral follicles with No. 5 fine needles (Sigma). Only COCs with a uniform covering of compacted cumulus cells were used in this study. The COCs were collected separately from the muGCs by pipette, pooled, and treated as described in the text. COCs were cultured in drops of medium under mineral oil. The basic culture medium for the COCs consisted of MEM-a supplemented with 3 mg/ml BSA, 1% sodium pyruvate (sigma), and 1% antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin; Life Technologies) at 37 C under 5% CO2 atmosphere for the times indicated in the text. For COC viral infection, 20 COCs were cultured in a 50 ll drop of basic culture medium supplemented with the phosphodiesterase inhibitor milrinone (10 lM) (Sigma) (to maintain the oocytes at the germinal vesicle stage) and 108 M 17b-estradiol (Sigma) together with of 2  106 ifu adenovirus/COC. After 24 h, the COCs were washed thoroughly in basic culture medium, then cultured in basic culture medium supplemented with 1% FBS and 10 ng/

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ml EGF to examine cumulus expansion and RNA expression at the indicated times. 2.11. Ovarian intrabursal injection Ovarian bursa adenovirus injection was performed as previously described (Carletti et al., 2010; Otsuka et al., 2008). Briefly, female mice (6 weeks of age) were injected i.p. with 10 IU eCG. Twenty-four hours later, the mice were anesthetized by i.p. injection of a 1:1 mixture of ketamine (0.75 mg/animal; Ketaset, Fort Dodge, IA) and xylazine (0.75 mg/animal, Anased, Lloyd Laboratories, Shenendoah, IA). A small dorsolateral incision was made in the lumber region on each side of the midline. Through the peritoneal incision, the ovaries and associated fat pad were gently exteriorized through incisions in the dorsal abdominal wall. Intrabursal injection of 2  109 ifu adenovirus was performed by inserting a 30-gauge needle through the ovarian fat pad into the ovarian bursa with a manual micro-injector (IM-9B, NARISHIGE, Tokyo, Japan) and a micro-manipulator (M-152, NARISHIGE, Tokyo, Japan) under a stereo microscope. The ovaries were returned to the abdominal cavity and the wound was sutured. The animals were then closed and 20–24 h later they were injected i.p. with 10 IU hCG (Sigma). Animals were sacrificed 14–16 h after hCG injection and the number of ovulated COCs in the oviduct were collected to determine the ovulation rate. For experiments to observe mating and embryo implantation, female mice were bred overnight to wild-type males with known fertility after hCG injection. The following morning (day 0.5 after coitus), the female mice were checked for a vaginal plug. The mice were sacrificed on day 1.5 and the embryos were collected at the two-cell stage. In addition, embryo implantation sites were identified on day 5 by intravenous injections of 100 ll of 1% Chicago blue dye (Sigma) in saline as described previously (Chen et al., 2011). Using the above adenovirus injection method, the number of offspring born to the group with bilateral ovarian bursa injected with the Ad-laminshRNA or Ad-miR-224 adenoviruses was also determined. 2.12. Statistical analysis Experiments were performed at least three times and the values are presented as means ± SEM. Means of groups were analyzed by Student’s t-test or the ANOVA with the Student–Newman–Keuls post hoc test when appropriate. A P value

MicroRNA-224 is involved in the regulation of mouse cumulus expansion by targeting Ptx3.

MicroRNAs (miRNAs) are indicated to regulate ovarian development in a cell- or stage-specific manner. Our previous study showed that miR-224 is involv...
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