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BIOLOGY OF REPRODUCTION (2013) 88(5):128, 1–11 Published online before print 17 April 2013. DOI 10.1095/biolreprod.112.105361

Identification of Differentially Expressed miRNAs and Their Potential Targets During Fish Ovarian Development1 Ame´lie Juanchich, Aure´lie Le Cam, Je´roˆme Montfort, Yann Guiguen, and Julien Bobe2 INRA, UR 1037 Laboratoire de Physiologie et Ge´nomique des Poissons (LPGP), Rennes, France

Oogenesis is a complex process requiring the coordinated sequential expression of specific genes and ultimately leading to the release of the female gamete from the ovary. In the present study we aimed to investigate the contribution of miRNAs to the regulation of this key biological process in teleosts using a model in which growing oocytes develop simultaneously. Taking advantage of the strong sequence conservation of miRNAs among phylogenetically distant species, we designed a generic microarray displaying most known chordate miRNAs. It allowed us to provide an overview of the ovarian miRNome during oogenesis for the first time in any vertebrate species. We identified 13 differentially expressed miRNAs, and a differential expression of at least one miRNA was observed at each step of oogenesis. A surprisingly high differential expression of several miRNAs was observed at several stages of oogenesis and subsequently confirmed using quantitative PCR. By refining in silico prediction of target genes with gene expression data obtained within the same sample set, we provide strong evidence that miRNAs target major players of oogenesis, including genes involved in rate-limiting steps of steroidogenesis and those involved in gonadotropic control of oocyte development, as well as genes involved in ovulation, oocyte hydration, and acquisition of the ability of the oocyte to support further development once fertilized (i.e., oocyte developmental competence). Together, these observations stress the importance of miRNAs in the regulation and success of female gamete formation during oogenesis. metazoans, microarray, microRNA, oogenesis, rainbow trout

INTRODUCTION In the last decade, miRNAs have emerged as important posttranscriptional regulators of gene expression (GE) in a wide variety of biological processes. MicroRNAs are short, endogenous noncoding RNA (ncRNA) present in a wide variety of organisms, including animals, plants, unicellular organisms, and viruses. Mature miRNAs derive from primary transcripts forming hairpins that are cleaved in both ends by RNAse III enzyme. One strand of the resulting miRNA duplex is loaded into an miRNA-induced silencing complex (miRISC) 1 Supported by an Institut National de la Recherche Agronomique (INRA) PHASE grant to J.B. Microarray data were deposited in the Gene Expression Omnibus (GEO) database under references GSE39587, GSE39588, and GSE39590. 2 Correspondence: Julien Bobe, INRA, LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Bat 16A, 35042 Rennes Cedex, France. E-mail: [email protected]

MATERIALS AND METHODS Animals and Tissue Collection Investigations were conducted according to the guiding principles for the use and care of laboratory animals and in compliance with French and European regulations on animal welfare (under approval no. 35-31 to J.B.). Female rainbow trout (O. mykiss) were obtained from PEIMA experimental fish farm at different periods during the reproductive cycle. Fishes were deeply anesthetized in 2-phenoxyethanol (10 mg/ml water), killed by a blow on the head, and bled by gill arch section. Tissues (gill, brain, spleen, muscles, kidney, liver, and intestine) were sampled from postovulatory female trout, and testes were collected from males at different stages. All tissues were sampled from three different individuals. All samples were immediately frozen in liquid

Received: 4 October 2012. First decision: 17 December 2012. Accepted: 10 April 2013. Ó 2013 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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by Argonaute proteins. The miRISC can bind to the 3 0 untranslated region (UTR) of its target mRNA and lead to degradation or repression of translation [1–3]. The role of miRNAs has been studied in several physiological and physiopathological processes. They are involved in animal developmental processes [4, 5], such as maternal transcript clearance [6] or axial patterning [7]. The miRNAs can control both temporal and spatial GE and are believed to regulate 30% to 70% of the genes [8]. Even though miRNAs are thought to be important players in spermatogenesis in mammals [9] and sex differentiation in chicken [10], existing data on their role in animal reproduction remain scarce. Oogenesis is a complex physiological process leading to the release of the female gamete at ovulation. It has been extensively studied in vertebrates, including in teleost fishes [11–13]. The molecular mechanisms involved in this key biological process, however, remain far from being fully understood. Previous studies have indeed been limited to the characterization of the miRNome in female reproductive tissues. A total of 155 miRNAs were identified in the adult mouse ovary [14], whereas 74 miRNAs were identified in the adult bovine ovary [15]. More recently, the emergence of deep sequencing approaches has led to the identification of 516 (including 118 novel) miRNAs in the newborn mouse ovary [16], 673 mature miRNAs in the adult porcine ovary [17], and 536 miRNAs in samples derived from human female reproductive organs under normal and pathological conditions [18]. Very recently, the rainbow trout egg miRNA transcriptome has been investigated using deep RNA sequencing [19]. The presence of these post-transcriptional regulatory elements in the ovary and in the egg suggests that miRNAs could play a role in coordinating mRNA stability and translation during oogenesis. Existing studies on the role of miRNAs during vertebrate oogenesis, however, remain extremely limited, especially when considering the dynamic of intrafollicular oocyte development that requires sequential GE in the ovary [20]. The aim of the present work was to study the dynamic expression of miRNAs and mRNAs in the ovary throughout oogenesis in order to identify differentially expressed miRNAs and their possible targets. The work was carried out using a teleost fish, the rainbow trout Oncorhynchus mykiss, in which a large number of follicles develop simultaneously throughout oogenesis.

ABSTRACT

JUANCHICH ET AL. nitrogen and subsequently stored at 808C until RNA extraction. In order to describe the reproductive cycle, ovaries from different females were collected at the following specific stages: previtellogenesis (5 mo old, perinucleolar stage), mid vitellogenesis (gonadosomatic index [GSI] ;3%–4%), late vitellogenesis (GSI ;10%), prior to meiosis resumption (i.e., before germinal vesicle breakdown), and after meiosis resumption (i.e., during oocyte maturation, GSI ;16%). Ovaries from at least three different females were collected for each stage studied.

instructions. Real-time PCR was performed using a Step One Plus thermocycler (Applied Biosystems). Reverse transcription products were diluted to 1:100, and 4 ll was used for each real-time PCR reaction. Duplicates were run for each RT product. Real-time PCR was performed using a real-time PCR kit provided with an SYBR Green fluorophore (Fast SYBR Green Master Mix kit; Applied Biosystems) according to the manufacturer’s instructions, with 100 and 500 nM each primer (Supplemental Table S1; all Supplemental Data are available online at www.biolreprod.org) for miRNAs and mRNAs, respectively. After a 30-sec incubation step at 958C, amplification was performed using the following cycle: 958C, 3 sec; 608C, 30 sec; 40 times. The relative abundance of target cDNA within the sample set was calculated from a serially diluted cDNA pool using the Step One Plus software. After amplification, a fusion curve was obtained in order to ensure that a single PCR product had been generated using the following protocol: 1 sec holding followed by a 0.58C increase, from 608C to 958C. For all miRNAs studied by quantitative PCR (QPCR), primers were designed using publicly available rainbow trout miRNA sequences [19]. The QPCR signal was normalized using a geometric mean of six genes identified from the microarray analysis and found to be stable throughout oogenesis (Supplemental Fig. S1), as previously recommended for optimized normalization [21, 22]. To further validate this procedure, an 18S profile was normalized using the same procedure and found to be stable throughout oogenesis (Supplemental Fig. S1).

RNA Extraction Tissues were homogenized in Tri reagent (Sigma) at a ratio of 100 mg of tissue per milliliter of reagent, and total RNA was extracted according to the manufacturer’s instructions. Because of the high egg yolk content of vitellogenic ovaries, all RNA samples were subsequently repurified using a NucleoSpin miRNA kit (isolation of small and large RNA) in order to obtain genomic-grade RNA quality. The RNA integrity was checked using an RNA 6000 Nano chip (Agilent), and the presence of miRNAs in the samples was validated using a small RNA chip (Agilent).

MicroRNA Microarray Hybridization and Data Processing The miRNA microarray 8x60K (Gene Expression Omnibus [GEO] platform GPL15841) was designed using an e-array custom platform (Agilent) with miRBase version 16.0. The miRNA microarray analysis was performed using the miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent v2.2) according to the manufacturer’s procedure, with minor modifications. Total RNA (200 ng) from ovaries at different stages with labeling Spike-In (Agilent) was dephosphorylated for 30 min at 378C. The RNA was denatured with dimethyl sulfoxide at 1008C for 7 min. The RNA was then labeled by terminal ligation of cyanine3-pCp using T4 ligase for 2 h at 168C. Purification of labeled RNA was done using a Micro Bio-Spin column (Bio-Rad) to eliminate free cyanine3-pCp. Hybridization was performed with hybridization Spike-In at 558C, 20 rpm for 20 h. Slides were washed for 5 min with wash buffer 1 (Agilent) at room temperature and wash buffer 2 (Agilent) at 378C and immediately scanned. Agilent C Scanner (Agilent DNA Microarray Scanner; Agilent Technologies) and Scan Control software were used with the following protocol: profile ¼ AgilentHD_miRNA, channels ¼ Green, resolution ¼ 3 lm, and TIFF ¼ 20 bit. Signal intensities were then quantified using Feature Extraction software (Agilent v10.5.1.1). Features with a low signal (lower than three times the background level) were excluded. Data were processed by GeneSpring software (Agilent v.11.5.0) using GmedianSignal values. Data were subsequently normalized using the 75th percentile of each array. Corresponding data were deposited in GEO database under the reference GSE39588.

MicroRNA Targets Prediction

Statistical Analysis An ANOVA analysis with Benjamin-Hochberg correction was performed to detect differential expression between stages in the microarray experiments for both miRNA and GE. All samples were tested against each other with a corrected P value of 0.05. Differences in expression with RT-QPCR were validated by a two-sample Wilcoxon test (P value ,0.05).

GE Microarray Hybridization and Data Processing

RESULTS

The exact same RNA samples (see above miRNA microarray section) were hybridized on an 8x60K trout microarray (GEO platform GPL15840) to characterize gene expression on the same ovarian sample set. Gene expression microarray analysis was performed using a One-Color Microarray-Based Gene Expression Analysis kit (Agilent v6.5) according to the manufacturer’s procedure. Briefly, for each sample, 150 ng of total RNA was amplified and labeled using Cy3-CTP. Yield (.0.825 lg of cRNA) and specific activity (.6 pmol of Cy3 per microgram of cRNA) of Cy3-cRNA produced were checked using a Nanodrop (Thermo Scientific). A total of 600 ng of Cy3-cRNA was fragmented and hybridized on a subarray. Hybridization was carried out for 17 h at 658C in a rotating hybridization oven prior to washing and scanning with an Agilent C Scanner (Agilent DNA Microarray Scanner) using the standard parameters for a GE 8x60K oligoarray (channels ¼ Green, resolution ¼ 3 lm, TIFF ¼ 20 bit). Data were then obtained with the Agilent Feature Extraction software (v10.5.1.1) according to the appropriate GE protocol (GE1_107_ Sep09). Before analysis, saturated spots, nonuniform spots, and spots not significantly different from background (k ¼ 5) were flagged using the Agilent GeneSpring GX software (v11.5.0). Probes were considered valid when corresponding spots remained present in at least 80% of the replicates of each experimental condition after the flagging procedure. Data were subsequently scale normalized using the median value of each array. Corresponding data were deposited in GEO database under the reference GSE39587.

Design of a Generic Metazoan miRNA Microarray Our current knowledge of the miRNA repertoire remains extremely limited in teleost fishes and suffers from the lack of extensive sequencing-based characterization. This is especially true in rainbow trout, in which only a limited repertoire of miRNAs has been described [24]. Taking advantage of the strong conservation of miRNA sequences among metazoans [25–27], we used a heterologous approach and designed an Agilent generic 8x60K miRNA microarray displaying most metazoan miRNAs present in miRBase v16.0 [28]. This platform was subsequently used to characterize miRNA expression profiles throughout oogenesis in rainbow trout. The miRNA microarrays have specific characteristics that are different from GE microarrays. For each target miRNA in miRBase, 16 probes are synthetized that are divided in two to four groups and have limited variations in sequence and size (one or two nucleotide differences at the 3 0 and 5 0 extremities). This leads to a robust miRNA expression analysis. The array was primarily designed to incorporate all known teleost sequences. All existing 246 zebrafish (D. rerio) sequences present in miRBase were deposited on the array. The array was then built by selecting in other species the miRNAs that were not already present on the array. New miRNAs from fish

MicroRNA Reverse Transcription and Quantitative PCR Analysis Total RNA (500 ng) was reverse transcribed using an NCode VILO miRNA cDNA synthesis kit (Invitrogen) according to the manufacturer’s

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As a rainbow trout genome sequence is not yet publically available, we used the zebrafish genome to predict miRNA target. Targets were predicted using miRanda algorithm (default presets) [23] for the miRNA candidates in the zebrafish genome (Zv8). The analysis was refined based on the differential expression of putative targets during oogenesis. A putative target was thus qualified as potentially true if expressed in the ovary and differentially abundant (with fold change .10) between at least two stages. A total of 1634 probes were found to be differentially expressed between at least two of the studied stages, with a fold change of at least 10. Theses probes were blasted on existing rainbow trout public contigs. A BlastX analysis (identity .85%, evalue ,0.05) was subsequently performed using identified rainbow trout contig sequences in order to identify corresponding zebrafish (Danio rerio) sequences.

miRNAs REGULATE FISH OVARIAN DEVELOPMENT

species were added (Fugu rubripes, Tetraodon nigroviridis, and Oryzias latipes). The miRNAs from mice (Mus musculus), frogs (Xenopus laevis and Xenopus tropicalis), birds (Gallus gallus and Taeniopygia guttata), and platypus (Ornithorhynchus anatinus) were subsequently added. The array was further extended to nonvertebrate metazoan lineages with the incorporation of miRNAs from Ciona intestinalis, Bronchiostoma floridae, Stongylocentrotus purpuratus, Nematostella vectensis, fruit fly (Drosophila melanogaster), or Caenorhabditis elegans, as shown in Figure 1. A total of 3800 miRNAs were deposited on the array.

parallel GE analysis in the same sample set. The RNA samples used for the miRNA microarray analysis were thus also used to monitor GE profiles using a GE microarray (Fig. 5). A total of 25 073 genes were expressed in the ovary, among which 15 710 exhibited a differential expression between at least two of the studied stages. Approximately 60% of differentially expressed genes (9391 genes) were highly expressed during previtellogenesis. A large number of genes were expressed only at maturation (1271 genes), whereas 584 genes were preferentially expressed during vitellogenic stages. Finally, 4464 genes were expressed throughout follicular development and oocyte maturation.

Thirteen miRNAs Are Differentially Expressed During Oogenesis

MicroRNA Target Prediction

Tissue Expression of Candidate miRNAs Expression of all candidate miRNAs (except miR-2184) was characterized by RT-QPCR in a wide variety of organs and tissues (gill, brain, liver, intestine, muscle, spleen, kidney, ovary, and testis). As shown in Figure 4, miR-15, miR-29, miR-92, miR-101, miR-126, and miR-221 are expressed in all tissues at varying levels. In contrast, miR-181*, miR-196, miR301, and miR-338 were highly expressed in one or two tissues, even though the expression remained detectable in most tissues. Finally, both miR-202 and miR-202* were strongly expressed in the testis and ovary but could not be detected in any other tissue.

DISCUSSION The present study provides, for the first time in any vertebrate species, an overview of the miRNAs expressed throughout oogenesis with a prediction of their targets based on their expression in the ovary during the same process.

GE Profiling In order to analyze the ovarian miRNome in light of the genes expressed in the ovary during oogenesis, we carried out a 3

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Because in silico target prediction sometimes results in a high level of false-positive candidates, we combined in silico prediction and GE data to identify strong miRNA targets among expressed ovarian genes. Prediction of target genes among the differentially expressed genes. Because of our limited knowledge of 3 0 UTRs in rainbow trout, putative miRNA targets were predicted with the miRanda algorithm using corresponding zebrafish 3 0 UTRs sequences. The analysis resulted in hundreds of putative target genes for each miRNA. In order to identify major targets for each miRNA (Fig. 6), the list of putative target genes was restricted to genes that had .10-fold differential expression during oogenesis. For each studied miRNA, between 10 and 28 strong targets were identified using this strategy (Supplemental Table S2). Some of these results are highlighted below for genes known to play an important role during oogenesis. Our results indicate that miR-202, miR-202*, and miR-338 target transforming growth factor b receptor II (tgfbr2), whereas miR-101 targets aquaporin 1a (aqp1a) and matrix metallopeptidase 30. We were also able to predict that miR-301 targets steroidogenic acute regulatory protein (star), whereas miR-126 targets luteinizing hormone/choriogonadotropin receptor (lhcgr). Finally, our results indicate that miR-301, miR101, and miR-126 target a disintegrin and metalloproteinase domain 8 (adam8). Genes targeted by several miRNAs with similar expression profiles. Based on the hypothesis that several miRNAs from the same expression cluster could regulate the same set of genes, we searched for genes predicted to be targeted by several miRNAs with similar expression profiles. The analysis was further limited to candidate genes expressed in at least one of the five ovarian stages. In clusters 2 and 3, which have very similar expression profiles (Fig. 2), miR-15, miR-101, and miR-202 shared four predicted targets expressed in the ovary (Fig. 7): factor in the germline a (figla), spermine oxydase, deoxyguanosine kinase, and leucine rich repeat and sterile alpha motif containing 1. miR-15, miR-92, and miR-202 share one target: ftsJ methyltransferase domain containing 1. miR92, miR-202, and miR-202* share one target: limb region 1 homolog (mouse). For clusters 1 (miR-181*, miR-196, and miR-301; Fig. 2) and 4 (miR-29, miR-126, and miR-338), no common targets were shared by the three miRNAs. In all clusters, no target was found to be shared by more than three miRNAs within an expression cluster (Supplemental Table S3).

The expression of most known metazoan miRNAs was screened using the miRNA generic metazoan microarray. As shown in Figure 2, 483 probes were differentially expressed during rainbow trout oogenesis. The miRNAs in which at least 8 of the 16 probes exhibited a statistically significant differential expression were considered as differentially expressed. Of the 3800 miRNAs that were present on the microarray, 13 miRNAs showed a significant differential expression during oogenesis. All 13 mature miRNA sequences were found to be 100% identical with recently released rainbow trout miRNA sequences identified from metaphase II oocytes [19]. The number of differentially expressed miRNAs did not change when the threshold of eight differentially expressed probes per miRNA was reduced to six or seven probes. The expression profiles of differentially expressed miRNAs could be divided into five clusters, as shown in Figure 2. In cluster 1 (miR-181*, miR-196, and miR-301), miRNA expression was high at the previtellogenic stage, dramatically decreased at the midvitellogenic stage, and remained low afterwards. In cluster 2 (miR-101 and miR-202), expression levels were high during vitellogenesis and sharply decreased at maturation. In cluster 3 (miR-15, miR-92, and miR-202*), expression levels increased from previtellogenic to late vitellogenic stages and slightly decreased at maturation stages. In cluster 4 (miR-29, miR-126, miR-338, and miR-2184), miRNA expression increased progressively throughout oogenesis. Finally, in cluster 5 (miR-221), expression levels remained low until the end of the vitellogenic process and sharply increased during maturation. All profiles were confirmed by RT-QPCR, as shown in Figure 3, with the exception of miR-2184, for which we failed to amplify a PCR product because of the low sequence complexity of this specific miRNA.

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could be detected for all studied stages, suggesting that miRNAs play a role throughout oogenesis. This is consistent with the changes in miRNA expression reported within the oocyte during meiotic maturation in the mouse [30] and in the cow [31]. To date, however, no study has provided a global view of the ovarian miRNome during oogenesis in any vertebrates. Fold changes in miRNA levels reported here were surprisingly high between studied ovarian stages. For instance, miR-202 expression was 28 times higher in the late vitellogenic ovary than in the previtellogenic ovary. Similarly, miR-29 was expressed 35 times more in the mature ovary than in the previtellogenic ovary. In contrast, most existing studies have reported much more limited changes in miRNA expression between stages within the same tissue [32–34]. So far, none of the 13 miRNAs reported here had previously been shown to be involved in ovarian development in any animal species. For instance, miR-301 mediates proliferation and invasion in human breast cancer [35] and directly targets MEOX2, which affects the ERK/CREB pathway [36]. miR-15, which forms a cluster with miR-107, is implicated in cell division and apoptosis by targeting BCL2 [37]. miR-196 is genomically located in the HOX cluster region and directly targets genes involved in early development, such as HOX or HMGA2 [38], and is essential for axial patterning in zebrafish [7]. miR-101 is involved in cancer by modulating the cancer epigenome via a direct repression of the polycomb group protein EZH2 [39].

Generic miRNA Microarray The use of a heterologous miRNA microarray displaying most known metazoan miRNAs to analyze the expression profile of trout miRNAs during oogenesis was successful and allowed us to monitor a large variety of miRNAs despite the lack of a complete miRNA repertoire characterization in rainbow trout [24] and the absence of trout miRNA sequences in miRBase. This is consistent with the strong conservation of miRNA sequences previously reported among animal species [26, 29]. This custom design led to a generic metazoan miRNA microarray that can be used to study miRNA expression in any animal species, even when the miRNA repertoire is unknown or poorly described. It is also a valuable tool to study evolution and conservation of miRNA expression among species. MicroRNA Expression During Oogenesis Using this generic microarray, we were able to identify 13 miRNAs exhibiting a differential expression pattern in the trout ovary throughout oogenesis (i.e., from immature to mature stages). In consistency with our observations, a recent study reported that all 13 miRNAs were expressed in the rainbow trout metaphase II oocyte [19]. This does not, however, rule out expression in somatic follicular cells or extrafollicular tissues. Interestingly, differential expression of at least one miRNA 4

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FIG. 1. Generic metazoan miRNA 8x60K microarray design. MicroRNA microarray 8x60K design was done using an e-array custom platform (Agilent) with miRBase version 16.0. Starting from the zebrafish miRNAs, a large number of miRNAs were added from different metazoan species.


Downloaded from www.biolreprod.org. FIG. 2. MicroRNA expression profiling during rainbow trout oogenesis. Supervised average linkage clustering analysis of 483 miRNA probes in rainbow trout ovary during oogenesis. Each row represents a single probe (16 probes per miRNA on the microarray), and each column an ovarian RNA sample. The 14 samples were supervised according to the natural time course of oogenesis: previtellogenic (IMM), midvitellogenesis (MV), late vitellogenesis (LV), prior to germinal vesicle breakdown (PV), and during meiotic maturation (MAT). Data were median centered prior to the clustering analysis. For each miRNA probe, the expression level within the sample set is indicated using a color density scale. Yellow and blue are used for overexpression and underexpression, respectively, whereas black is used for median expression. Expression profiles of differentially expressed miRNAs during oogenesis can be divided into five clusters (C1 to C5). C1 corresponds to miR-181*, miR-196, and miR-301. C2 corresponds to miR-101 and miR-202. C3 corresponds to miR-15, miR-92, and miR-202*. C4 corresponds to miR-29, miR-126, miR-338, and miR-2184. C5 corresponds to miR-221.

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Downloaded from www.biolreprod.org. FIG. 3. MicroRNA expression profiles through oogenesis. Expression profiles of all candidate miRNAs were monitored by RT-QPCR in rainbow trout ovarian samples. The samples are ordered according to the natural time course of oogenesis: previtellogenic (IMM; n ¼ 3), midvitellogenesis (MV; n ¼ 3), late vitellogenesis (LV; n ¼ 4), prior to germinal vesicle breakdown (PV; n ¼ 3), and during meiotic maturation (MAT; n ¼ 4). C1 to C5 refer to the miRNA expression cluster number: C1, cluster 1; C2, cluster 2; C3, cluster 3; C4, cluster 4; and C5, cluster 5. Data were normalized using the geometric mean of six genes identified from the microarray analysis and found to be stable throughout oogenesis. Mean values (6SD) are displayed on the graphs. Expression was arbitrarily set to 1 at the previtellogenic stage (IMM).

comparison with ovaries. This difference between fish and mammals could, however, be related to the reproductive stages used for analysis. In our study, miR-202 expression was 28 times higher in the late vitellogenic ovary in comparison with the previtellogenic ovary. Moreover, miR202* had a dimorphic expression in chicken gonads undergoing differentiation [10]. Its expression was higher in male gonads than in female gonads. Also, miR-202* expression was altered by estrogen treatment in chicken

miR-202, a Gonad-Specific miRNA As reported here, miR-202 and miR-202* are specifically expressed in rainbow trout gonads and could not be detected in any other studied tissue. This is in full agreement with previous observations made in frog [40], Atlantic halibut [41], human, mouse, and rat [42, 43]. In rainbow trout, expression levels are similar in ovary and testes, whereas in mammals both forms of miR-202 are highly expressed in testes in 6

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Downloaded from www.biolreprod.org. FIG. 4. MicroRNA tissue distribution. Expression profiles of all candidate miRNAs were studied by RT-QPCR in rainbow trout tissues: gill (Gi), brain (Br), liver (Li), intestine (In), white muscle (Mu), spleen (Sp), head kidney (hK), testis (Te), and vitellogenic ovary (Ov). For each studied tissue, RNA samples originating from three different individuals were pooled before RT. Expression levels were measured in triplicates in each pool, and mean values (6SD) are displayed on the graphs. C1 to C5 refer to the miRNA expression cluster number: C1, cluster 1; C2, cluster 2; C3, cluster 3; C4, cluster 4; C5, cluster 5. Data were normalized to the abundance of 18S. Expression was arbitrarily set to 100 for the tissue with the highest miRNA expression. #Expression levels that are not significantly different from background signal.

gonads [44]. To date, however, no direct targets have been characterized for miR-202 or miR-202*. Together, these findings strongly suggest that gonad-specific expression of miR-202 duplex is conserved among vertebrates, including teleost fish, and that miR-202/miR-202* plays an important role in gametogenesis.

MicroRNA Target Prediction Experimental validation of miRNA targets remains a major challenge and has led to the development of in silico prediction to identify potential miRNA targets [45]. To date, many algorithms have been developed using several properties 7

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Downloaded from www.biolreprod.org. FIG. 5. Gene expression profiling during rainbow trout oogenesis. Supervised average linkage clustering analysis of 15 710 probes in rainbow trout ovary during oogenesis. Each row represents an mRNA probe, and each column an ovarian RNA sample. The 16 samples were supervised according to the natural time course of oogenesis: previtellogenic (IMM), midvitellogenesis (MV), late vitellogenesis (LV), prior to germinal vesicle breakdown (PV), and during meiotic maturation (MAT). Data were median centered prior to the clustering analysis. For each mRNA probe, the expression level within the sample set is indicated using a color density scale. Yellow and blue are used for overexpression and underexpression, respectively, whereas black is used for median expression. On the right of the figure, specific gene expression profiles are highlighted.

knowledge of the rainbow trout 3 0 UTR sequences is currently extremely limited. Furthermore, using microarray GE data from the same samples, we identified several putative genes targeted by differentially expressed miRNAs during oogenesis. The use of GE data eliminates nonexpressed false-positive targets predicted by computational analyses and significantly increases the level of confidence for remaining putative targets.

needed for the pairing between miRNA and targeted mRNA [46]. In this study, we used a miRanda algorithm that has been developed for target prediction in fruit fly [23] and then extended to several organisms, including zebrafish. This algorithm evaluates the energy of physical interaction among analyzed miRNA:mRNA duplexes. In our study, the prediction was made using zebrafish 3 0 UTR sequences because our 8

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FIG. 6. MicroRNA target prediction using gene expression data. Pipeline used to predict strong miRNA targets using gene expression data and miRanda in silico prediction.

FIG. 7. MicroRNA target prediction using miRNA expression data. Venn diagrams showing targets shared by several miRNAs within the same expression cluster in Figure 2. The analysis was restricted to targets expressed in the ovary in at least one of the five stages. The table shows targets that are shared by three miRNAs within the same expression cluster (Fig. 2).

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Among predicted targets, we identified genes that are known to be key players of oogenesis, such as star, figla, slc26a4 (also known as pendrin), and adam8, as discussed below. MicroRNAs regulating final oocyte maturation. Steroidogenic acute regulatory protein (Star) plays a key role in enhancing the transport of cholesterol into the mitochondria for steroid biosynthesis. Star is necessary for the normal ovarian steroid production needed for many steroid-mediated processes, such as follicular growth and oocyte maturation [47]. In rainbow trout, star mRNA is sharply up-regulated in the ovarian follicle at the time of oocyte maturation [48]. Our results show that star is predicted to be a target of miR-301, an miRNA down-regulated in the ovary during oogenesis. Interestingly, miR-301 is also predicted to target ctsd (cathepsin d), a gene known to exhibit an increased expression in the ovarian follicle at the time of oocyte maturation [48] similarly to star. In addition, our results show that adam8 could also be a target of miR-301. adam8 is expressed in murine granulosa cells, and mRNA expression is induced by the ovulatory surge of luteinizing hormone. To date, adam8 is the only member of the Adam family that has been shown to be hormonally regulated [49]. The expression profile of adam8 reported here (Fig. 5) is consistent with an up-regulation during final oocyte maturation and a participation in the ovulatory

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ACKNOWLEDGMENT The authors are grateful to the experimental facility staff of the Institut National de la Recherche Agronomique Laboratoire de Physiologie et Ge´nomique des Poissons (INRA LPGP) laboratory for fish rearing, and to Thaovi Nguyen for excellent technical assistance.

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process, as in mammals. Moreover, smad7, which has an expression profile similar to that of adam8 (Fig. 5), is targeted by miR-181a*, miR-92, and miR-101. Smad7 is an inhibitory Smad of the TGFb pathway that blocks the downstream signal transduction by inhibiting the activating Smads, Smad2 and Smad3 [50, 51]. Recently, Smad7 was shown to be involved in apoptosis in primary murine granulosa cells [52]. Several other factors involved in oocyte hydration and ovulation, such as aquaporin 1 and several solute carrier proteins (including slc26a4 [53]), are also putatively targeted by several miRNAs (Supplemental Fig. S1). Together, these results suggest an important role for miRNA-mediated gene regulation during final oocyte maturation. MicroRNAs regulating oocyte growth and developmental competence. We also identified other target genes known to play important roles in ovarian physiology. Our data indicate that miR-15, miR-101, and miR-202 can target figla. Figla is a germline-specific transcription factor implicated in postnatal oocyte-specific GE in mammals. It plays a key regulatory role in the expression of multiple oocyte-specific genes, including those that initiate folliculogenesis and those required for fertilization and early embryonic survival [54]. figla has been localized to the ooplasm in medaka [55]. aqp1a (aquaporin1a) is also putatively targeted by miR-101. It belongs to the aquaporin family that allows water flow through the cytoplasmic membrane. aqp1a expression is high during vitellogenesis stages, which is consistent with the increase in the size of the oocyte at these stages. More than 50% of the final size of the oocyte is directly connected to the water storage during oocyte growth [12]. Furthermore, high choriolytic enzyme or hatching enzyme 2 (he2), which is necessary for the digestion of the egg envelope [56], is putatively targeted by miR-101 as well. Its expression increased during late stages of ovarian development (Fig. 5). Together, these results suggest a role for miRNA within the oocyte to regulate genes important for oocyte growth and oocyte developmental competence (i.e., the oocyte’s ability to be fertilized and subsequently support embryonic development). In the present study, we provide an overview of the ovarian miRNome during oogenesis for the first time in any vertebrate species, and we identify 13 differentially expressed miRNAs. The occurrence of the differential expression of at least one miRNA at all steps of oogenesis, and the unexpectedly high differential expression of several miRNAs suggest an important role for miRNAs in the regulation of oogenesis. By refining in silico prediction of target genes with GE data obtained in parallel in the same samples, we provide strong evidence that miRNAs target major players of oogenesis, including genes involved in rate-limiting steps of steroidogenesis, those involved in gonadotropic control of oocyte development, as well as genes involved in ovulation, oocyte hydration, and acquisition of the ability of the oocyte to support further development once fertilized (i.e., oocyte developmental competence). Together, these observations stress the importance of miRNAs in the regulation and success of female gamete formation during oogenesis.


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