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The microARN-202 (miR-202) controls female fecundity by regulating oogenesis in medaka

ABSTRACT: In fish, female fecundity is tightly linked to the proper completion of oogenesis that takes place in the ovary. These cellular processes have been shown to involve both endocrine and intra-ovarian factors. However, the role of small non-coding miRNAs remains largely unknown. Here, we analyzed the role of miR-202, a miRNA specifically expressed in the vertebrate gonads. We first used fluorescent in situ hybridization to determine the precise cellular expression of miR-202 in the medaka ovary. We then generated a mutant fish line (using CRISPR/Cas9 technology) to determine its role in female fecundity and oogenesis. We performed quantitative image analyses to analyze cellular modifications and a genome-wide transcriptomic approach to analyze gene expression modifications. Our results show that miR-202-5p is predominantly expressed in granulosa cells. In addition, mutant females display a drastically reduced fecundity. Our cellular and molecular analyses of ovaries from mutant females indicate that miR-202 deficiency impairs the early steps of oogenesis/folliculogenesis, which ultimately reduce female fecundity. This provides the first functional evidence that miR-202 is necessary for the female reproductive success, in particular the female fecundity, and shed new light on the regulatory mechanisms that control the early steps of follicular development.

ORGANISM(S): Oryzias latipes

PROVIDER: GSE111388 | GEO | 2018/08/27

REPOSITORIES: GEO

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Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

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The microARN-202 (miR-202) controls female fecundity by regulating oogenesis in medaka

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