Abstract
The success of female reproduction relies on high quality oocytes, which is determined by well-organized cooperation between granulosa cells (GCs) and oocytes during folliculogenesis. GC growth plays a crucial role in maintaining follicle development. Herein, miR-135a was identified as a differentially expressed microRNA in pre-ovulatory ovarian follicles between Large White and Chinese Taihu sows detected by Solexa deep sequencing. We found that miR-135a could significantly facilitate the accumulation of cells arrested at the G1/S phase boundary and increase apoptosis. Mechanically, miR-135a suppressed transforming growth factor, beta receptor I (Tgfbr1) and cyclin D2 (Ccnd2) expression by targeting their 3′UTR in GCs. Furthermore, subcellular localization analysis and a chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR) assay demonstrated that the TGFBR1-SMAD3 pathway could enhance Ccnd2 promoter activity and thus upregulate Ccnd2 expression. Finally, estrogen receptor 2 (ESR2) functioned as a transcription factor by directly binding to the miR-135a promoter region and decreasing the transcriptional activity of miR-135a. Taken together, our study reveals a pro-survival mechanism of ESR2/miR-135a/Tgfbr1/Ccnd2 axis for GC growth, and also provides a novel target for the improvement of female fertility.
Highlights
Folliculogenesis, from primordial follicle activation to oocyte release, is a complex and dynamic process that relies on synchronization between the oocyte maturation and the neighboring granulosa cell growth
Ki67 staining and Cell Counting Kit-8 (CCK-8) assay demonstrated that miR-135a overexpression in murine granulosa cells (mGCs) resulted in a reduction of Ki67-positive cells and a decline of cell vitality, which was in accordance with the observation of high proliferative activity and cell vitality in miR-135a inhibited mGCs (Figure 1A–C)
Fluorescenceactivated cell sorting (FACS) analysis revealed that miR-135a overexpression obviously increased the cell population of the G1 phase, accompanied by an elevated rate of G1/S and a decreased proliferation index (PI, Propidium Iodide (PI) = (G2 + S)/G1) in mGCs (Figure 1D)
Summary
Folliculogenesis, from primordial follicle activation to oocyte release, is a complex and dynamic process that relies on synchronization between the oocyte maturation and the neighboring granulosa cell growth. During this process, ovarian granulosa cells (GCs) provide necessary nutrients and steroids to the oocytes, and play vital roles in ovarian follicle development [1]. In the awakening stage of mammalian oocytes, the flattened GCs from primordial follicles first proliferate and differentiate into cuboidal granulosa cells. This is followed by the dramatic growth of oocytes [2]. GC growth is necessary for follicular maturation and ovulation, whereas GC apoptosis would lead to follicular atresia and degradation [6,7,8]
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