Many members of the TGF-beta superfamily are indicated to play important roles in ovarian follicular development, such as affecting granulosa cell function and oocyte maturation. Abnormalities associated with TGF-beta1 signaling transduction could result in female infertility. MicroRNAs (miRNAs), as small noncoding RNAs, were recently found to regulate gene expression at posttranscriptional levels. However, little is known about the role of miRNAs in TGF-beta-mediated granulosa cell proliferation and granulosa cell function. In this study, the miRNA expression profiling was identified from TGF-beta1-treated mouse preantral granulosa cells (GCs), and three miRNAs were found to be significantly up-regulated and 13 miRNAs were down-regulated. Among up-regulated miRNAs, miR-224 was the second most significantly elevated miRNA. This up-regulation was attenuated by treatment of GCs with SB431542 (an inhibitor of TGFbeta superfamily type I receptors, thus blocking phosphorylation of the downstream effectors Smad2/3), indicating that miR-224 expression was regulated by TGF-beta1/Smads pathway. The ectopic expression of miR-224 can enhance TGF-beta1-induced GC proliferation through targeting Smad4. Inhibition of endogenous miR-224 partially suppressed GC proliferation induced by TGF-beta1. In addition, both miR-224 and TGF-beta1 can promote estradiol release from GC, at least in part, through increasing CYP19A1 mRNA levels. This is the first demonstration that miRNAs can control reproductive functions resulting in promoting TGF-beta1-induced GC proliferation and ovarian estrogen release. Such miRNA-mediated effects could be potentially used for regulation of reproductive processes or for treatment of reproductive disorders.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

Our previous studies have shown that microRNA-383 (miR-383) is one of the most down-regulated miRNA in TGF-β1-treated mouse ovarian granulosa cells (GC). However, the roles and mechanisms of miR-383 in GC function during follicular development remain unknown. In this study, we found that miR-383 was mainly expressed in GC and oocytes of mouse ovarian follicles. Overexpression of miR-383 enhanced estradiol release from GC through targeting RNA binding motif, single stranded interacting protein 1 (RBMS1). miR-383 inhibited RBMS1 by affecting its mRNA stability, which subsequently suppressed the level of c-Myc (a downstream target of RBMS1). Forced expression of RBMS1 or c-Myc attenuated miR-383-mediated steroidogenesis-promoting effects. Knockdown of the transcription factor steroidogenic factor-1 (SF-1) significantly suppressed the expression of Sarcoglycan zeta (SGCZ) (miR-383 host gene), primary and mature miR-383 in GC, indicating that miR-383 was transcriptionally regulated by SF-1. Luciferase and chromatin immunoprecipitation assays revealed that SF-1 specifically bound to the promoter region of SGCZ and directly transactivated miR-383 in parallel with SGCZ. In addition, SF-1 was involved in regulation of miR-383- and RBMS1/c-Myc-mediated estradiol release from GC. These results suggest that miR-383 functions to promote steroidogenesis by targeting RBMS1, at least in part, through inactivation of c-Myc. SF-1 acts as a positive regulator of miR-383 processing and function in GC. Understanding of regulation of miRNA biogenesis and function in estrogen production will potentiate the usefulness of miRNA in the control of reproduction and treatment of some steroid-related disorders.

Background: MicroRNAs are small noncoding RNAs associated with ovarian follicle development and female fertility. Results: miR-320 inhibited estradiol synthesis and proliferation of granulosa cells (GCs) through targeting E2F1 and SF-1 in vivo and in vitro. Conclusion: E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. Significance: These will potentiate the usefulness of miRNA in the treatment of some steroid-related disorders.