FMR1 expression in human granulosa cells and variable ovarian response: control by epigenetic mechanisms

Abstract

In humans, FMR1 (Fragile X mental retardation 1) is strongly expressed in granulosa cells (GCs) of the female germline and apparently controls efficiency of folliculogenesis. Major control mechanism(s) of the gene transcription rate seem to be based on the rate of CpG-methylation along the CpG island promoter. Conducting CpG-methylation-specific bisulfite-treated PCR assays and subsequent sequence analyses of both gene alleles, revealed three variably methylated CpG domains (FMR1-VMR (variably methylated region) 1, -2, -3) and one completely unmethylated CpG-region (FMR1-UMR) in this extended FMR1-promoter-region. FMR1-UMR in the core promoter was exclusively present only in female GCs, suggesting expression from both gene alleles, i.e., escaping the female specific X-inactivation mechanism for the second gene allele. Screening for putative target sites of transcription factors binding with CpG methylation dependence, we identified a target site for the transcriptional activator E2F1 in FMR1-VMR3. Using specific electrophoretic mobility shift assays, we found E2F1 binding efficiency to be dependent on CpG-site methylation in its target sequence. Comparative analysis of these CpGs revealed that CpG 94-methylation in primary GCs of women with normal and reduced efficiency of folliculogenesis statistically significant differences. We therefore conclude that E2F1 binding to FMR1-VMR3 in human GCs is part of an epigenetic mechanism regulating the efficiency of human folliculogenesis. Our data indicate that epigenetic mechanisms may control GC FMR1-expression rates.

FMR1 and AKT/mTOR Signaling in Human Granulosa Cells: Functional Interaction and Impact on Ovarian Response

“…Additionally, variable FMR1 genotypes with CGG repeats below or above 26-34 impact ovarian reserve [18][19][20]. Recently, we discovered that the FMR1 transcription rate in human GCs is dependent on epigenetic factors, namely, CpG methylation patterns in three distinct genomic regions, designated as VMR1-3 (variably methylated regions) [21].…”

Activation of AKT/mammalian target of rapamycin signaling in the peripheral blood of women with premature ovarian insufficiency and its correlation with FMR1 expression

“…The expression of FMR1 /FMRP is controlled through a negative feedback loop [ 14 ] and FMRP, as part of the RISC, interacts with other proteins from the Argonaute family as well as with coding and noncoding RNAs to regulate the translation of various proteins [ 21 , 22 ]. In the female germline, FMRP is a major regulator of folliculogenesis, and changes in gene expression, genotype, and epigenetics (variable methylation status) are associated with disordered folliculogenesis from diminished ovarian reserve until POI [ 13 , 18 , 19 , 30 ].…”

Expression of FMRpolyG in Peripheral Blood Mononuclear Cells of Women with Fragile X Mental Retardation 1 Gene Premutation

“…PBMCs and human granulosa cells (hGCs) were IF-stained using established methods [ 33 ]. PBMCs were freshly collected and hGCs were collected from follicular fluid following oocyte retrieval, as previously described [ 34 ]. Cells were suspended in PBS, layered on glass coverslips at the bottom of the six-well culture plate and allowed to stand for 30 min to enable cells to adhere to the coverslip through gravity sedimentation.…”