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

Abstract

We aimed to determine whether a functional link with impact on female ovarian reserve exists between FMR1 expression and expression ratios of AKT/mTOR signaling genes in human granulosa cells in vivo, as suggested from prior in vitro data. Three hundred and nine women, who were classified as normal (NOR; n = 225) and poor (POR; n = 84) responders based on their ovarian reserve, were recruited during stimulation for assisted reproductive techniques. Expressions of FMR1 and of key genes of the AKT/mTOR and AKT/FOXO1/3 signaling pathways were comparatively analyzed in their granulosa cells. FMR1 expression in granulosa cells of NOR and POR correlated significantly with AKT1, TSC2, mTOR, and S6K expression. No correlation was found between FMR1 and FOXO1 in all, and FOXO3 expression in POR, patients. AKT1 expression was significantly higher and FOXO1 expression lower in POR samples, whereas AKT1 expression was lower and FOXO1 expression was higher in NOR samples. In human native granulosa cells, FMR1 expression significantly correlated with the expression of key genes of the AKT/mTOR signaling pathway, but not with the FOXO1/3 signaling pathway. Our data point to a functional link between FMR1 expression and expression of the AKT/mTOR signaling pathway genes controlling human follicular maturation.

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

“…In addition, we observed that mTOR and S6K was correlated with FMR1 expression in both groups, but significantly correlated in patients with POI. These results further supported the mechanism observed in experimental animals [ 34 ] and were consistent with our data from GCs of women with poor ovarian response, in whom mTOR signaling was similarly linked to FMR1 and particularly to S6K [ 25 ]. As expected from the findings of previous studies, the expression of FMR1 was significantly more abundant in patients with POI and PM than with POI without PM [ 9 ].…”

“…We believe that FMR1/ FMRP regulates proper follicular maturation not only via its premutated CGG repeat length, but also through multiple other ways. In this context, we presumed a functional linkage between FMR1 and the AKT/mTOR signaling pathway based on the findings of previous studies [ 10 , 17 , 23 – 25 ]. In the present study, we demonstrated upregulated AKT/mTOR signaling in patients with POI that might reflect a pathognomonic reason for POI development or represent a compensatory mechanism for POI.…”

“…TaqMan predesigned gene expression assays for FMR1 (Hs00924544_m1), AKT1 (Hs00178289_m1), mTOR (Hs00234508_m1), S6K (Hs00177357_m1), TSC2 (Hs01020387_m1), two housekeeping genes HPRT and TBP (Hs99999909_m1; Hs00427620_m1, respectively), and TaqMan universal PCR master mix were obtained from Thermo Fisher Scientific Inc. and performed as per the manufacturer’s protocol and as described previously [ 25 ]. All samples were analyzed in triplicates under standard qPCR conditions on a Fast Forward 7500 real-time PCR system (Thermo Fisher Scientific Inc.).…”

“…We previously identified a putative functional linkage of FMR1 /FMRP expression with mTOR/AKT signaling under maintenance of the FMR1 /FMRP negative feedback loop, which can be altered by specific inhibition of mTOR [ 24 ]. In addition, we recently identified significant correlations between the expression of FMR1 and that of AKT1 , TSC2 , mTOR , and S6K (encoding ribosomal protein S6 kinase) in fresh GCs from women undergoing controlled ovarian stimulation for in vitro fertilization/intracytoplasmic sperm injection with either a normal or a poor response [ 25 ]. However, these relationships have not yet been evaluated in the peripheral blood of patients with POI and might differ according to tissue-specific expression and disease-specific alterations.…”

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

“…Briefly, DNA samples were obtained from 10 mL blood samples collected in ethylenediaminetetraacetic acid (EDTA), as described previously [ 29 ]. The CGG repeat length of the 5′UTR of FMR1 exon 1 was analyzed using polymerase chain reaction (PCR) analysis.…”