Ectopic expression of CGG-repeats alters ovarian response to gonadotropins and leads to infertility in a murine FMR1 premutation model

Abstract

Women heterozygous for an expansion of CGG repeats in the 5’UTR of FMR1 risk developing Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and/or Tremor and Ataxia Syndrome (FXTAS). We show that expanded CGGs, independent of FMR1, are sufficient to drive ovarian insufficiency and that expression of CGG-containing mRNAs alone or in conjunction with a polyglycine-containing peptide translated from these RNAs contribute to dysfunction. Heterozygous females from two mouse lines expressing either CGG RNA-only (RNA-only) or CGG RNA and the polyglycine product FMRpolyG (FMRpolyG+RNA) were used to assess ovarian function in aging animals. The expression of FMRpolyG+RNA led to early cessation of breeding, ovulation, and transcriptomic changes affecting cholesterol and steroid hormone biosynthesis. Females expressing CGG RNA-only did not exhibit decreased progeny during natural breeding, but their ovarian transcriptomes were enriched for alterations in cholesterol and lipid biosynthesis. The enrichment of CGG RNA-only ovaries for differentially expressed genes related to cholesterol processing provided a link to the ovarian cysts observed in both CGG-expressing lines. Early changes in transcriptome profiles led us to measure ovarian function in prepubertal females that revealed deficiencies in ovulatory responses to gonadotropins. These include impairments in cumulus expansion and resumption of oocyte meiosis, as well as reduced ovulated oocyte number. Cumulatively, we demonstrated the sufficiency of ectopically expressed CGG repeats to lead to ovarian insufficiency and that co-expression of CGG-RNA and FMRpolyG lead to premature cessation of breeding. However, the expression of CGG RNA-alone was sufficient to lead to ovarian dysfunction by impairing responses to hormonal stimulation.

Evidence for an FMR1 mRNA Gain-Of-Function Toxicity Mechanism in the Pathogenesis of Fragile-X Associated Premature Ovarian Insufficiency

“…In murine models of FXTAS, the expression of FMRpolyG was pathogenic, with these mice exhibiting inclusion formation, motor phenotypes and reduced lifespan, while the sole expression of CGG-repeat RNA did not induce any of these features 33 ; however, no examination of ovarian tissue was carried out. In contrast, Shelly et al recently studied the fertility phenotype of two premutation mouse models, but in these the expression of coding Fmr1 was not altered and therefore there was no interference due to decreased FMRP expression 39 . Only expression of both CGG RNA and FMRpolyG led to a progressive loss of fertility with age 39 although expression of CGG-repeat RNA alone was su cient to impair key ovulatory processes in response to exogenous hormones.…”

“…In contrast, Shelly et al recently studied the fertility phenotype of two premutation mouse models, but in these the expression of coding Fmr1 was not altered and therefore there was no interference due to decreased FMRP expression 39 . Only expression of both CGG RNA and FMRpolyG led to a progressive loss of fertility with age 39 although expression of CGG-repeat RNA alone was su cient to impair key ovulatory processes in response to exogenous hormones. CGG RNA aggregates have not been reported in women with FXPOI, however FMRpolyG inclusions have been observed in the ovarian stroma of one woman with FXPOI 37 and the mural granulosa cells of six premutation carriers 38 .…”

“…While investigations for CGG RNA aggregates in human FXPOI ovarian tissue have not been undertaken, it is possible that the cellular stress induced by CGG-repeat RNA and FMRpolyG expression causes atresia of ovarian follicles before RNA aggregates are visible by FISH or immunostaining. Derivation of ovarian somatic cells from FXPOI patient iPSCs may be informative as a model, as these cells will carry the human genetic landscape as well as the disease 39 .…”

“…Intranuclear inclusions of FMRpolyG have been detected in the brains of FXTAS patients 31,33−35 , as well as in non-CNS tissues 36 , including the ovarian stroma of a woman with FXPOI 37 , and in mural granulosa cells from six FMR1 premutation carriers 38 . Furthermore, FMRpolyG protein has been detected in the ovarian stroma of mice expressing expanded CGG repeats 37,39 . Collectively these data suggest that RAN translation may be involved in FXPOI.…”

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

“…The colocalization of ubiquitin and FMRpolyG has recently been demonstrated in ovarian stromal and mural granulosa cells from FMR1 PM carriers [ 18 , 20 ]. Shelly et al [ 21 ] demonstrated impaired response to gonadotropin stimulation in two mouse models; one produced FMRpolyG and CGG-RNA, whereas the other only expressed CGG-RNA (99 CGG repeats). However, only the expression of both FMRpolyG and CGG-RNA resulted in a progressive loss of fertility with age.…”