Presence of inclusions positive for polyglycine containing protein, FMRpolyG, indicates that repeat-associated non-AUG translation plays a role in fragile X-associated primary ovarian insufficiency

Abstract

Study question: Does repeat-associated non-AUG (RAN) translation play a role in fragile X-associated primary ovarian insufficiency (FXPOI), leading to the presence of polyglycine containing protein (FMRpolyG)-positive inclusions in ovarian tissue?

Summary answer: Ovaries of a woman with FXPOI and of an Fmr1 premutation (PM) mouse model (exCGG-KI) contain intranuclear inclusions that stain positive for both FMRpolyG and ubiquitin.

What is known already: Women who carry the FMR1 PM are at 20-fold increased risk to develop primary ovarian insufficiency (FXPOI). A toxic RNA gain-of-function has been suggested as the underlying mechanism since the PM results in increased levels of mRNA containing an expanded repeat, but reduced protein levels of fragile X mental retardation protein (FMRP). Recently, RAN translation has been shown to occur from FMR1 mRNA that contains PM repeat expansions, leading to FMRpolyG inclusions in brain and non-CNS tissues of fragile X-associated tremor/ataxia syndrome (FXTAS) patients.

Study design, size, duration: Ovaries of a woman with FXPOI and women without PM (controls), and ovaries from wild-type and exCGG-KI mice were analyzed by immunohistochemistry for the presence of inclusions that stained for ubiquitin and FMRpolyG . The ovaries from wild-type and exCGG-KI mice were further characterized for the number of follicles, Fmr1 mRNA levels and FMRP protein expression. The presence of inclusions was also analyzed in pituitaries of a man with FXTAS and the exCGG-KI mice.

Participants/materials, setting, methods: Human ovaries from a woman with FXPOI and two control subjects and pituitaries from a man with FXTAS and a control subjects were fixed in 4% formalin. Ovaries and pituitaries of wild-type and exCGG mice were fixed in Bouin's fluid or 4% paraformaldehyde. Immunohistochemistry was performed on the human and mouse samples using FMRpolyG, ubiquitin and Fmrp antibodies. Fmr1 mRNA and protein expression were determined in mouse ovaries by quantitative RT-PCR and Western blot analysis. Follicle numbers in mouse ovaries were determined in serial sections by microscopy.

Main results and the role of chance: FMRpolyG-positive inclusions were present in ovarian stromal cells of a woman with FXPOI but not in the ovaries of control subjects. The FMRpolyG-positive inclusions colocalized with ubiquitin-positive inclusions. Similar inclusions were also observed in the pituitary of a man with FXTAS but not in control subjects. Similarly, ovaries of 40-week-old exCGG-KI mice, but not wild-type mice, contained numerous inclusions in the stromal cells that stained for both FMRpolyG- and ubiquitin, while the ovaries of 20-week-old exCGG-KI contained fewer inclusions. At 40 weeks ovarian Fmr1 mRNA expression was increased by 5-fold in exCGG-KI mice compared with wild-type mice, while Fmrp expression was reduced by 2-fold. With respect to ovarian function in exCGG-KI mice: (i) although the number of healthy growing follicles did not differ between wild-type and exCGG-KI mice, the number of atretic large antral follicles was increased by nearly 9-fold in 40-week old exCGG-KI mice (P < 0.001); (ii) at 40 weeks of age only 50% of exCGG-KI mice had recent ovulations compared with 89% in wild-type mice (P = 0.07) and (iii) those exCGG-KI mice with recent ovulations tended to have a reduced number of fresh corpora lutea (4.8 ± 1.74 versus 8.50 ± 0.98, exCGG-KI versus wild-type mice, respectively, P = 0.07).

Limitations, reasons for caution: Although FMRpolyG-positive inclusions were detected in ovaries of both a woman with FXPOI and a mouse model of the FMR1 PM, we only analyzed one ovary from a FXPOI subject. Caution is needed to extrapolate these results to all women with the FMR1 PM. Furthermore, the functional consequence of FMRpolyG-positive inclusions in the ovaries for reproduction remains to be determined.

Wider implications of the findings: Our results suggest that a dysfunctional hypothalamic-pituitary-gonadal-axis may contribute to FXPOI in FMR1 PM carriers.

Study funding/competing interests: This study was supported by grants from NFXF, ZonMW, the Netherlands Brain Foundation and NIH. The authors have no conflict of interest to declare.

Keywords: CGG-repeat; FMR1 premutation; FMRpolyG; FXPOI; FXTAS; HPG-axis; RAN translation; inclusions; ovarian failure; trinucleotide repeat expansion.

Figure 1

Intranuclear inclusions in ovarian stromal cells of a fragile X-associated primary ovarian insufficiency (FXPOI) patient. (A) Ubiquitin- and FMRpolyG-positive inclusions (indicated with arrows) are detected in stromal cells of a FXPOI patient, while two control samples were negative. (B) Co-localization of ubiquitin (green) and FMRpolyG (red) in stromal cells of an ovary from a FXPOI patient, as shown by double immunofluorescence. Nuclei are shown in blue (Hoechst stain); the square indicates of blow up of an inclusion.

Figure 2

Intranuclear inclusions in ovarian stromal cells of 40-week-old Fmr1 premutation mouse model (exCGG-KI) mice. Immunohistochemistry revealed ubiquitin- and FMRpolyG-positive inclusions (indicated with arrows) in ovarian stromal cells of exCGG-mice (right panels) but not in wild-type mice (left panels). The square indicates of blow up of an inclusion.

Figure 3

Ovarian Fmr1 mRNA and fragile X mental retardation protein (Fmrp) expression. (A) Fmr1 mRNA expression in whole ovaries of 40-week-old wild-type (n = 5) and Fmr1 premutation mouse model (exCGG-KI) mice (n = 10). Results are expressed as percentage expression relative to wild-type mice. (B) Western blot analysis of Fmrp expression in 40-week-old whole ovaries of wild-type and exCGG-KI mice. Expression was quantified and expressed relative to wild-type mice (n = 5 per group). (C) Immunohistochemical analysis of Fmrp expression in ovaries of 20-week-old wild-type (WT) and exCGG-KI. Fmrp is expressed in granulosa and theca cells of all growing follicles at different stages. In addition, expression was observed in stromal cells. Ovaries of Fmr1-KO (KO) mice were included as a negative control and did not show specific staining for Fmrp (lower left panel), although some nonspecific staining of the zona pellucida was occasionally seen (lower right panel).

Figure 4

Follicle numbers in Fmr1 premutation mouse model (exCGG-KI) mice. The number of follicles in different size classes was determined in both ovaries for wild-type (open bars) and exCGG-KI (black bars) mice. (A) Number of primordial, healthy and atretic follicles at 20 weeks of age (upper panel) and 40 weeks of age (lower panel). Data represent mean ± SEM (n = 6–9 mice). **P < 0.01; ***P < 0.001. (B) The mRNA expression of FSH and LH receptors (FSHR, LHR) was determined in whole ovaries of 40-week-old wild-type (n = 5) and exCGG-KI (n = 10) mice. Results are expressed as percentage expression relative to wild-type mice. Data represent mean ± SEM.

Figure 5

Intranuclear inclusions in Fmr1 premutation mouse model (exCGG-KI) mouse pituitary. Ubiquitin- and FMRpolyG-positive intranuclear inclusions (indicated with arrows) in exCGG-KI mouse pituitary (right panel). Wild-type mouse pituitary was negative for these two markers (left panel). The square indicates of blow up of an inclusion.

Figure 6

Intranuclear inclusions in human fragile X-associated tremor/ataxia syndrome (FXTAS) pituitary. (A) Ubiquitin- and FMRpolyG-positive intranuclear inclusions (indicated with arrows) in human FXTAS pituitary (right panel). The control pituitary stains negative for the two markers (left panel). (B) Co-localization of ubiquitin (green) and FMRpolyG (red) in stromal cells of an ovary from a FXPOI patient, as shown by double immunofluorescence. Nuclei are shown in blue (Hoechst stain); the square indicates of blow up of an inclusion.