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

Hum Reprod. 2016 Jan;31(1):158-68. doi: 10.1093/humrep/dev280. Epub 2015 Nov 3.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Animals
  • Ataxia / genetics*
  • Disease Models, Animal
  • Female
  • Fragile X Mental Retardation Protein / genetics*
  • Fragile X Syndrome / genetics*
  • Humans
  • Intranuclear Inclusion Bodies / genetics*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Middle Aged
  • Mutation
  • Peptides
  • Primary Ovarian Insufficiency / genetics*
  • Tremor / genetics*
  • Trinucleotide Repeat Expansion / genetics*

Substances

  • Fmr1 protein, mouse
  • Peptides
  • Fragile X Mental Retardation Protein
  • polyglycine

Supplementary concepts

  • Fragile X Tremor Ataxia Syndrome