title: 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: OBJECTIVE
  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?
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  Our results suggest that a dysfunctional hypothalamic-pituitary-gonadal-axis may contribute to FXPOI in FMR1 PM carriers.
  
  
  BACKGROUND
  This study was supported by grants from NFXF, ZonMW, the Netherlands Brain Foundation and NIH. The authors have no conflict of interest to declare.
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
authors.forename: R A M
authors.initials: RA
authors.lastname: Buijsen
authors.affiliation: Department of Internal Medicine, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Visser
authors.affiliation: Department of Internal Medicine, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Kramer
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
authors.forename: E A W F M
authors.initials: EA
authors.lastname: Severijnen
authors.affiliation: Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Gearing
authors.affiliation: Department of Neurobiology and Genetics, IGBMC, INSERM U964, CNRS UMR7104, University of Strasbourg, Illkirch, France.
authors.forename: N
authors.initials: N
authors.lastname: Charlet-Berguerand
authors.affiliation: Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Sherman
authors.affiliation: Department of Neurological Surgery, UC Davis, Davis 95618, CA, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Berman
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Willemsen
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, 3015 CE Rotterdam, The Netherlands r.hukema@erasmusmc.nl.
authors.forename: R K
authors.initials: RK
authors.lastname: Hukema
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001259:Ataxia / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics*; D006801:Humans; D045586:Intranuclear Inclusion Bodies / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009154:Mutation; D010455:Peptides; D016649:Primary Ovarian Insufficiency / Q000235:genetics*; D014202:Tremor / Q000235:genetics*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D010455:Peptides; C497684:Fmr1 protein, mouse; C011080:polyglycine
keywords: CGG-repeat; FMR1 premutation; FMRpolyG; FXPOI; FXTAS; HPG-axis; RAN translation; inclusions; ovarian failure; trinucleotide repeat expansion