title: Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency.
abstract: We hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse model of fragile X premutation (FXPM).
  
  
  
  Mice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corpora lutea with a gene dosage effect manifesting in decreased litter size(s). Furthermore, granulosa cells (GC) and oocytes of FXPM mice have decreased mitochondrial content, structurally abnormal mitochondria, and reduced expression of critical mitochondrial genes. Because this mouse allele produces the mutant Fragile X mental retardation 1 (Fmr1) transcript and reduced levels of wild-type (WT) Fmr1 protein (FMRP), but does not produce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary.
  
  
  
  Mitochondrial dysfunction has been detected in somatic cells of human and mouse FX PM carriers and mitochondria are essential for oogenesis and ovarian follicle development, FX-associated primary ovarian insufficiency (FXPOI) is seen in women with FXPM alleles. These alleles have 55-200 CGG repeats in the 5' UTR of an X-linked gene known as FMR1. The molecular basis of the pathology seen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNA with long CGG-repeat tracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic.
  
  
  
  Analysis of ovarian function in a knock-in FXPM mouse model carrying 130 CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corpora lutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantified in oocytes and GC using quantitative PCR, and cumulus granulosa mitochondrial content was measured by flow cytometric analysis after staining of cells with Mitotracker dye. Transmission electron micrographs were prepared of GC within small growing follicles and mitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recycling was performed.
  
  
  
  A defect was found in follicle survival at the large antral stage in PM/+ and PM/PM mice. Litter size was significantly decreased in PM/PM mice, and corpora lutea were significantly reduced in mice of both mutant genotypes. Mitochondrial DNA copy number was significantly decreased in GC and metaphase II eggs in mutants. Flow cytometric analysis revealed that PM/+ and PM/PM animals lack the cumulus GC that harbor the greatest mitochondrial content as found in wild-type animals. Electron microscopic evaluation of GC of small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrant mitochondrial gene expression was detected. Mitofusin 2 (Mfn2) and Optic atrophy 1 (Opa1), genes involved in mitochondrial fusion and structure, respectively, were significantly decreased in whole ovaries of both mutant genotypes. Mitochondrial fission factor 1 (Mff1) was significantly decreased in PM/+ and PM/PM GC and eggs compared with wild-type controls.
  
  
  
  Data from the mouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition.
  
  
  
  Our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeats is intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlying mechanism.
  
  
  
  Not applicable.
  
  
  
  Studies were supported by NIH R21 071873 (J.J./G.H), The Albert McKern Fund for Perinatal Research (J.J.), NIH Intramural Funds (K.U.), and a TUBITAK Research Fellowship Award (B.U.). No conflict(s) of interest or competing interest(s) are noted.
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Reproductive Endocrinology and Infertility, Yale School of Medicine, New Haven, CT, USA Vita-Salute San Raffaele University/IRCCS San Raffaele Hospital, Milan, Italy.
authors.forename: Carola
authors.initials: C
authors.lastname: Conca Dioguardi
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Reproductive Endocrinology and Infertility, Yale School of Medicine, New Haven, CT, USA.
authors.forename: Bahar
authors.initials: B
authors.lastname: Uslu
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Reproductive Endocrinology and Infertility, Yale School of Medicine, New Haven, CT, USA.
authors.forename: Monique
authors.initials: M
authors.lastname: Haynes
authors.affiliation: Department of Histology & Embryology, Izmir Katip Celebi University School of Medicine, Izmir, Turkey.
authors.forename: Meltem
authors.initials: M
authors.lastname: Kurus
authors.affiliation: Department of Histology & Embryology, Inonu University School of Medicine, Malatya, Turkey.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Gul
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Reproductive Endocrinology and Infertility, Yale School of Medicine, New Haven, CT, USA.
authors.forename: De-Qiang
authors.initials: DQ
authors.lastname: Miao
authors.affiliation: Department of Obstetrics & Gynecology, IVF Unit, Vita-Salute San Raffaele University/IRCCS San Raffaele Hospital, Milan, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: De Santis
authors.affiliation: Laboratory of Clinical Molecular Biology and Cytogenetics, Vita-Salute San Raffaele University/IRCCS San Raffaele Hospital, Milan, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Gynecologic Oncology, Yale School of Medicine, New Haven, CT, USA.
authors.forename: Stefania
authors.initials: S
authors.lastname: Bellone
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Gynecologic Oncology, Yale School of Medicine, New Haven, CT, USA.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Santin
authors.affiliation: Department of Molecular Biosciences, M.I.N.D. Institute, University of California-Davis, Davis, CA, USA.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Giulivi
authors.affiliation: Department of Biology, Morgan State University, Baltimore, MD, USA.
authors.forename: Gloria
authors.initials: G
authors.lastname: Hoffman
authors.affiliation: Laboratory of Cellular and Molecular Biology, NIH/NIDDK, Bethesda, MD, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Usdin
authors.affiliation: Department of Obstetrics, Gynecology, & Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA Division of Reproductive Endocrinology and Infertility, Yale School of Medicine, New Haven, CT, USA josh.johnson@yale.edu.
authors.forename: Joshua
authors.initials: J
authors.lastname: Johnson
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006107:Granulosa Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D009865:Oocytes / Q000378:metabolism* / Q000473:pathology; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism; D010053:Ovary / Q000378:metabolism / Q000473:pathology; D016649:Primary Ovarian Insufficiency / Q000378:metabolism* / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1
keywords: atresia; fertility; follicle; fragile X premutation; fragile X primary ovarian insufficiency; mitochondria; oocyte; ovary