Granulosa cellandoocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency

Study hypothesis:We hypothesized that the mitochondria of granulosa cells (GC) and/or oocytes might be abnormal in a mouse modelof fragile X premutation (FXPM).study finding: Mice heterozygous and homozygous for the FXPM have increased death (atresia) of large ovarian follicles, fewer corporalutea 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 notproduce a Repeat Associated Non-ATG Translation (RAN)-translation product, our data lend support to the idea that Fmr1 mRNA withlarge numbers of CGG-repeats is intrinsically deleterious in the ovary.What is known already: Mitochondrial dysfunction has been detected in somatic cells of human and mouse FXPM carriers and mitochondriaare 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 pathologyseen in this disorder is unclear but is thought to involve either some deleterious consequence of overexpression of RNAwith long CGG-repeattracts or of the generation of a repeat-associated non-AUG translation (RAN translation) product that is toxic.Study design, samples/materials, methods: Analysis of ovarian function in a knock-in FXPM mouse model carrying 130CGG repeats was performed as follows on WT, PM/+, and PM/PM genotypes. Histomorphometric assessment of follicle and corporalutea numbers in ovaries from 8-month-old mice was executed, along with litter size analysis. Mitochondrial DNA copy number was quantifiedin 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 andmitochondrial architecture was compared. Quantitative RT-PCR analysis of key genes involved in mitochondrial structure and recyclingwas performed.main results and the role of chance: Adefect was found in follicle survival at the large antral stage in PM/+ andPM/PMmice.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 GCof small growing follicles revealed mitochondrial structural abnormalities, including disorganized and vacuolar cristae. Finally, aberrantmitochondrial 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.Limitations, reasons for caution: Datafromthemouse model used for these studies should be viewed with some caution when considering parallels to the human FXPOI condition.Wider implications of the findings: Our data lend support to the idea that Fmr1 mRNA with large numbers of CGG-repeatsis intrinsically deleterious in the ovary. FXPM disease states, including FXPOI, may share mitochondrial dysfunction as a common underlyingmechanism.large scale data: Not applicable.study funding and competing interest(s): 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 interestor competing interest(s) are noted.