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. 2012 Jun;60(6):439-56.
doi: 10.1369/0022155412441002. Epub 2012 Apr 2.

Ovarian Abnormalities in a Mouse Model of Fragile X Primary Ovarian Insufficiency

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Free PMC article

Ovarian Abnormalities in a Mouse Model of Fragile X Primary Ovarian Insufficiency

Gloria E Hoffman et al. J Histochem Cytochem. .
Free PMC article

Abstract

FMR1 premutation (PM) alleles have 55-200 CGG·CCG-repeats in their 5' UTR. PM carriers are at risk of fragile X-associated tremor and ataxia syndrome (FXTAS). Females are also at risk for FX primary ovarian insufficiency (FXPOI). PM pathology is generally attributed to deleterious properties of transcripts with long CGG-tracts. For FXPOI, hormone changes suggest a reduced residual follicle pool. Whether this is due to a smaller than normal original follicle pool or an increased rate of follicle depletion is unclear. A FX-PM mouse the authors generated with 130 CGG·CCG-repeats in the endogenous Fmr1 gene recapitulates features of FXTAS. Here the authors demonstrate that the gross development of the ovary and the establishment of the primordial follicle pool is normal in these mice. However, these animals show a faster loss of follicles of all follicle classes, suggesting that the problem is intrinsic to the ovary. In addition, many oocytes show aberrant nuclear accumulation of FMRP and elevated levels of ubiquitination. Furthermore, PM follicles are smaller and have fewer granulosa cells (GCs) than normal. Thus, these animals have ovarian abnormalities involving both the oocytes and GCs that may shed light on the molecular basis of FXPOI in humans.

Conflict of interest statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Negative controls for FMRP localization. (A) No primary antibody and (B) substitution of normal rabbit serum in place of the primary antibody. The arrows in both figures show follicles. Bars = 100 µm.
Figure 2.
Figure 2.
FMRP immunoreactivity in the ovaries of wild-type (WT) and premutation (PM) mice. FMRP immunoreactivity in WT (A, B) and PM (C, D) ovaries from animals five to seven weeks of age. A and C, bars = 500 µm; B and D, bars = 10 µm.
Figure 3.
Figure 3.
Changes in FMRP and Fmr1 expression. (A) Intensity measures of FMRP in somatic cells of the ovaries of five- to seven-week-old mice. Wild-type (WT), black bar; premutation (PM), open bar (*p<0.05, analysis of variance [ANOVA]). (B) Total ovarian Fmr1 mRNA (mean ± SEM) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in WT (white bars) and PM mice (black bars) at 6, 12, and 18 months of age. Bars with different letters are significantly different by two-way ANOVA.
Figure 4.
Figure 4.
(A) In situ Fmr1 mRNA hybridization in eight-month-old wild-type (WT) (A, B, and F) and premutation (PM) (C, D, E, and G) mice, showing primordial oocytes (A–E) and the oocytes and granulosa cells (GCs) of early antral follicles (F, G). The mRNA is visible as black spots that are predominantly cytoplasmic. Bars = 10 µm.
Figure 5.
Figure 5.
FMRP expression in oocytes of mice seven months of age. (A–D) Examples of FMRP staining (black stain) in wild-type (WT) oocytes from animals seven months of age. Sections are counterstained with Neutral Red. Bars = 10 µm. (E–H) Follicles from seven-month-old premutation (PM) mice. The micrographs show primordial (A, E), primary (B, F) and preantral follicles (C, D, G, and H). Bars = 10 µm.
Figure 6.
Figure 6.
Bar graph showing the mean number of oocytes with nuclear concentrations of FMRP (± SEM) in every sixth section from each ovary in nine wild-type (WT) mice (open bars) and nine premutation (PM) mice (black bars). *p=0.0001.
Figure 7.
Figure 7.
Changes in ubiquitin. Staining for ubiquitin in wild-type (A) and premutation (B–D) oocytes. Bars = 100 µm.
Figure 8.
Figure 8.
(A) Numbers of follicles in wild-type (WT) and premutation (PM) mice that expressed elevated or abnormally distributed ubiquitin. Numbers of oocytes in WT (open bar) and PM (black bar) mice had either visibly increased ubiquitin throughout the cytoplasm or high nuclear or perinuclear concentrations of ubiquitin. *p<0.005. (B) Relationship between numbers of oocytes with nuclear concentrations of FMRP and oocytes with elevated ubiquitin immunoreactivity (WT, black dots; PM, red dots).
Figure 9.
Figure 9.
Follicle changes in wild-type (WT) and premutation (PM) ovaries with age. Bar graphs illustrating (A) the changes in total follicle numbers with age in WT (white bars) and PM (black bars) mice. (B–E) Changes in follicle numbers with age for (B) primordial, (C) primary, (D) preantral, and (E) antral follicles. Values represent the average total counts/ovary ± SEM. A two-way analysis of variance was first conducted followed by post hoc analyses. Bars with different letters are significantly different by Tukey honestly significant difference (HSD) post hoc comparisons (p<0.05).
Figure 10.
Figure 10.
Corpora lutea (CL) numbers in wild-type (WT) and premutation (PM) mice. (A) Bar graphs illustrating the CL number/ovary at 4, 7 to 9, and 10 to 12 months of age in WT (white bars) and PM mice (black bars). Effect of genotype in a two-way analysis of variance (ANOVA; indicated by different letters) was significant. (B) Expression of the CL numbers relative to the number of advanced follicles (preantral + antral) in each ovary. Bars with different letters are significantly different by two-way ANOVA and paired t-test post hoc comparisons (p<0.05).
Figure 11.
Figure 11.
Histological changes in the ovaries with age in wild-type (WT) and premutation (PM) mice. (A) Hematoxylin and eosin (H&E)–stained sections from the ovaries of a four-month-old WT mouse and (B) a four-month-old PM mouse. The asterisk in the PM ovary marks a small cyst. A large fat deposit just outside the ovary of the PM mouse would not be counted as ovarian parenchyma. (C) H&E staining of the ovary of a one-year-old WT mouse and (D) a one-year-old PM mouse. CL, corpora lutea; AF, atretic follicle, Bars = 500 µm. (E) H&E-stained section of an ovary of an 8.5-month-old PM mouse showing an example of the ovarian cysts that occur more commonly in PM mice. Asterisks indicate the cysts. Bar = 500 µm. (F) Ovarian atrophy and tubulostromal hypertrophy in a 12-month-old PM mouse. An asterisk indicates a large cyst. Bar = 500 µm.
Figure 12.
Figure 12.
Changes in the size of the ovary and its follicles. (A) Bar graph depicting the changes in ovarian volume with age in wild-type (WT) mice (white bars) and premutation (PM) mice (black bars). Bars with different letters are significant at p<0.05 by analysis of variance (ANOVA) followed by t-test post hoc. (B) Antral follicle area in four-month-old WT and PM mice (ANOVA, p<0.003). Antral follicle areas were measured at each follicle’s maximal diameter. (C) Granulosa cell (GC) number in these follicles (ANOVA, p<0.002). (D) The relationship between antral follicle area and GC number. Values for WT mice are represented by black dots; values for PM mice are represented by red dots.
Figure 13.
Figure 13.
Coronal abnormalities and premature loss of prophase I arrest in wild-type (WT) and premutation (PM) mice. (A) Hematoxylin and eosin (H&E) staining of a follicle from a four-month-old WT mouse shows a normal graafian follicle. Bar = 100 µm. (B) PM follicle with a partial corona (insert) or absent corona typical of atresia. Bar = 100 µm. (C) Example of a PM antral follicle with an incomplete corona that has a spindle and chromosomes in anaphase.
Figure 14.
Figure 14.
Coronal abnormalities, loss of prophase arrest, and atresia in wild-type (WT) and premutation (PM) mice. (A) Bar graph showing the number of follicles/ovary with either partial or absent corona (one-way analysis of variance [ANOVA], p=0.01). (B) Follicles in four-month-old WT (white bar) and PM mice (black bar) that have escaped meiotic arrest (one-way ANOVA, p=0.04). (C) Relationship between numbers of follicles with coronal abnormalities and those with signs of advancing meiosis in WT (black dots) and PM (red dots) mice. The diagonal lines in the figures represent the condition where all the follicles with abnormal coronas escaped meiotic arrest. (D) Atretic follicles in mice that have (+) and have not (–) recently ovulated. Bars with different letters are significantly different (p<0.05; two-way ANOVA, t-test pairwise comparison). (E) The relationship between repeat number and atresia. The number of atretic follicles in six-week-old mice homozygous for a PM allele were determined as described in the Materials and Methods and plotted as a function of the total number of repeats in both alleles. Different letters indicate clusters of animals whose values are statistically different (ANOVA, p<0.05).
Figure 15.
Figure 15.
Fallopian tubes showing two wild-type (WT) eggs with a normal cumulus cloud (left panel) and a premutation (PM) egg without such a cloud (right panel). Bars = 100 µm.

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