White spotting variant mouse as an experimental model for ovarian aging and menopausal biology

Abstract

Objective: Menopause is a unique phenomenon in modern women, as most mammalian species possess a reproductive period comparable with their life span. Menopause is caused by the depletion of germ cell-containing ovarian follicles and in laboratory studies is usually modeled in animals in which the ovarian function is removed through ovariectomy or chemical poisoning of the germ cells. Our objective was to explore and characterize the white spotting variant (Wv) mice that have reduced ovarian germ cell abundance, a result of a point mutation in the c-kit gene that decreases kinase activity, as a genetic model for use in menopause studies.

Methods: Physiological and morphological features associated with menopause were determined in female Wv/Wv mice compared with age-matched wildtype controls. Immunohistochemistry was used to evaluate the presence and number of follicles in paraffin-embedded ovaries. Bone density and body composition were evaluated using the PIXImus x-ray densitometer, and lipids, calcium, and hormone levels were determined in serum using antigen-specific enzyme immunoassays. Heart and body weight were measured, and cardiac function was evaluated using transthoracic echocardiography.

Results: The ovaries of the Wv/Wv females have a greatly reduced number of normal germ cells at birth compared with wildtype mice. The remaining follicles are depleted by around 2 months, and the ovaries develop benign epithelial lesions that resemble morphological changes that occur during ovarian aging, whereas a normal mouse ovary has numerous follicles at all stages of development and retains some follicles even in advanced age. Wv mice have elevated plasma gonadotropins and reduced estrogen and progesterone levels, a significant reduction in bone mass density, and elevated serum cholesterol and lipoprotein levels. Moreover, the Wv female mice have enlarged hearts and reduced cardiac function.

Conclusions: The reduction of c-kit activity in Wv mice leads to a substantially diminished follicular endowment in newborn mice and premature depletion of follicles in young mice, although mutant females have a normal life span after cessation of ovarian function. The Wv female mice exhibit consistent physiological changes that resemble common features of postmenopausal women. These alterations include follicle depletion, morphological aging of the ovary, altered serum levels of cholesterol, gonadotropins and steroid hormones, decreased bone density, and reduced cardiac function. These changes were not observed in male mice, either age-matched male Wv/Wv or wildtype mice, and are improbably caused by global loss of c-kit function. The Wv mouse may be a genetic, intact-ovary model that mimics closely the phenotypes of human menopause to be used for further studies to understand the mechanisms of menopausal biology.

Figure 1. Ovarian germ cell phenotype in Wv/Wv mice

Ovaries were collected from wildtype and Wv/Wv mice at ages ranging from newborn (1 day) to 2 years. (A) Wildtype ovaries retain follicular structues in advanced age. Ovaries were collected from wildtype females at the indicated ages and stained with H&E. A newborn ovary is shown at 200X magnification; other images are shown at 40X magnification. (B) Ovaries from wildtype and Wv/Wv mice were collected and stained with the germ cell marker, PGC7. Representative images are shown for newborn (200X magnification) and 1.5 and 3 months (40X). The far right panel enlarges on follicles, including primary and secondary, present in 3-month wildtype ovaries. Wv/Wv ovaries at this age completely lack follicles.

Figure 2. Serum hormone levels in wildtype and Wv/Wv mice

Blood was collected from wildtype and Wv/Wv mice at 3 months of age to determine hormone levels: (A) FSH (ng/mL) (n=5) (mean +/− s.d.), (B) progesterone (ng/mL) (WT, n=8; Wv/Wv, n=5), and (C) beta-estradiol (pg/mL) (WT, n=7; Wv/Wv, n=8). The means are indicated for progesterone and beta-estradiol. Significant difference (*) between Wv/Wv and WT was calculated using Student’s t-test and Wilcoxon-Mann-Whitney analysis and set as P < 0.05. All hormone levels were determined by specific ELISA based assays.

Figure 3. Serum cholesterol and lipoprotein levels in Wv/Wv female and male mice

Serum cholesterol (CHOL), triglycerides (TG), high density lipoprotein (HDL), very low density lipoprotein (VLDL), and low density lipoprotein (LDL) levels were determined in serum samples from 3 month old female and male Wv/Wv and WT mice. Samples from 4 animals in each group were analyzed. Results are reported as mean +/− s.d., with significance set as P < 0.05 (*). LDL levels were not detectable (N.D.) in female WT or any male mice.

Figure 4. Body composition and bone density in wildtype and Wv/Wv mice

The PIXImus small animal DEXA system (PIXImus^™, Fitchburg, WI) was used to assess whole body area for body composition of wildtype and Wv/Wv mice at 3 months of age. (A) Representative examples of wildtype (n=5) and Wv/Wv (n=6) females are shown. (B) Both female and male mice (n=5–6) were measured for body weight (g), percent body fat (%), and bone mineral density (BMD) (g/cm²). Results are reported as mean +/− s.d., with P < 0.05 (*), calculated using unpaired Student’s t-test, and confirmed using one-way ANOVA and Wilcoxon-Mann-Whitney analyses. The variation of the PIXImus for BMD is <1%. BMD is 0.0565 g/cm² in wildtype and 0.0470 g/cm² in Wv/Wv. Wv/Wv females had significantly lower body weight and BMD than wildtype controls at 3 months. Male wildtype and Wv/Wv males showed no significant differences in body composition. (C) Stiffness of femurs and 4^th and 5^th lumbars from Wv/Wv and wildtype females was determined using a biomechanical fracture test. Wv/Wv femurs had approximately 23% reduced stiffness.

Figure 5. Heart enlargement in Wv/Wv mice

(A) The ratio of heart weight (mg) to body weight (g) [HW:BW ratio] was determined for 5–6 female mice at 3, 6, and 12 months of age. Values are expressed as mean +/− s.d. Significant difference (* P < 0.05) was determined using both the unpaired Student’s t-test and Wilcoxon-Mann-Whitney test, and was P = 0.0001 at 12 months. (B) Examples of representative HW:BW for 12 month female wildtype and Wv/Wv mice. The mean HW:BW ratios for WT (n=5) and Wv/Wv (n=6) were 3.5 +/− 0.5 and 7.5 +/− 1.0, respectively. Hearts were photographed at equal magnification using a Zeiss stereodissection microscope.

Figure 6. Cardiac function in Wv/Wv mice

Transthoracic echocardiography was performed on 3 month-old female and male Wv/Wv and wildtype mice (n=6 mice, each) to determine basic cardiac function. In (A), heart function was assessed as left ventricle ejection fraction (EF), fractional shortening (FS), and stroke volume (SV). Wv/Wv females had significantly lower EF and FS (mean +/− s.d., P < 0.05). In (B), the range of % ejection fractions determined is shown for female and male mice. The mean ejection fraction percent for wildtype females, wildtype males, and Wv/Wv males was 80%, with minimal variation, whereas the mean ejection fraction percent for Wv/Wv females was 63%, and values ranged from 57–68%.