Modeling perimenopause in Sprague-Dawley rats by chemical manipulation of the transition to ovarian failure

Abstract

Various age-related diseases increase in incidence during perimenopause. However, our understanding of the effects of aging compared with hormonal changes of perimenopause in mediating these disease risks is incomplete, in part due to the lack of an experimental perimenopause model. We therefore aimed to determine whether manipulation of the transition to ovarian failure in rats via the use of 4-vinylcyclohexene diepoxide (VCD) could be used to model and accelerate hormonal changes characteristic of perimenopause. We examined long-term (11 to 20 mo), dose-dependent effects of VCD on reproductive function in 1- and 3-mo-old female Sprague-Dawley rats. Twenty-five daily doses of VCD (80 or 160 mg/kg daily compared with vehicle alone) depleted ovarian follicles in a dose-dependent fashion in rats of both ages, accelerated the onset of acyclicity, and caused dose-dependent increases in follicle-stimulating hormone that exceeded those naturally occurring with age in control rats but left serum levels of 17β-estradiol unchanged, with continued ovarian production of androstenedione. High-dose VCD caused considerable nonovarian toxicities in 3-mo-old Sprague-Dawley rats, making this an unsuitable model. In contrast, 1-mo-old rats had more robust dose-dependent increases in follicle-stimulating hormone without evidence of systemic toxicity in response to either VCD dose. Because perimenopause is characterized by an increase in follicle-stimulating hormone with continued secretion of ovarian steroids, VCD acceleration of an analogous hormonal milieu in 1-mo-old Sprague-Dawley rats may be useful for probing the hormonal effects of perimenopause on age-related disease risk.

Figure 1.

Mortality and body weights of juvenile and adult Sprague–Dawley rats treated with VCD. (A) Kaplan–Meier survival curve of juvenile rats during injection with vehicle (n = 24), low-dose VCD (n = 18), or high-dose VCD (n = 29). There were no significant differences between groups. Inset, Average body weights of juvenile rats. No significant differences between groups by ANOVA. (B) Kaplan–Meier survival curve of adult rats during injection with vehicle (n = 38), low-dose VCD (n = 11), or high-dose VCD (n = 40). ‡, P < 0.001 compared with control by log-rank testing. Inset, Average body weights of adult rats. ‡, P < 0.001 compared with controls over time by ANOVA with Newman–Keuls testing. (C) Kaplan–Meier survival curve of juvenile rats after completion of treatment with vehicle (n = 10), low-dose VCD (n = 12), or high-dose VCD (n = 12). There were no significant differences between groups. Inset, Average body weights of juvenile rats. There were no significant differences between groups by ANOVA. (D) Kaplan–Meier survival curve of adult rats after completion of treatment with vehicle (n = 17), low-dose VCD (n = 5), or high-dose VCD (n = 10). ‡, P < 0.001 compared with controls by log-rank testing. Inset, Average body weights of adult rats in same treatment groups. No significant differences between groups by ANOVA.

Figure 2.

Dose-dependent depletion of ovarian follicles by VCD in juvenile (1 mo) and adult (3 mo) female Sprague–Dawley rats. Follicle number per ovary (mean ± SEM) are reported for end of VCD treatment or end of the experiment. (A) Ovarian follicle counts in juvenile rats immediately posttreatment with vehicle (n = 5), low-dose VCD (n = 6), or high-dose VCD (n = 6). *, P < 0.05 compared with vehicle; $, P < 0.05 compared with vehicle or 80 mg/kg/d VCD. (B) Ovarian follicle counts in adult rats immediately after treatment with vehicle (n = 17), low-dose VCD (n = 5), or high-dose VCD (n = 17). *, P < 0.05 compared with vehicle. (C) Ovarian follicle counts in 20-mo-old juvenile rats, 18 mo after completion of VCD treatment with vehicle (n = 3), low-dose VCD (n = 4), or high-dose VCD (n = 4). *, P < 0.05 compared with vehicle. (D) Ovarian follicle counts in 12-mo-old adult rats at 8 mo after completion of VCD treatment with vehicle (n = 15), low-dose VCD (n = 5), or high-dose VCD (n = 7). *, P < 0.05 compared with vehicle.

Figure 3.

Effects of VCD on reproductive function and hormones when administered to 1-mo-old Sprague–Dawley rats. (A) Cyclicity in juvenile rats treated with vehicle (n = 3 to 17), low-dose VCD (n = 10 to 12), or high-dose VCD (n = 4 to 12), expressed as the incidence of persistent estrus (at least 75% of days in epithelial phase determined by days in proestrus and estrus). (B) Serum FSH levels in rats treated as juveniles with vehicle (n = 3 to 10), low-dose VCD (n = 5 to 12), or high-dose VCD (n = 4 to 12). Within each treatment group, FSH levels in aging rats were significantly (P < 0.05) increased over baseline (2.4 mo), except for high-dose VCD rats at 13 and 20 mo. *, P < 0.05 compared with vehicle; †, P < 0.01 compared with vehicle; ‡, P < 0.001 compared with vehicle. (C) Serum 17β-estradiol levels in rats treated as juveniles with vehicle (n = 3 to 10), low-dose VCD (n = 5 to 10), or high-dose VCD (n = 4 to 6). No significant differences were observed between treatment groups by ANOVA or unpaired t test as appropriate, nor were significant differences detected within any treatment group as compared with baseline (2.4 mo cycling animals). (D) Serum androstenedione levels in rats treated as juveniles with vehicle (n = 3 to 10), low-dose VCD (n = 5 to 10), or high-dose VCD (n = 4 to 6). No significant difference was observed between or within groups.

Figure 4.

Effects of VCD on reproductive function and hormones when administered to 3-mo-old Sprague–Dawley rats. (A) Cyclicity in adult rats treated with vehicle (n = 3 to 17), low-dose VCD (n = 5), or high-dose VCD (n = 7 to 10), expressed as incidence of persistent estrus. (B) Serum FSH levels in rats treated as adults with vehicle (n = 5 to 11), low-dose VCD (n = 5), or high-dose VCD (n = 5 to 8). In control rats, FSH levels did not change over time (compared with 4.1 mo), as assessed by ANOVA. *, P < 0.05 compared with vehicle. (C) Serum 17β-estradiol (E₂) levels in rats treated as adults with vehicle (n = 5 to 15), low dose VCD (n = 5), or high dose VCD (n = 3 to 10). No significant differences were observed between treatment groups, nor were significant differences detected within any treatment group as compared with initial values at 4.1 mo. (D) Serum androstenedione levels in rats treated as adults with vehicle (n = 5 to 17), low-dose VCD (n = 5), or high-dose VCD (n = 7 to 10). In control rats, androstenedione levels did not change over time (compared with levels at 4.1 mo), as assessed by Student t test. Within each VCD treatment group, androstenedione levels declined at the later time point (12 mo, P < 0.01) but were no different than those in age-matched controls. At 4.1 mo, androstenedione levels in high-dose VCD rats were increased as compared with age-matched controls. ‡, P < 0.001.