CALORIC RESTRICTION INCREASES OVARIAN FOXO3A EXPRESSION IN A MOUSE MODEL OF DELAYED AGING: IMPLICATIONS FOR REPRODUCTIVE LONGEVITY

Abstract

Ovarian hormonal and reproductive function are dependent on the primordial follicle pool. The exit of follicles from the primordial follicle pool is the critical event that depletes the ovarian reserve; therefore, an understanding of the mechanisms that regulate the primordial pool is critical for the development of interventions to prolong the reproductive lifespan. Moderate caloric restriction (CR) without malnutrition has been shown to increase ovarian reserve and prolong the reproductive lifespan in mouse models; however, the mechanism by which it does so remains under investigation. The aim of this study was to evaluate the nutritional triggers, specifically the contribution of amino acid composition during CR, as well as the molecular mechanisms, by which CR impacts ovarian reserve during physiologic aging in mice. Twenty-six 9-month-old female C57BL/6 mice were divided into 4 groups: (1) ad libitum feeding, (2) 40% caloric restriction (CR), (3) 40% CR + non-essential glycine amino acid supplementation (CR + Gly), and (4) 40% CR + sulfur amino acid supplementation (CR + SAA). CR was continued for 12 weeks. Following CR, the ovarian reserve was quantified. Formalin-fixed ovaries were sectioned and stained with hematoxylin and eosin for primordial follicle counts. Quantitative polymerase chain reaction (qPCR) was used to quantify levels of ovarian expression of forkhead box protein O3A (FOXO3A), an important suppressor of follicular activation, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-1). Primordial follicle counts increased in the 40% CR (53 vs 41, p = 0.7) and CR + Gly supplementation groups (82 vs 41, p = 0.029) compared to the ad-lib fed group. There was no increase in primordial follicles in the CR + SAA group (44 vs 41, p = 0.99). The relative expression of FOXO3A was increased by 10-fold in the CR group (p = 0.3) and 20-fold in the CR + Gly group compared to the ad lib group (p = 0.016). There was no increase in FOXO3A expression in the CR + SAA group. Relative GHR expression was decreased 4-fold in the CR + Gly compared to the ad-lib-fed animals (p = 0.07). There was no difference in IGF-1 levels between the CR + Gly group and controls; however, levels of IGF-1 in the CR + SAA group increased dramatically by 100-fold (p = 0.001). CR (40%) with Gly supplementation significantly increases ovarian reserve in a mouse model of physiologic aging. The improvement in ovarian reserve appears to be mediated by an increase in the expression of FOXO3A, an important suppressor of follicular activation. Consistent with observations that GHR knockout mice have a larger ovarian reserve, the CR + Gly group also showed a decrease in GHR expression.