Canagliflozin treatment prevents follicular exhaustion and attenuates hallmarks of ovarian aging in genetically heterogenous mice

Abstract

Ovarian aging is characterized by declines in follicular reserve and the emergence of mitochondrial dysfunction, reactive oxygen species production, inflammation, and fibrosis, which eventually results in menopause. Menopause is associated with increased systemic aging and the development of numerous comorbidities; therefore, the attenuation of ovarian aging could also delay systemic aging processes in women. Recent work has established that the anti-diabetic drug Canagliflozin (Cana), a sodium-glucose transporter 2 inhibitor, elicits benefits on aging-related outcomes, likely through the modulation of nutrient-sensing pathways and metabolic homeostasis. Given that nutrient-sensing pathways play a critical role in controlling primordial follicle activation, we sought to determine if chronic Cana administration would delay ovarian aging and curtail the emergence of pathological hallmarks associated with reproductive senescence. We found that mice receiving Cana maintained their ovarian reserve through 12 months of age, which was associated with declines in primordial follicles FoxO3a phosphorylation, a marker of activation, when compared to the age-matched controls. Furthermore, Cana treatment led to decreased collagen, lipofuscin, and T cell accumulation at 12 months of age. Whole ovary transcriptomic and proteomic analyses revealed subtle improvements, predominantly in mitochondrial function and the regulation of cellular proliferation. Pathway analyses of the transcriptomic data revealed a downregulation in cell proliferation and mitochondrial dysfunction signatures, with an upregulation of oxidative phosphorylation. Pathway analyses of the proteomic data revealed declines in signatures associated with PI3K/AKT activity and lymphocyte accumulation. Collectively, we demonstrate that Cana treatment can delay ovarian aging in mice and could potentially have efficacy for delaying ovarian aging in women.

Keywords: Fertility; Fibrosis; Glucose; Insulin; Ovary; SGLT2.

Fig. 1

Cana treatment reduces fasting glucose in breeding age female mice. A Fasting glucose (n = 6/group), B fasting insulin (n = 5–6/group), and C Fasting IGF-1 (n = 5/group) in 12-month-old control and Cana-treated mice. All data are presented as mean ± SEM and were analyzed by Student’s t-tests. *p < 0.05

Fig. 2

Cana treatment attenuates primordial follicle activation and preserves follicular reserve in breeding age female mice. A Representative immunofluorescence images of pFoxO3a, DDX4, and merged pFoxO3a:DDX4 (magnification = 100 × ; scale bar = 20 μm), B quantified immunofluorescent intensity of merged pFoxO3a:DDX4 (n = 5/group), C estimated number of follicles in each stage (n = 6/group), and D primordial-to-primary follicle ratio (n = 6/group) in 6-month-old control, 12-month-old control, and 12-month-old Cana-treated mice. All data are presented as mean ± SEM and were analyzed by one-way ANOVA with Tukey post hoc analyses *p < 0.05; **p < 0.01

Fig. 3

Cana treatment attenuates age-related ovarian fibrosis and MNGC accumulation in breeding age female mice. A Representative Picrosirius Red stained images (magnification = 4 × ; scale bar = 500 μm), B Quantified Picrosirius Red positivity (n = 6/group), C representative lipofuscin stained images (magnification = 4 × ; scale bar = 500 μm), and D quantified lipofuscin positivity; E representative CD3 (green) and DAPI (blue) immunostained images (magnification = 10 × ; scale bar = 200 μm); and F quantified immunofluorescent intensity of CD3 (n = 5–6/group) in 6-month-old control, 12-month-old control, and 12-month-old Cana-treated mice. All data are presented as mean ± SEM and were analyzed by one-way ANOVA with Tukey post hoc analyses. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Cana treatment attenuates transcriptional signatures of age-related mitochondrial dysfunction in ovaries from breeding age female mice. A Volcano plot showing upregulated and downregulated transcripts, B principal component analysis (PCA) of the transcriptomic data, C IPA canonical pathway analysis, and D IPA upstream regulator analysis showing inhibition or activation of pathways associated with mitochondrial function and cell proliferation from bulk ovarian RNAseq from 12-month-old control (n = 5) and Cana-treated (n = 5) mice. Statistical analysis was performed by t-test. p-values < 0.05 and log2 change > 1 and <  − 1 were considered significant

Fig. 5

Cana treatment attenuates proteomic signatures of age-related proinflammatory and ER stress in ovaries from breeding age female mice. A Volcano plot showing upregulated and downregulated proteins, B principal component analysis (PCA) of proteomic data, C IPA canonical pathway analysis, and D IPA upstream regulator analysis showing inhibition or activation of pathways associated with cell proliferation, metabolism, endoplasmic reticulum (ER) stress, and inflammation/immune responses from ovarian proteomic analyses from 12-month-old control (n = 7) and Cana-treated (n = 6) mice. Statistical analysis was performed by t-test. p-values < 0.05 and log2 change > 0.25 and <  − 0.25 were considered significant

Fig. 6

Cana treatment only elicits mild changes in metabolic and antioxidant response pathway in ovaries from breeding age female mice. A Ovarian NADH oxidase activity in 12-month-old control (n = 7) and 12-month-old Cana-treated mice (n = 6). Heatmaps showing minor to no changes in protein panels associated with B beta oxidation, C glycolysis, D mitochondrial markers, and E antioxidants and stress responses from ovarian proteomic analyses in 12-month-old control (n = 7) and 12-month-old Cana-treated mice (n = 6). Data is presented as mean ± SEM. * < 0.05 by t-test