Developmental programming by androgen affects the circadian timing system in female mice

Abstract

Circadian clocks play essential roles in the timing of events in the mammalian hypothalamo-pituitary-ovarian (HPO) axis. The molecular oscillator driving these rhythms has been localized to tissues of the HPO axis. It has been suggested that synchrony among these oscillators is a feature of normal reproductive function. The impact of fertility disorders on clock function and the role of the clock in the etiology of endocrine pathology remain unknown. Polycystic ovarian syndrome (PCOS) is a particularly devastating fertility disorder, affecting 5%-10% of women at childbearing age with features including a polycystic ovary, anovulation, and elevated serum androgen. Approximately 40% of these women have metabolic syndrome, marked by hyperinsulinemia, dyslipidemia, and insulin resistance. It has been suggested that developmental exposure to excess androgen contributes to the etiology of fertility disorders, including PCOS. To better define the role of the timing system in these disorders, we determined the effects of androgen-dependent developmental programming on clock gene expression in tissues of the metabolic and HPO axes. Female PERIOD2::luciferase (PER2::LUC) mice were exposed to androgen (dihydrotestosterone [DHT]) in utero (Days 16-18 of gestation) or for 9-10 wk (DHT pellet) beginning at weaning (pubertal androgen excess [PAE]). As expected, both groups of androgen-treated mice had disrupted estrous cycles. Analysis of PER2::LUC expression in tissue explants revealed that excess androgen produced circadian misalignment via tissue-dependent effects on phase distribution. In vitro treatment with DHT differentially affected the period of PER2::LUC expression in tissue explants and granulosa cells, indicating that androgen has direct and tissue-specific effects on clock gene expression that may account for the effects of developmental programming on the timing system.

Keywords: PCOS; PER2::luciferase; androgens/androgen receptors; circadian rhythm; clock genes; developmental origins of health and disease; female reproductive tract; fertility; mechanisms of hormone action; mouse; reproduction.

Fig. 1

Developmental programming by excess androgen produces metabolic and reproductive features of PCOS in PER2::LUC mice. a) Weekly body weights of PER2::LUC mice exposed to PAE or placebo pellet and fed a standard diet ad libitum. PAE mice (n = 8) gained more weight over the course of treatment than controls (n = 8). b) Weekly body weights of PNA mice (n = 18) or controls (n = 14) fed a standard diet ad libitum. c and d) Estrous cycle patterns from representative PAE and controls (c) or PNA mice and vehicle-treated animals (d). PNA and PAE mice displayed intermittent or irregular cycles marked by persistent metestrus (M) or diestrus (D). e and f) Reproductive cycles from PNA (n = 18), oil control (n = 14), PAE (n = 8), and placebo pellet (n = 8) mice analyzed as a percentage of total time spent in each day of the 4-day cycle. Both PNA and PAE mice spent more time in M and D relative to controls. PNA mice, but not PAE mice, displayed a significant drop in the percent time spent in estrus (E) and proestrus (P; P < 0.05 for both). Data in a, b, e, and f are presented as the mean ± SEM. In a, *P < 0.001; in e and f, *P < 0.05.

Fig. 2

Gestational androgen excess has tissue-dependent effects on the phase of PER2::LUC expression in central and peripheral oscillators. a) Rayleigh plots showing the peak phase of PER2::LUC expression within individual explant cultures from control (top row) and PNA (bottom row) mice. Each gray dot plotted at the edge of the outer circle in a represents the peak phase of a different tissue explant. The outer circle represents the 24-h day, with ZT0 (lights-on) located at 1200 h and ZT12 (lights-off) located at 0600 h. The inner circle represents the statistical threshold for significant phase cohesion (P < 0.05). The vector in each plot represents the arithmetic mean phase of each group of tissues. Only when this vector crosses the significance threshold is the group of oscillators considered to have significant phase synchrony (indicated by asterisks). The numbers inside each plot indicate how many cultures for that tissue were included in the analysis. b and c) Phase distribution maps for individual tissues from control (b) and PNA (c) mice. Individual symbols represent tissues from different animals. The gray area indicates the 12-h dark phase. Dashed lines connect tissues from the same mouse.

Fig. 3

Excess androgen during late adolescence and puberty has tissue-dependent effects on the phase of PER2::LUC expression in central and peripheral oscillators. a) Rayleigh plots showing the peak phase of PER2::LUC expression within individual explant cultures from control (top row) and PAE (bottom row) mice. Each gray dot plotted at the edge of the outer circle in a represents the peak phase of a different tissue explant. The outer circle represents the 24-h day, with ZT0 (lights-on) located at 1200 h and ZT12 (lights-off) located at 0600 h. The inner circle represents the statistical threshold for significant phase cohesion (P < 0.05). The vector in each plot represents the arithmetic mean phase of each group of tissues. Only when this vector crosses the significance threshold is the group of oscillators considered to have significant phase synchrony (indicated by asterisks). The numbers inside each plot indicate how many cultures for that tissue were included in the analysis. b and c) Phase distribution maps for individual tissues from control (b) and PAE (c) mice. Individual symbols represent tissues from different animals. The gray area indicates the 12-h dark phase. Dashed lines connect tissues from the same mouse.

Fig. 4

Treatment with DHT in vitro has tissue-specific and dose-dependent effects on the period of PER2::LUC expression in central and peripheral oscillators. The effects of DHT on the period of PER2::LUC expression in isolated tissue explants of the liver (a), lung (b), pituitary (c), ovarian follicles (d), oviduct (e), WAT (f), and SCN (g). Significant effects of DHT were detected in liver, follicle, and oviduct. A small but significant period shortening was also detected among SCN explants between 100 and 500 nM DHT. For a–g, n = 3–5 per treatment group. In each graph, differing letters above bars indicate differences between adjacent treatment groups (e.g., 100 nM DHT vs. 500 nM DHT), and an asterisk indicates differences between vehicle- and DHT-treated explants (both P < 0.05).

Fig. 5

Dose-dependent effects of DHT on period of PER2::LUC expression in cultured ovarian GCs are attenuated by an AR antagonist. a) Representative luminescence traces of PER2::LUC expression in isolated GCs. The timing of peak PER2::LUC expression in both the 100 nM DHT- and 1 μM DHT-treated cultures peaked later on the first full day in culture but appeared to have an advancing peak, indicative of a short period, such that the treated cultures peaked at the same time as (or earlier, for 100 nM DHT) than the control. b) The period of PER2::LUC expression as a function of treatment. DHT dose-dependently shortened the period of PER2::LUC expression relative to vehicle-treated controls. c) The effects of DHT on the period of PER2::LUC expression were attenuated when DHT was added in the presence of the AR antagonist BIC (100 nM). In b and c, *P < 0.05. In b, n = 4–8 cultures per treatment group, and in c, n = 5–8 per treatment group.