Long-term environmental exposure of darkness induces hyperandrogenism in PCOS via melatonin receptor 1A and aromatase reduction

Abstract

Polycystic ovary syndrome (PCOS) is a common and complex disorder impairing female fertility, yet its etiology remains elusive. It is reported that circadian rhythm disruption might play a crucial role in PCOS pathologic progression. Here, in this research, we investigated the effect of environmental long-term circadian rhythm dysfunction and clarified its pathogenic mechanism in the development of PCOS, which might provide the targeted clinical strategies to patients with PCOS. Female SD rats were used to construct a circadian rhythm misalignment model with constant darkness (12/12-h dark/dark cycle), and the control group was kept under normal circadian rhythm exposure (12/12-h light/dark cycle) for 8 weeks. We measured their reproductive, endocrinal, and metabolic profiles at different zeitgeber times (ZTs). Different rescue methods, including melatonin receptor agonist and normal circadian rhythm restoration, and in vitro experiments on the KGN cell line were performed. We found that long-term darkness caused PCOS-like reproductive abnormalities, including estrous cycle disorder, polycystic ovaries, LH elevation, hyperandrogenism, and glucose intolerance. In addition, the expression of melatonin receptor 1A (Mtnr1a) in ovarian granulosa cells significantly decreased in the darkness group. Normal light/dark cycle and melatonin receptor agonist application relieved hyperandrogenism of darkness-treated rats. In vitro experiments demonstrated that decreased MTNR1A inhibited androgen receptor (AR) and CYP19A1 expression, and AR acted as an essential downstream factor of MTNR1A in modulating aromatase abundance. Overall, our finding demonstrates the significant influence of circadian rhythms on PCOS occurrence, suggests that MTNR1A and AR play vital roles in pathological progression of hyperandrogenism, and broadens current treatment strategies for PCOS in clinical practice.

Keywords: androgen receptor; constant darkness; hyperandrogenism; melatonin receptor 1A; polycystic ovary syndrome.

FIGURE 1

Long-term continuous darkness leads to estrous cycle disorder and polycystic ovary in rats. (A) Diagram of the experimental design. (B,C) Body weight of two groups of rats. (D) Ovarian weight of each group of rats. (E) Representative estrous cycles of two groups. The upper and lower panels represent the control and darkness groups, respectively. D, diestrus; P, proestrus; E, estrus; M, metestrus. (F) Representative examples of ovarian HE-stain histology of two groups. The upper and lower panels represent the control and darkness groups, respectively. N = 30/group. Values are expressed as means ± SEM. Scale bar: 200 μm.

FIGURE 2

Serum hormone profiles and glucose metabolism of rats. (A) Serum concentration of LH. (B) Serum concentration of FSH. (C) LH/FSH ratio. (D) Serum concentration of testosterone. (E) Serum concentration of SHBG. (F) Free androgen index calculated by total testosterone plus 100 and divided by SHBG. (G) Fasting blood glucose concentration. (H) Glucose tolerance test. (I) Area under the curve of the GTT test. N = 5/group. Values are expressed as means ± SEM. Significant differences between the two groups are indicated by asterisks (*p < 0.05 and **p < 0.01).

FIGURE 3

Gene expression patterns of Cyp17a1, Cyp19a1, and Ar in rat ovaries. (A) Relative mRNA abundance of Cyp17a1 in rat ovaries. (B) Relative mRNA abundance of Cyp19a1 in rat ovaries. (C) Relative mRNA abundance of Ar in rat ovaries. N = 5/group. Values are expressed as means ± SEM. Significant differences between the two groups are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001).

FIGURE 4

Melatonin level in serum and its receptor expression in rat ovaries. (A) Serum level of melatonin at ZT16. (B) Relative mRNA abundance of Mtnr1a in rat ovaries. (C) Relative mRNA abundance of Mtnr1b in rat ovaries. (D) Relative mRNA abundance of Rora in rat ovaries. (E) Immunostaining score of MT1 in ovarian GCs of rats. (F) Representative photomicrographs of MT1 in follicular GCs. Scale bar: 50 μm. N = 5/group. Values are expressed as means ± SEM. Significant differences between the two groups are indicated by asterisks (*p < 0.05 and **p < 0.01).

FIGURE 5

Effects of RMT and circadian rhythm restoration in constant darkness induced-PCOS rats. (A) Diagram of the experimental design. (B,C) Body weight of rats during the experimental progression. (D) Representative examples of ovarian HE-stain histology of each group of rats. (E) Representative estrous cycles of each group of rats. N = 10/group. Values are expressed as means ± SEM. Scale bar: 200 μm. RMT, ramelteon.

FIGURE 6

Influences of RMT and circadian rhythm restoration on the reproductive endocrine system and gene expression abundance. (A) Serum concentration of testosterone. (B) Free androgen index. (C) Serum estradiol content. (D) Serum concentration of melatonin. (E) Mtnr1a, Ar, and Cyp19a1 mRNA levels in ovarian GCs of each group of rats. (F) Mtnr1a, Ar, and Cyp19a1 mRNA levels in whole ovaries of each group of rats. N = 5/group. Values are expressed as means ± SEM. Significant differences between the two groups are indicated by asterisks (*p < 0.05 and **p < 0.01).

FIGURE 7

Downregulated MTNR1A and AR reduced aromatase expression in the KGN cell line and decreased estradiol concentration in the culture medium. (A,B) Efficiency of MTNR1A-knockdown in mRNA and protein levels, respectively. (C,D) Western blot analysis (C) and quantification (D) of AR, aromatase, and MT1 after MTNR1A-knockdown and incubation with or without 10 nmol/L melatonin or 10 ng/ml FSH. (E) Estradiol level after MTNR1A-knockdown and incubation with or without 10 nmol/L testosterone. (F,G) Efficiency of AR-knockdown in mRNA and protein levels, respectively. (H,I) mRNA and protein abundance of AR and CYP19A1 after AR-knockdown and incubation with or without 10 nmol/L testosterone, respectively. Values are expressed as means ± SEM. These results are representative of at least three independent experiments. Significant differences between the two groups are indicated by asterisks (*p < 0.05, **p < 0.01, and ***p < 0.001).

FIGURE 8

MTNR1A regulated aromatase expression through AR and PKA/cAMP pathway. (A,B) Efficiency of AR-overexpression in mRNA and protein levels, respectively. (C,D) mRNA and protein abundance of MTNR1A, AR, and CYP19A1 after MTNR1A-knockdown and AR-overexpression, respectively. (E) Protein abundance of MT1, AR, and aromatase after MTNR1A-knockdown and incubation with 100 μmol/L cAMP for 24 h. Values are expressed as means ± SEM. These results are representative of at least three independent experiments. Significant differences between the two groups are indicated by asterisks (*p < 0.05 and **p < 0.01).