Adropin ameliorates reproductive dysfunctions in letrozole-induced PCOS mouse

Abstract

Polycystic ovary syndrome (PCOS) is the most common cause of infertility in reproductive-age women, and its etiology and exact treatment are not yet established. Adropin is a unique hepatokine involved in maintaining energy homeostasis, and its level has been reported to decline in serum and follicular fluid of PCOS women. Thus, present study was designed to investigate the effect of adropin on hormonal and reproductive abnormalities in PCOS mice. PCOS was induced in adult mice by administering letrozole (6 mg/kg body weight) orally for 21 days. PCOS mice were subsequently treated with adropin (450 nmol/kg body weight) for 15 days. Adropin treatment drastically decreased serum testosterone by suppressing the ovarian expression of 17β-HSD in PCOS mice. It also improved the follicular proliferation and survival by enhancing the ovarian expression of PCNA and BCL2 and suppressing the BAX, cleaved caspase 3, and TUNEL-positive cells in PCOS mice. Most of the effects of adropin are comparable to metformin (current PCOS treatment). Notably, adropin shows more efficacy than metformin in treating reproductive abnormalities in PCOS mice, as evidenced by early regularization of cyclicity and enhanced ovarian expression of 3β-HSD and aromatase proteins. Thus, adropin may be an alternative therapeutic option for managing PCOS.

Keywords: Adropin; Fertility; GPR19; Metformin; PCOS.

Fig. 1

Ovarian expression of Adropin in VC and PCOS mice. (A–C) Representative histological sections showing strong adropin immunoreactivity in the CL, while moderate immunoreactivity in the TICs of the VC ovary. (D–F) Moderate adropin immunoreactivity is noted in the TICs of PCOS ovary. (A) and (D) are shown at 10x magnification, (B) and (E) are shown at 40x magnification whereas (C) and (F) are shown at 100x magnification. VC vehicle-treated control, PCOS polycystic ovary, CL corpus luteum, Oo oocyte, GC granulosa cell, TIC Theca interstitial cell.

Fig. 2

Vaginal smear and estrous cycle pattern in VC, PCOS, PCOS + ADR and PCOS + MET mice. (A) Representative vaginal smears from the VC mice showing different estrous cycle phases including proestrus with nucleated epithelium, estrus with cornified epithelium, metestrus with both cornified epithelium and neutrophils, and diestrus with neutrophils. (B) Representative vaginal smear from the PCOS mice showing the diestrus phase. (C) Schematic diagram showing animal experimentation. (D) The normal estrous cycle of the VC mice. (E) Persistent diestrus phase of the estrous cycle in the PCOS mice. Recovery of the estrous cycle in (F) PCOS + ADR mice and (G) PCOS + MET mice. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice.

Fig. 3

Ovary mass and histoarchitectural changes in different experimental groups. (A) Effect of exogenous treatment of adropin and metformin on ovary mass. (B) Representative photographs of the ovary with uterus of the mice of different experimental groups. Data are expressed as mean ± SEM (n = 3) and were analyzed by one-way ANOVA followed by the post hoc Bonferroni test. (C–F) Representative images of VC mice ovary; (C) ovarian section showing the presence of large CL as well as numerous small and large antral follicles; (D) healthy ovarian follicles with the presence of CL; (E) Antral follicle (AnF) with healthy GCs and oocyte (Oo); (F) luteal cells of the CL. (G–I) Representative images of PCOS mice ovary; (G) ovarian section showing presence of numerous cystic follicles (CF) and absence of CL; (H) presence of numerous CFs with thin layers of GCs; (I) cystic follicle having a large antrum and sparsely distributed GCs. (J–M) Representative images of adropin-treated PCOS mice ovary; (J) section of the ovary showing healthy follicles and CL; (K) ovarian antral follicles and CL; (L) antral follicle with healthy GCs; (M) luteal cells of CL. (N–P) Representative images of metformin-treated PCOS mice ovary; (N) section of the ovary showing numerous antral follicles and few atretic follicles; (O) antral follicles with healthy GCs; (P) antral follicle showing densely aggregated GCs. (C, G, J, N) are shown at 4x magnification, (D, H, K, O) are shown at 10x magnification, (E, I, L, and P) are shown at 40x magnification whereas (F, M) are shown at 100x magnification. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice, CL corpus luteum, GC granulosa cell, Oo oocyte. ^ap < 0.01; ^bp < 0.001.

Fig. 4

Representative images of PCNA immunolocalization in the ovarian sections in different experimental groups. Arrowhead indicates nuclear PCNA immunostaining in GCs of ovarian follicle. (B) Antral follicle of PCOS ovary is showing few immunopositive GCs, whereas antral follicle of (A) VC, (C) PCOS + ADR, (D) PCOS + MET ovaries are showing the majority of PCNA immunopositive GCs. Figures (A–D) are shown in 40x magnification. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice.

Fig. 5

Western blot analysis of apoptosis-related proteins and detection of apoptotic cells in ovarian sections of different experimental groups. (A) Representative immunoblots and densitometric analysis of ovarian BAX & BCL2; (C) Caspase 3 & cleaved caspase 3 protein expressions. (B) The bar graphs showing the ratio of BAX/BCL2; (D) Cleaved caspase3/caspase 3 in different experimental groups. Data are expressed as IRDV ± SEM analyzed by one-way ANOVA followed by the post hoc Bonferroni test. β-actin is used to normalize the results of immunoblots. Representative histological sections showing the TUNEL-positive apoptotic cells in the ovary of (E, F) VC, (G, H) PCOS, (I, J) PCOS + ADR, and (K, L) PCOS + MET mice. Apoptotic granulosa cells are shown by an arrowhead. Figures (A, C, E, G) are shown in 40x magnification, whereas figures (B, D, F, H) are shown in 100x magnification. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice, IRDV integrated relative density value. ^ap < 0.01; ^bp < 0.001; ns—not significant.

Fig. 6

Hormone levels and ovarian expression of steroidogenic proteins in different experimental groups. Circulating (A) progesterone, (B) testosterone, and (C) estradiol concentration in the different groups of experiment. Data are expressed as mean ± SEM (n = 10), and were analyzed by one-way ANOVA followed by the post hoc Bonferroni test. (D) Representative immunoblots and densitometric analysis of ovarian steroidogenic proteins, namely steroidogenic acute regulatory protein (StAR), Cytochrome P450 11A1 (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in different experimental groups. Data are expressed as IRDV ± SEM (n = 3), analyzed by one-way ANOVA followed by the post hoc Bonferroni test. β-actin is used to normalize the results of immunoblots. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice, IRDV integrated relative density value. ^ap < 0.05; ^bp < 0.01; ^cp < 0.001; ns—not significant.

Fig. 7

Evaluation of fertility. (A) Vaginal plug of the mice showing the presence of spermatozoa, a sign of successful mating (Day 1). (B) Representative photographs of a mouse uterus showing implantation sites (Day 10) in different experimental groups. The arrowhead indicates implantation sites. VC vehicle-treated control mice, PCOS polycystic mice, PCOS + ADR adropin-treated PCOS mice, PCOS + MET metformin-treated PCOS mice.