Ovarian Rev-erbα: a central regulator of fertility following chronodisruption

Abstract

Introduction: Circadian rhythm disruption caused by shift work, nutritional imbalance, and the stresses of modern life can alter hormone secretion, lead to menstrual irregularities, impair follicle development, and contribute to ovarian hypoplasia. The mechanistic role of circadian rhythm disruption in reproductive disorders has been thoroughly investigated. Nuclear receptors are known to play an important role in female reproduction and in maintaining pregnancy. Rev-erbα, a circadian nuclear receptor, is a key component of the circadian clock and helps sustain circadian rhythm.

Materials and methods: We evaluated the potential prophylactic and therapeutic functions of Rev-erbα in supporting female fertility by orchestrating a series of events that culminate in successful pregnancy. We generated a circadian rhythm-disrupted female mouse model to study fertility.

Results: The Rev-erbα ligand SR9011 improved the fertility index in these circadian rhythm-disrupted female mice. Moreover, SR9011 treatment restored impaired ovarian follicular cell proliferation and division, regulated steroidogenesis and steroid hormone production, enhanced progesterone and melatonin secretion, and mitigated the adverse effects of circadian disruption on folliculogenesis. SR9011 also reduced follicular atresia and promoted follicle development in CR-disrupted mice. Rev-erbα is a key regulator influencing oocyte retrieval, as demonstrated in mice treated with the Rev-erbα antagonist SR8278.

Discussion/conclusion: These findings suggest that targeting Rev-erbα signaling and elucidating its mechanistic role in female reproduction could inform the development of more effective strategies for treating female infertility. As a sought-after druggable target, Rev-erbα has a broad range of potential therapeutic applications and has recently attracted considerable attention in the context of female infertility treatment.

Keywords: REV-ERBα; clock genes; folliculogenesis; superovulation; therapeutic.

Graphical abstract

Figure 1

Effect of Rev-erbα on fertility. Breeding efficiency was assessed by examining the impact of circadian rhythm disruption on fertility outcomes, and the effectiveness of SR9011 on breeding was evaluated using both prophylactic (A, B) and therapeutic (C, D) approaches. Each litter is indicated on the x-axis according to the number of days elapsed since the mating trial commenced. At the occurrence of each litter, the plot exhibits a vertical shift corresponding to the cumulative number of offspring produced. The graph presents data from individual cages, illustrating the following outcomes: (A) CR-disrupted mouse pairs produced nine live litters over 275 days. The effectiveness of SR9011 on breeding was assessed, revealing that (B) CR-disrupted mice treated with SR9011 produced 12 live litters over 341 days, whereas (C) untreated CR-disrupted mouse pairs produced eight live litters over 345 days. (D) CR-disrupted pairs treated with SR9011 produced nine live litters over 377 days. The mean litter interval is presented as the mean time interval between litters ± SD.

Figure 2

Prophylactic effect of Rev-erbα on clock genes, steroidogenesis, folliculogenesis, and cell division. The prophylactic effects of SR9011 (A–C) were evaluated. (A) The expression levels of Amh, Star, Cyp11a1, Per2, Cry1, and FSHR were analyzed in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011 using qRT-PCR. (B) Western blotting was performed to examine the expression of CYP11A1, STAR, PER2, CRY1, FSHR, P27, and CYCLIN D2 in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. (C) Quantification of protein bands is shown. Asterisks represent significant differences compared with the control group or as indicated (^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05 or p = 0.05). Data are presented as the average (A, C) or as representative images (B) from three independent experiments. Results in (A) are expressed as mean ± SD, and results in (C) are expressed as mean ± SEM.

Figure 3

Therapeutic effect of Rev-erbα on clock genes, steroidogenesis, folliculogenesis, and cell division. The therapeutic effects of SR9011 (A–C) were evaluated. (A) The expression levels of Amh, Star, Cyp11a1, Per2, Cry1, and FSHR were analyzed in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011 using qRT-PCR. (B) Western blotting was performed to examine the expression of CYP11A1, STAR, PER2, CRY1, FSHR, P27, and CYCLIN D2 in the ovaries of control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. (C) Quantification of protein bands is shown. Asterisks represent significant differences compared with the control or as indicated (^****p < 0.0001, ^**p < 0.01, ^*p < 0.05 or p = 0.05). Data are presented as the average (A, C) or as representative images (B) from three independent experiments. Results in (A) are expressed as mean ± SD, and results in (C) are expressed as mean ± SEM.

Figure 4

Effect of Rev-erbα on follicular growth and development. Hematoxylin and eosin staining was performed on ovarian sections from control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. Treatment with SR9011 in both prophylactic (A) and therapeutic (B) modes effectively inhibited follicle degeneration caused by CR disruption. Data shown are representative images (A, B) from five mice in each group. Representative mouse ovary sections illustrate different stages of follicular development. A black arrow indicates a primary follicle. A black arrow marked with one asterisk (^*) denotes a secondary follicle. A red arrow highlights the fluid-filled antral cavity. An arrow marked with three asterisks (^***) indicates the oocyte. A yellow arrow denotes a Graafian (mature) follicle. A red arrow marked with one asterisk (^*) indicates the corpus luteum. A black arrow marked with two asterisks (^**) indicates a large antral follicle undergoing atresia, characterized by apoptotic granulosa cells. A blue arrow indicates the follicular cells. Representative ovarian sections were acquired at × 20 magnification (scale bar: 100 µm) and × 4 magnification (scale bar: 500 µm).

Figure 5

Effect of Rev-erbα on ovarian follicle development. Immunohistochemical detection of Ki67 and p27 was performed in ovarian sections from control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011. The use of SR9011 in prophylactic (A–D) and therapeutic (E–H)modes restored follicular growth impaired by CR disruption and promoted follicular cell proliferation. This effect is supported by increased Ki67 expression and decreased p27 expression compared with CR disruption alone. Data shown are representative images (A, C, E, G) from five mice in each group. Ki67 and p27 images were acquired at × 40 magnification (scale bar: 50 µm) in both the prophylactic and therapeutic groups. Graphs shown (B, D, F, H) are representative of different ovarian sections analyzed for quantification of the percentage area fraction of Ki67 and p27 staining (mean ± SEM). Asterisks represent significant differences as compared to control or as indicated (*** indicates P<0.001, ** indicates P<0.01, * indicates P<0.05 or P=0.05).

Figure 6

Effect of SR9011 on follicle quantification. Ovarian sections from control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011 were subjected to hematoxylin and eosin staining to assess follicle numbers at each stage of development. Treatment with SR9011 under prophylactic (A, B) and therapeutic (C, D) conditions restored follicular growth and reduced the number of atretic follicles compared with CR disruption alone. Asterisks represent significant differences compared with the control group, and hash symbols indicate significant differences compared with the CR-disrupted group (^**/##p < 0.01, ^*/#p < 0.05 or p = 0.05). Data are presented as the average (A, C) from three independent experiments (mean ± SEM) and as representative images (B, D) from three mice in each group.

Figure 7

Rev-erbα: a key regulator of progesterone and melatonin synthesis and oocyte retrieval. The efficacy of SR9011 on melatonin and progesterone secretion following CR disruption was evaluated using both prophylactic (A, B) and therapeutic (C, D) approaches. Competitive ELISA was performed on serum samples isolated from control mice, CR-disrupted mice, and CR-disrupted mice treated with SR9011 to measure melatonin (A, C) and progesterone (B, D) levels. (E) Oocytes were collected from the oviducts and manually counted following superovulation. Each data point represents the oocyte yield of an individual mouse within the respective groups, with the horizontal line indicating the mean value for each group. The number of mice included in each group was as follows: control (n = 4), CR-disrupted only (n = 4), CR-disrupted + superovulation (n = 4), and CR-disrupted + SR8278 + superovulation (n = 4). Data are presented as the averages (A–D) from three independent experiments or from four mice in each group (mean ± SD). Asterisks represent significant differences as compared to control or as indicated (**** indicates P<0.0001, *** indicates P<0.001, ** indicates P<0.01, * indicates P<0.05 or P=0.05), and ns shows non-significant.