Sleep Deprivation Triggers the Excessive Activation of Ovarian Primordial Follicles via β2 Adrenergic Receptor Signaling

Abstract

Sleep deprivation (SD) is observed to adversely affect the reproductive health of women. However, its precise physiological mechanisms remain largely elusive. In this study, using a mouse model of SD, it is demonstrated that SD induces the depletion of ovarian primordial follicles, a phenomenon not attributed to immune-mediated attacks or sympathetic nervous system activation. Rather, the excessive secretion of stress hormones, namely norepinephrine (NE) and epinephrine (E), by overactive adrenal glands, has emerged as a key mediator. The communication pathway mediated by the KIT ligand (KITL)-KIT between granulosa cells and oocytes plays a pivotal role in primordial follicle activation. SD heightened the levels of NE/E that stimulates the activation of the KITL-KIT/PI3K and mTOR signaling cascade in an β2 adrenergic receptor (ADRB2)-dependent manner, thereby promoting primordial follicle activation and consequent primordial follicle loss in vivo. In vitro experiments further corroborate these observations, revealing that ADRB2 upregulates KITL expression in granulosa cells via the activation of the downstream cAMP/PKA pathway. Together, these results reveal the significant involvement of ADRB2 signaling in the depletion of ovarian primordial follicles under sleep-deprived conditions. Additionally, ADRB2 antagonists are proposed for the treatment or prevention of excessive activation of primordial follicles induced by SD.

Keywords: KIT ligand; ovarian function; primordial follicles; sleep deprivation; β2 adrenergic receptor.

Figure 1

SD induces the loss of primordial follicles. A) Experimental design diagram of three groups of 6–8 week‐old female mice subjected to SD experiments, with mice deprived of sleep for 0, 3, and 6 days, respectively. B) Significant decrease in body weight of mice after SD over time (CON n = 6; SD3D n = 6; SD6D n = 6). Statistical method: One‐way ANOVA with LSD Test. C) Representative images of mouse ovaries after SD treatment, showing a significant reduction in ovarian weight in sleep‐deprived mice (CON n = 14; SD3D n = 14; SD6D n = 14). Statistical method: One‐way ANOVA with LSD Test. D) The serum AMH levels in mice significantly increased after 6 days of sleep deprivation (CON n = 5; SD3D n = 5; SD6D n = 5). Statistical method: One‐way ANOVA with LSD Test. E‐F) There were no significant differences in serum E2 and FSH levels after sleep deprivation (CON n = 6; SD3D n = 5; SD6D n = 6). Statistical method: One‐way ANOVA. G) Number of primordial follicles, antral follicles, total follicles count in mice after SD treatment (CON n = 5; SD3D n = 5; SD6D n = 5). Statistical methods: One‐way ANOVA with LSD Test. Data are presented as mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates not significant. CON, control; SD, sleep deprivation.

Figure 2

Single‐cell transcriptome profiling of mouse ovarian cells after SD and the role of KIT in activating ovarian primordial follicle. A) Uniform Manifold Approximation and Projection (UMAP) plot of different cell clusters in mouse ovaries. B) The proportion of stromal cells, granulosa cells, endothelial cells, immune cells, and epithelial cells in mouse ovarian cells after 0 and 6 days of SD. C) Representative KEGG enrichment pathways for endothelial cells, epithelial cells, granulosa cells, and stromal cells. D) UMAP plot of granulosa cells showing three subpopulations: granulosa cells 0, 1, and 2. E) Expression markers of the three granulosa cell subpopulations. F) Significantly upregulated differentially expressed genes (DEGs) enriched GO terms (biological processes) of DEGs in granulosa cell cluster 0. G) Significantly downregulated enriched GO terms (biological processes) of DEGs in granulosa cell cluster 0. H) Expression of KITL in granulosa cells across different clusters. I) Western Blot (WB) analysis revealing the high expression of KIT/KITL in ovarian tissue after SD. J) Immunofluorescence analysis of ovarian tissue showing overexpression of KIT in primordial follicles after SD. K) Expression analysis graph of KIT/KITL in ovarian tissue after SD via WB Experiment (CON n = 4; SD3D n = 4; SD6D n = 4). Statistical method: One‐way ANOVA with LSD Test. L) HE staining and proportion analysis of developing follicles in ovarian tissue of 6 days old mice after adding KITL (500 ng mL⁻¹) to the culture medium for 4 days. Scale bar, 100 µm. (CON n = 3; KITL n = 3), Statistical method: Independent Samples Test. M) Immunofluorescence analysis showing increased expression of KIT in oocytes of ovarian tissue after adding KITL to the culture medium. Scale bar, 100 µm. Data are presented as mean with SEM. *p < 0.05, and **p < 0.01. CON, control; SD, sleep deprivation; KITL, KIT ligand.

Figure 3

A) The protein expression of AMH and ZP3 increase in mice ovaries after SD by Western blots. B, C) Western blots analysis of the activation of phosphorylated PI3K/AKT and mTOR pathways in ovarian tissue. D) Immunostaining analysis of ovarian tissue showing overexpression of p‐AKT^Ser473, p‐mTOR^Ser2481, and p‐FOXO3a^Ser253 in primordial follicles after SD. E–J) Protein expression analysis chart of AMH, ZP3, phosphorylated PI3K/AKT, and mTOR (E–G, I, J: CON n = 4; SD3D n = 4; SD6D n = 4. H: CON n = 4; SD3D n = 3; SD6D n = 3). Statistical method: E, H–J: One‐way ANOVA with LSD Test; F, G: Independent‐Samples Kruskal‐Wallis Test. Data are presented as mean with SEM. *p < 0.05, **p < 0.01, and ns indicates not significant. CON, control; SD, sleep deprivation.

Figure 4

The loss of primordial follicles induced by SD is dependent on the adrenal glands. A) Experimental design for SD in sham‐operated mice and ADX mice. B) Representative images of ovaries and changes in ovarian weight after experimentation in four groups: Sham, Sham + SD6D, ADX, and ADX + SD6D mice (Sham, n = 12; Sham + SD6D n = 10; ADX n = 8; ADX + SD6D n = 10). Statistical method: One‐way ANOVA with Tamhane's T2 Test. C) Changes in body weight after experimentation in the four groups of mice, showing a significant increase in body weight in SD mice after adrenal gland removal compared to Sham + SD6D mice (Sham, n = 6; Sham + SD6D n = 5; ADX n = 4; ADX + SD6D n = 5). Statistical method: One‐way ANOVA with LSD Test. D) Number of primordial follicles, antral follicles and total follicle count after experimentation in the four groups of mice (Sham, n = 6; Sham + SD6D n = 5; ADX n = 4; ADX + SD6D n = 5). Statistical method: One‐way ANOVA with LSD Test. Data are presented as mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates not significant. SD, sleep deprivation; ADX, adrenalectomy.

Figure 5

Both noradrenaline (NE) and epinephrine (E) induce the loss of primordial follicles. A) Adrenal gland weight significantly increased in mice after 0, 3, and 6 days of SD (CON n = 14; SD3D n = 14; SD6D n = 14). Statistical method: One‐way ANOVA with LSD Test. B‐E) Levels of stress hormones NE, E, CORT, and COR in mice after 0, 3, and 6 days of SD (NE: CON n = 5; SD3D n = 6; SD6D n = 4) (E, CORT, and COR: CON n = 5; SD3D n = 5; SD6D n = 4). Statistical methods: NE, E, One‐way ANOVA with LSD Test; CORT, COR, Nonparametric tests. F) Representative images of ovaries and changes in ovarian weight in mice after treatment with vehicle, NE (0.2 and 2 mg kg⁻¹, intraperitoneal injections for 6 days), and E (0.2 and 2 mg kg⁻¹ intraperitoneal injections for 6 days) (CON n = 12, NE‐0.2 n = 12, NE‐2 n = 12, E‐0.2 n = 12, E‐2 n = 12). Statistical method: One‐way ANOVA with LSD Test. G) Number of primordial follicles, antral follicles and total follicle count in mice after each experimentation (CON n = 8, NE‐0.2 n = 8, NE‐2 n = 8, E‐0.2 n = 8, E‐2 n = 8). Statistical method: Primordial follicles: One‐way ANOVA with Tamhane's T2 Test; Antral follicles: One‐way ANOVA with LSD Test; Total follicles: Independent‐Samples Kruskal‐Wallis Test. Data are presented as mean with SEM. In all panels, *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates not significant. CON, control; SD, sleep deprivation; NE, noradrenaline; E, epinephrine; CORT, corticosterone; COR, Cortisol.

Figure 6

Activation of the ADRB2 signaling pathway induces the loss of primordial follicles. A) Significant reduction in ovarian weight in mice after administering water containing ADRB2 agonist CLEN for 6 days (CON n = 22, CLEN n = 22). Statistical method: Mann‐Whitney test. B) Number of primordial follicles, antral follicles, and total follicle count in mice after treatment with ADRB2 agonist CLEN (CON n = 5, CLEN n = 5). Statistical method: Independent Samples Test. C) Significant reduction in ovarian weight in mice after intraperitoneal administration of another ADRB2 agonist SAL for 6 days (CON n = 12, SAL n = 12). Statistical method: Independent Samples Test. D) Number of primordial follicles, antral follicles and total follicle count in mice after treatment with ADRB2 agonist SAL (CON n = 5, SAL n = 5). Statistical method: Independent Samples Test. E) ADRB2 antagonist ICI was intraperitoneally injected to rescue SD mice, showing significant recovery of ovarian weight in mice after addition of ICI to SD6D mice (CON n = 12, SD6D n = 12, SD6D + ICI n = 12, ICI n = 12). Statistical method: Independent‐Samples Kruskal‐Wallis Test. F) Number of primordial follicles, antral follicles and total follicle count in mice after treatment with ADRB2 antagonist ICI (CON n = 5, SD6D n = 5, SD6D + ICI n = 5, ICI n = 5). Statistical methods: Primordial follicles: Independent‐Samples Kruskal‐Wallis Test; Antral follicles: One‐way ANOVA with LSD Test; Total follicles: Independent‐Samples Kruskal‐Wallis Test. Data are presented as mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicates not significant. CON, control; SD, sleep deprivation; CLEN, Clenbuterol; SAL, Salbutamol; ICI, ICI‐118551.

Figure 7

Activated ADRB2 upregulates KITL via the cAMP/PKA pathway. A) After treating mice with the ADRB2 agonist CLEN, Western blot analysis revealed high expression of KIT/KITL in ovarian tissue (KIT: CON n = 3, CLEN n = 4), (KITL: CON n = 5, CLEN n = 6). Statistical methods: KIT: Independent Samples Test; KITL: Mann‐Whitney Test. B) Similarly, after treatment with another ADRB2 agonist SAL, Western blot analysis showed high expression of KIT/ KITL in ovarian tissue (CON n = 3, SAL n = 3). Statistical methods: KIT: Independent Samples Test; KITL: Mann‐Whitney Test. C) Stimulation of the human granulosa cell line KGN with CLEN and SAL at a concentration of 1 and 100 µm, respectively, resulted in a significant upregulation of KITL mRNA expression (CON n = 3, CLEN n = 3) (CON n = 3, SAL n = 3). Statistical method: Independent Samples Test. D,E) Following treatment with CLEN and SAL, Western blot analysis revealed a significant increase in KITL protein levels in KGN cells (CON n = 5, CLEN n = 4) (CON n = 4, SAL n = 4). Statistical method: Independent Samples Test. F) After treatment with CLEN and SAL, KGN cells were collected and subjected to cAMP ELISA testing, which showed a significant increase in cellular cAMP content compared to control cells (CON n = 4, CLEN n = 4; SAL n = 4). Statistical method: One‐way ANOVA with LSD Test. G,H) Following treatment with CLEN and SAL, Western blot analysis showed a significant upregulation of p‐PKA levels in KGN cells (CON n = 3, CLEN n = 3) (CON n = 4, SAL n = 4). Statistical method: Independent Samples Test. I) Correlation analysis of the protein levels of KITL and p‐PKA in KGN cells revealed a strong correlation between the expression levels of these two proteins, with a Pearson correlation of 0.925 (n = 8, p = 0.001**). J) After treating KGN cells with 20 µm of cAMP, the expression level of KITL significantly increased (CON n = 4, cAMP n = 4). Statistical method: Mann‐Whitney Test. K) Treatment of KGN cells with 10 nm concentration of the PKA activator BC resulted in a significant upregulation of KITL levels (CON n = 4, BC n = 5). Statistical method: Independent Samples Test. L,M) Treatment of KGN with the PKA antagonists STA and H89 in a 100 nm concentration led to a significant downregulation of KITL levels (CON n = 5, STA n = 4) (CON n = 5, H89 n = 4). Statistical method: Independent Samples Test. Data are presented as mean with SEM. In all panels, *p < 0.05, **p < 0.01, and ***p < 0.001. CON, control; CLEN, Clenbuterol; SAL, Salbutamol; BC, Bucladesine sodium; STA, Staurosporine; H89, H‐89 dihydrochloride; KITL, KIT ligand.

Figure 8

A schematic model depicting the mechanism of activation of ovarian primordial follicles induced by SD. SD leads to adrenal hyperactivity, resulting in the secretion of NE and E, which act on the ADRB2 of granulosa cells in primordial follicles of the ovary. This activation of ADRB2 triggers the cAMP/PKA and cAMP/mTOR signaling pathway in granulosa cells, leading to increased expression of KITL which initiating the activating of primordial follicles. Overactivation of primordial follicles accelerates the depletion of ovarian reserves and ultimately reduces the reproductive longevity. SD, sleep deprivation; NE, norepinephrine; E, epinephrine; ADRB2, β2‐adrenergic receptor.