The effect of norepinephrine on ovarian dysfunction by mediating ferroptosis in mice model

Abstract

Studies have shown that stress is associated with ovarian dysfunction. Norepinephrine (NE), a classic stress hormone involved in the stress response, is less recognized for its role in ovarian function. In this study, an NE-treated mouse model is induced by intraperitoneal injection of NE for 4 weeks. Compared with normal control mice, NE-treated mice show disturbances in the estrous cycle, decreased levels of anti-Mullerian hormone (AMH) and estradiol (E2), and increased level of follicle-stimulating hormone (FSH). Additionally, the numbers of primordial follicles, primary follicles, secondary follicles, and antral follicles are decreased, whereas the number of atretic follicles is increased in NE-treated mice, indicating NE-induced ovarian dysfunction. RNA sequencing further reveals that genes associated with ferroptosis are significantly enriched in NE-treated ovarian tissues. Concurrently, the levels of reactive oxygen species (ROS), ferrous ions, and malondialdehyde (MDA) are increased, whereas the expression level of glutathione peroxidase 4 (GPX4) is decreased. To elucidate the mechanism of NE-induced ferroptosis in ovaries and the potential reversal by Coenzyme Q10 (CoQ10), an antioxidant, we conduct both in vitro and in vivo experiments. In vitro, the granulosa cell line KGN, when treated with NE, shows decreased cell viability, reduced expression of GPX4, elevated levels of ferrous ion and ROS, and increased MDA level. However, these NE-induced changes are reversed by the addition of CoQ10. Compared with the NE group, the NE-treated mice supplemented with CoQ10 present increased GPX4 level and decreased iron, ROS, and MDA levels. Moreover, the differential expression of genes associated with ferroptosis induced by NE is ameliorated by CoQ10 in NE-treated mice. Additionally, CoQ10 improves ovarian function, as evidenced by increased ovarian weight, more regular estrous cycles, and an increase in follicles at various stages of growth in NE-treated mice. In conclusion, NE induces ovarian dysfunction by triggering ferroptosis in ovarian tissues, and CoQ10 represents a promising approach for protecting reproductive function by inhibiting ferroptosis.

1. Introduction

Premature ovarian insufficiency (POI) refers to a decline in ovarian function in women before the age of 40 and is characterized by elevated gonadotropin level and fluctuating low estrogen level. The global incidence is approximately 3.5% and is increasing [1–3]. This condition leads not only to infertility but also to severe health consequences, including hot flashes, depression, headaches, cognitive decline, vaginal atrophy, cardiac risks, and osteoporosis [1].

The etiology of POI is complex, with an increasing number of studies suggesting that stress plays a crucial role in human reproductive health. Clinical studies have demonstrated that stress can lead to abnormal reproductive function [4,5]. For example, a study evaluating 7352 women attending infertility clinics reported that 56% exhibited significant depressive symptoms and that 76% presented significant anxiety symptoms [6]. The effects of stress on reproductive function are multifaceted and include the inhibition of ovulation, a reduction in the fertilization rate, and the hindrance of fertilized egg implantation [7]. Recent research from our group revealed that POI patients experienced a greater incidence of adverse life events related to work stress, family stress, and sleep problems before diagnosis, suggesting that these patients might have been exposed to long-term chronic stress prior to illness onset [8]. Stress can activate the hypothalamic-pituitary-adrenal axis, leading to prolonged abnormal endocrine hormone levels in women, ultimately resulting in menstrual disorders, infertility, and other reproductive dysfunctions [9]. In animal experiments, chronic stress has been shown to disrupt the estrous cycle, alter hormone levels, and decrease follicle numbers in mice [10,11]. Stress can also induce cytoplasmic fragmentation, apoptosis, spindle disorders, and oxidative stress, accelerating the aging of mouse oocytes and increasing the risk of early pregnancy failure [12].

Norepinephrine (NE), a key catecholamine neurotransmitter involved in stress signaling, has been implicated in various psychological disorders resulted from stress, including cardiovascular disease, affective disorders, post-traumatic stress disorder, and cancer [13]. NE is involved in the regulation of ovarian functions. NE is linked to reactive oxygen species (ROS)-regulated events in ovarian physiology, including ovulation [14]. Increased NE content has been detected in the follicular fluid of patients with polycystic ovary syndrome [15]. Wang et al. [16] reported that glutamine and NE in follicular fluid synergistically enhance the antioxidant capacity of human granulosa cells and may pred