During stress conditions, the activation of the HPA axis increases the plasmic content of glucocorticoids, which inhibit the synthesis and release of reproductive hormones [29, 30], leading to impaired follicular development and decreased ovulation in female animals [31]. LH is the key gonadotrophin that promotes ovulation. In the normal estrous cycle, the pituitary gland will secrete LH, which functions by binding to its receptor LHR in the ovary [32]. Studies have shown that when glucocorticoid exceeds the normal level, it will work through GR, which acts as a nuclear transcription factor to regulate the transcription of target genes. As the main component of glucocorticoids, CORT was recognized to negatively affect the ovulation process in our previous studies, although the underlying mechanism remains unclear. Here, we demonstrated that continuous intraperitoneal injection of CORT on mice downregulates LHR expression in ovarian GCs. In addition, our findings showed for the first time that CORT inhibits LHR expression by activating the GR-CREB/AP1 axis.
Impact of CORT on LHR expression
During the process of follicular dominance, the level of LHR is gradually increased. At the end of the follicular phase, the surge of LH,stimulates ovulation by binding to LHR in ovarian GCs [33]. The decrease of LHR expression in GCs causes ovulation failure due to insufficient LH stimulation [34, 35]. Although CORT has been reported to inhibit ovulation, it is still unclear whether CORT might directly regulate the expression of LHR in GCs. In this study, we demonstrated that continuous intraperitoneal injection of CORT into mice reduced LHR expression in GCs collected from developing follicles, especially the preovulatory follicles, indicating that CORT might impede ovulation in females by inhibiting LHR expression in GCs. On the other hand, studies have shown that stress-induced CORT accumulation can inhibit GnRH secretion and reduce LH levels [36]. It is conceivable that the downregulation of gonadotropins levels might affect GCs survival and proliferation during CORT stimulation. Interestingly, our results showed that CORT treatment itself could repress GCs.viability. The results thus suggested that CORT-induced unovulation might also be attributed to the developing failure of ovarian follicles caused by GCs damage.
LHR transcriptional regulation
The transcription of LHR is regulated by a variety of transcription factors [37, 38]. Previous studies have detected widespread distribution of AP1 in the follicular membrane and GCs within growing follicles and corpus luteum [39]. In the ovary, gonadotropins can induce a rapid and transient phosphorylation of the CREB protein in GCs, leading to the activation of transcription of many gonadotropin-regulated ovarian genes [40]. In the case of cell damage, AP1 cannot be activated, and the phosphorylation of CREB is also inhibited, resulting in the inability to activate gene transcription [41]. Considering that CORT might cause GCs damage as mentioned previously, we asked whether AP1 and CREB are involved in CORT-regulated LHR expression in GCs. Our in vivo and in vitro results both showed that CORT treatment reduced the levels of CREB, phosphorylated CREB (Ser133), c-Fos, and c-Jun in mouse ovarian GCs. In addition, the expression of LHR was suppressed when GCs were treated with inhibitors of AP1 and CREB, Therefore, our data suggested that the inhibitory effects of CORT on LHR expression might be dependent on the downregulation of AP1 and CREB in mouse ovarian GCs.
Effect of GR on LHR transcription
The feedback mechanism of hypothalamic-pituitary-adrenal axis involves the activation of glucocorticoids receptors (GR) by glucocorticoids [42]. GR belongs to the superfamily of ligand-induced transcription factors. After binding to corticosterone, these receptors are homodimerized or heterodimerized [43], and transferred to the nucleus to bind with other transcription factors or DNA response elements, leading to altered gene transcription [44–46]. However, there is no definitive evidence as to whether GR can specifically regulate LHR expression in GCs under stress conditions. Our in vivo and in vitro results showed that CORT activated GR expression in ovarian GCs, which was associated with decreased LHR expression. In contrast, inhibition of GR restored LHR expression in CORT-treated GCs. These data thus suggested that GR activation is required for CORT-repressed LHR expression.
GR can both positively or negatively regulate gene transcription by affecting the functions of transcription factors [47]. Therefore, under stress conditions, the CORT-GR axis might act through certain transcription factors to regulate LHR expression. Our results showed that the activation of GR reduced the expression of c-Fos, c-Jun, and CREB. In contrast, the GR inhibitor RU486 can block CORT-induced down-regulation of c-Fos, c-Jun, and CREB. These findings thus further demonstrated that CORT-mediated transcription regulation of LHR might depend on GR-induced suppression of AP1 and CREB.