Stress increases putative gonadotropin inhibitory hormone and decreases luteinizing hormone in male rats

Abstract

The subjective experience of stress leads to reproductive dysfunction in many species, including rodents and humans. Stress effects on reproduction result from multilevel interactions between the hormonal stress response system, i.e., the hypothalamic-pituitary-adrenal (HPA) axis, and the hormonal reproductive system, i.e., the hypothalamic-pituitary-gonadal (HPG) axis. A novel negative regulator of the HPG axis known as gonadotropin-inhibitory hormone (GnIH) was recently discovered in quail, and orthologous neuropeptides known as RFamide-related peptides (RFRPs) have also been identified in rodents and primates. It is currently unknown, however, whether GnIH/RFRPs influence HPG axis activity in response to stress. We show here that both acute and chronic immobilization stress lead to an up-regulation of RFRP expression in the dorsomedial hypothalamus (DMH) of adult male rats and that this increase in RFRP is associated with inhibition of downstream HPG activity. We also show that adrenalectomy blocks the stress-induced increase in RFRP expression. Immunohistochemistry revealed that 53% of RFRP cells express receptors for glucocorticoids (GCs), indicating that adrenal GCs can mediate the stress effect through direct action on RFRP cells. It is thought that stress effects on central control of reproduction are largely mediated by direct or indirect effects on GnRH-secreting neurons. Our data show that stress-induced increases in adrenal GCs cause an increase in RFRP that contributes to hypothalamic suppression of reproductive function. This novel insight into HPA-HPG interaction provides a paradigm shift for work on stress-related reproductive dysfunction and infertility, and indicates that future work on stress and reproductive system interactions must include investigation of the role of GnIH/RFRP.

Fig. 1.

Acute stress effects on RFRP expression. (A) Experimental time line. (B) Stressed rats showed higher RFRP mRNA expression levels (mean ± SEM) than controls immediately after stress (P = 0.002). (C) Stressed rats showed more RFRP peptide positive cells (mean ± SEM) than controls immediately after stress (P = 0.006). (D) Representative images of RFRP mRNA positive cells in the DMH. (E) Representative images of RFRP-ir cells in the DMH. (F) There was a significant negative correlation between fold change in RFRP mRNA and plasma-luteinizing hormone (r = −0.920, P = 0.003). *P < 0.05 compared with no-stress and acute stress, 24-hour-delay controls combined. °P < 0.05 correlation. Scale bar, 40 μM.

Fig. 2.

Chronic stress effects on RFRP expression. (A) Experimental time line. (B) Gene expression changes in the hypothalamus (solid bars), pituitary (lined bar), and testes (cross-hatched bars) after chronic immobilization, normalized to no-stress control levels. Immobilization led to an increase in hypothalamic RFRP mRNA expression (mean ± SEM, P = 0.007). No change was seen in hypothalamic, pituitary, or testicular RFRP receptor (OT7T022) or testicular RFRP expression (all P > 0.10). (C) Chronic immobilization led to an increase in hypothalamic RFRP-ir cell number in the DMH (mean ± SEM, P = 0.041). (D) Immobilization decreased luteinizing hormone (LH) on the last day of the stressor. Plasma LH levels after stress were significantly lower than those at baseline on day 14 (P = 0.023). *P < 0.05 no stress versus chronic stress or effect of time immobilized within day, as appropriate. ^†P < 0.10 effect of time immobilized within day. Scale bar, 40 μM.

Fig. 3.

HPA responsivity of RFRP cells. (A) Three hours of immobilization led to a rapid increase in plasma corticosterone (mean ± SEM; P = 0.001). (B) Poststress corticosterone difference from baseline. Daily immobilization led to an increase in plasma corticosterone (P < 0.001 effect of time immobilized within day; P < 0.001 time by day interaction). Immobilization led to a significant increase in corticosterone (mean difference from day 1 baseline) on days 1 (P = 0.022), 3 (P < 0.001), and 14 (P = 0.030). The increase on day 7 was not significant (P = 0.054). (C) Percentage of RFRP cells co-expressing CRH-R1 and GR. (D) An RFRP-ir cell (red, first panel) co-expressing CRH-R1 (blue, second panel) in the adult rat DMH. (E) An RFRP-ir cell (red, first panel) co-expressing GR (blue, second panel). *P < 0.05 effect of immobilization over baseline day 1. ^†P < 0.10 effect of time immobilized over baseline day 1. Scale bar, 20 μM.

Fig. 4.

Adrenalectomy prevents stress-induced increase in hypothalamic RFRP expression. (A) Stress led to a significant increase in plasma corticosterone in sham-operated solid line, (P = 0.001) but not ADX rats dashed line, (P = 0.891). (B) Two weeks of daily immobilization led to a significant increase in RFRP mRNA expression in the hypothalamus of sham operated (P = 0.050) but not ADX (P = 0.794) rats. Open bar = control; closed bar = stress. * P < 0.05 stress versus no stress or 0 hours versus 3 hours of stress as appropriate.

Fig. 5.

Model of how stress may affect the HPG axis through GnIH. (A) Previously, it was known that both GCs and GnIH inhibited the HPG axis independently. These interactions are shown in blue. (B) We propose that GCs released in response to stress act on GnIH via GR to increase the inhibitory actions of GnIH on GnRH secretion and/or pituitary sensitivity, resulting in decreased LH release. Our proposed pathway of GC–GnIH interaction is represented in red, joining the previously established independent effects of GCs and GnIH on reproduction. Arrows represent stimulation; Ts represent inhibition. CNS, central nervous system; PNS, peripheral nervous system.