Mechanisms and mediators involved in regulating reproduction during stress in ewes

Stress suppresses a vast array of physiological systems including the reproductive system, and can have a profound inhibitory effect on neuroendocrine function and sexual behaviour. While several of the mediators involved have been identified, such as the glucocorticoids, the mechanisms and central pathways by which they act to inhibit reproduction during stress remain to be elucidated. This thesis set out to contribute to the key gaps in knowledge on how stress inhibits reproductive processes in females. This was accomplished by testing the unifying hypothesis that the mediators of the stress-induced inhibition of reproduction in the ewe include cortisol, neural inputs from the brain stem and gonadotropin-inhibitory hormone (GnIH). Three major studies were conducted to test this hypothesis in gonadectomised female sheep. In the first of the 3 studies, the actions of the glucocorticoid, cortisol, on sexual behaviour were measured. Stress-like levels of cortisol have been shown to inhibit aspects of sexual behaviour in ewes, primarily sexual receptivity. The mechanisms by which cortisol acts to inhibit this behaviour are unknown, however, it is plausible that cortisol interferes with the actions of oestradiol which are necessary to induce oestrus and the display of sexual receptivity. Two experiments were conducted to test this hypothesis in ovariectomised ewes artificially induced into oestrus by 12 days of treatment with progesterone followed by a single intramuscular injection of oestradiol benzoate (ODB). In the first experiment, vehicle (saline) or cortisol was infused for 40 h (chronic), beginning at the time of the ODB injection. Two doses of ODB were used, a normal physiological dose (25 µg) and a higher dose (50 µg), which is known to reduce the time to onset and extend the duration of oestrus in ewes. In the second experiment, an acute (5 h) dose of cortisol was infused around the time of the ODB injection (25 µg only) to determine whether acute rises in cortisol at the time of the oestradiol stimulus are sufficient to inhibit sexual receptivity. For both experiments, blood samples were collected and 3 well characterised sexual behaviours were measured; attractivity, proceptivity and receptivity. In both experiments, treatment with cortisol significantly elevated plasma concentrations of cortisol and inhibited the amplitude of luteinising hormone (LH) pulses. In addition, treatment with cortisol inhibited sexual receptivity in ewes, irrespective of the dose of ODB (Experiment 1). Importantly, the acute dose of cortisol (Experiment 2) had the same inhibitory effect on sexual receptivity. These results suggest that cortisol interferes with actions of oestradiol to induce sexual receptivity in ewes. It is clear from these studies that acute rises in plasma cortisol at the critical time of the oestradiol stimulus can hinder sexual receptivity and greatly reduce the chances of successful conception. Although cortisol robustly inhibits some aspects of reproduction, such as LH secretion and sexual behaviour, it has been shown that cortisol does not act centrally to regulate gonadotrophin-releasing hormone (GnRH) secretion during stress. Factors other than cortisol, therefore, must be involved in regulating GnRH secretion during stress. Little is known about the central pathways that are activated during stress and this thesis (Chapter 5) aimed to determine whether projections from the brain stem to the preoptic are (POA) activated during stress. It was hypothesised that catecholaminergic cells in the ventro-lateral medulla (A1) nucleus of the tractus solitaries (A2) and locus coeruleus (A6) regions of the brain stem that project to the POA are activated during psychosocial stress in ovariectomised ewes. The retrograde tracer dye fluorogold (FG) was injected into the POA and used to identify neuronal pathways that project to this region. Ewes were subjected to 90 min of psychosocial stress and the brains were immediately collected. Immunohistochemistry for FG and Fos identified that between 75% to 85% of all FG cells that projected to the POA were activated by stress. Furthermore, these cells were found to be catecholaminergic. These results suggest that catechomlamic cells in these brain stem nuclei project to the POA and could potentially relay inhibitory effects on GnRH neurones during stress. Finally, the role of RF-amide related peptide-3 (RFRP-3) in regulating reproduction during stress was investigated. Recently RFRP-3, the mammalian ortholog of GnIH, was suggested as an important regulator of LH secretion during stress. This neuropeptide was shown to inhibit LH secretion during restraint stress in male rats, however, whether RFRP-3 has the same role in female mammals has yet to be explored. The role of RFRP-3 in regulating stress-induced gonadotrophin secretion was investigated in ewes, in Chapter 6, by testing the hypothesis that the expression of RFRP mRNA and RFRP-3 peptide will be higher in ewes exposed to 90 min of isolation/restraint stress than in controls. Stress ewes were subjected to isolation/restraint stress while control ewes had no stress imposed. Blood samples were collected and subsequent to the stress, brains were collected. Immunohistochemistry for RFRP-3 and Fos showed no change in peptide levels between control and stress ewes. Similarly there was no change in RFRP mRNA levels, following in situ hybridisation, between control and stress ewes. In contrast, following isolation/restraint stress, plasma concentrations of cortisol were significantly higher and plasma concentrations of LH were significantly reduced in stress ewes than in controls. These results strongly suggest that RFRP-3 does not mediate stress-induced suppression of gonadotrophin secretion, at least in ewes following isolation/restraint stress. Overall, the findings from the studies presented in this thesis do not support the unifying hypothesis completely, however, they provide important insight into the mechanisms and mediators that may be involved in how stress inhibits reproductive processes in female sheep. Future research should focus on determining the mechanisms by which cortisol interferes with oestradiol to inhibit sexual receptivity and whether catecholaminergic cells in the brain stem synapse with, and influence the activity of, GnRH neurones in the POA. The role of RFRP-3 needs to be further addressed in female mammals although it appears that its role in regulating reproductive processes in the ewe during stress is negligible.