RF-amide related peptide neurons as modulators of stress-induced reproductive suppression
Reproduction is influenced by the physiological and environmental modulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neuronal network. GnRH hypothalamic activity is reflected in the systemic circulation by its downstream effector, luteinising hormone (LH). During stress, the GnRH-dependent LH activity that is required for adequate gonadal development and function is suppressed by glucocorticoids secreted by the adrenal glands. Emerging research in the field of stress-induced infertility show glucocorticoids do not directly inhibit GnRH activity and instead may act via the inhibitory RFamide-related peptide (RFRP) neurons, which suppress GnRH secretion. Thus far, this intermediary inhibitory role of RFRP neurons between the stress and reproductive axes is mainly supported by the upregulated expression of hypothalamic RFRP activity with stress in both sexes. Using a transgenic mouse model, the experiments in this thesis examine whether hypothalamic RFRP neuronal signalling is required for stress-induced infertility and whether increased RFRP activity is sufficient in mediating downstream suppression of LH in vivo. The chemogenetic approach of Cre-dependent DREADD (designer receptors exclusively activated by designer drugs) expression was used to selectively manipulate hypothalamic RFRP neuronal signalling via the administration of the DREADD agonist, clozapine-n-oxide (CNO). With the use of Fos-related antigens as the neuronal activity marker, immunohistochemical staining of hypothalamic brain sections confirmed significant suppression of RFRP activity in DREADD-silenced mice. Similarly, a significant upregulation of RFRP activity was confirmed in DREADD-activated mice in comparison to control mice. Additional data also showed that this increase in RFRP activity also caused a marked release of glucocorticoids in both sexes. To assess the requirement of RFRP neurons in stress-induced infertility, glucocorticoid-mediated inhibition of reproductive activity was achieved by long-term subcutaneously-placed corticosterone implants, with subsequent CNO-induced RFRP neuronal silencing. The suppression of LH pulse frequency and amplitude by glucocorticoids was observed for female control mice, but strikingly not for the RFRP-silenced mice. However, no significant effect of RFRP silencing was seen on pulsatile LH secretion in males. Similarly, hyperactivation of the RFRP neuronal population was sufficient to suppress LH secretion in females, but not in males. Attempts were also made to develop an in vivo model where the effect of RFRP neuronal signalling could be measured on the pre-ovulatory LH surges in females. The naturally occurring LH surge in intact female mice and the artificially-induced LH surge in gonadectomised mice were assessed, but an adequate control for glucocorticoid treatment could not be configured. These results showed a need for further optimisation before the effect of RFRP neuronal manipulation could be measured on glucocorticoid-induced inhibition of the LH surge. Nonetheless, in this thesis I have demonstrated using Cre-dependent DREADD technology, a novel, sex specific physiological necessity of RFRP neurons for stress-induced suppression of pulsatile reproductive hormone secretion. This testing of physiological properties of RFRP neurons may lead to clinical trials of RFRP antagonists, at least in females, to restore homeostasis in reproductive hormone secretion affected by stress related disorders.
Advisor: Anderon, Greg
Degree Name: Master of Science
Degree Discipline: Anatomy
Publisher: University of Otago
Keywords: RFRP; LH; Glucocorticoid; Stress; Fertility; Reproduction; Cre-dependent; DREAD
Research Type: Thesis