PROJECT SUMMARY Stress disturbs many homeostatic functions, including reproduction. While previous studies have extensively examined the role of physical, psychosocial, and immunological stressors on reproduction, to date, the mechanisms remain unknown. Stress impacts multiple levels of the hypothalamic-pituitary-gonadal (HPG) axis, decreasing Gnrh expression and basal gonadotropin (luteinizing hormone) secretion. Recent advances in the field have allowed for an ultra-sensitive LH assay that will be used in the proposed Aims to examine changes in LH pulses induced by stress. The proposed Aims will examine if upstream neuroendocrine peptides regulate the reproductive axis during stress. Two neuropeptides of interest are RFRP-3 and kisspeptin. RFRP-3 is an inhibitory neuropeptide that inhibits production of the HPG axis via a central inhibition of GnRH neurons and thus decreased LH levels. By contrast, kisspeptin is a stimulatory neuropeptide that functions as the pulse generator to increase GnRH output. Together, RFRP-3 and kisspeptin are important regulators of hypothalamic GnRH neurons and may be potential targets during stress. To determine if RFRP-3 and kisspeptin regulate reproduction during stress, it is necessary to examine both males and females. The stress response is highly sexually dimorphic, with females being more sensitive than males. To date, there are no studies that examine sex differences in stress and reproduction. Therefore, the objective of this project is to examine the role of restraint stress in mediating reproduction, namely through RFRP-3 and kisspeptin neurons and LH pulsatility, in both males and females. Aim 1 examines if there is a sex difference in the effects of restraint stress on RFRP-3 and kisspeptin neurons and LH pulses. Aim 2 explores if RFRP-3 neurons are necessary to inhibit reproduction during restraint stress. As previously stated, RFRP-3 neurons are inhibitory to the HPG axis. Thus, this Aim will determine if ablation of an inhibitory signal will be important in mediating stress inhibition of reproduction as well. Aim 3 will identify if stress functions through glucocorticoid receptor (GR) localized on RFRP-3 or kisspeptin neurons. This Aim will provide a potential mechanism if selective ablation of GR on RFRP-3 or kisspeptin neurons abolishes the inhibition of reproduction by stress. The crosstalk of stress and reproduction is critical in identifying mechanisms of reproductive problems associated with stress including metabolic disease (obesity, anorexia nervosa), infertility during in vitro fertilization treatment, and additional reproductive problems such as hypogonadotropic hypogonadism and polycystic ovarian syndrome. Furthermore, identifying if sex differences exist in stress-induced inhibition of reproduction is critical to future experiments as well as treatment options in humans. Overall, this work is critical to understand and improving our understanding of reproductive physiology and its disruption by stress.

PROJECT NARRATIVE It is well appreciated that stress inhibits reproduction through the hypothalamic pituitary gonadal (HPG) axis, including actions in the brain, though the molecular and physiological mechanisms underlying this inhibition are largely unknown. This proposal will examine the role of RFRP-3 and kisspeptin neurons, which regulate gonadotropin releasing hormone (GnRH) neurons, in mediating the effects of psychosocial stress on reproduction in both sexes. This work may provide important novel information relevant to treatments of infertility, anovulation, amenorrhea, and idiopathic hypogonadotropic hypogonadism, in addition to offering new insight into the interplay between stress and reproductive function.