bioRxiv preprint doi: https://doi.org/10.1101/2020.10.08.330209; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Isolating the role of corticosterone in the hypothalamic-pituitary-gonadal 2 genomic stress response 3 4 By: Suzanne H. Austin1, *, Rayna Harris1, April M. Booth1, Andrew S. Lang2, Victoria S. Farrar1, 5 Jesse S. Krause1, 3, Tyler A. Hallman4, Matthew MacManes2, and Rebecca M. Calisi1 6 7 1Department of Neurobiology, Physiology, and Behavior, University of California Davis, 1 8 Shields Avenue, Davis, CA, USA, 95616 9 2 Department of Molecular, Cellular and Biomedical Sciences, The University of New 10 Hampshire, 434 Gregg Hall, Durham, NH, USA, 03824 11 3 Department of Biology, University of Nevada, Reno, 1664 N. Virginia Street, Reno, NV, USA, 12 89557-0314 13 4 Department of Fisheries and Wildlife, Oregon State University, 104 Nash Hall, Corvallis, OR 14 97331 15 16 *Current address: 17 Department of Integrative Biology, Oregon State University, 3029 Cordley Hall, Corvallis, OR, 18 USA, 97331; Department of Fisheries and Wildlife, Oregon State University, 104 Nash Hall, 19 Corvallis, OR 97331 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.08.330209; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 24 ABSTRACT: 25 The negative impacts of stress on reproduction have long been studied. A large focus of 26 investigation has centered around the effects of the adrenal steroid hormone corticosterone 27 (CORT) on a system of tissues vital for reproduction, the hypothalamus of the brain, the pituitary 28 gland, and the gonads (the HPG axis). Investigations of the role of CORT on the HPG axis have 29 predominated the stress and reproductive biology literature, potentially overshadowing other 30 influential mediators. To gain a more complete understanding of how elevated CORT, 31 characteristic of the stress response, affects the activity of the HPG axis, we experimentally 32 examined its role at the level of the genome in both male and female rock doves (Columba livia). 33 We exogenously administrated CORT to mimic circulating levels during the stress response, 34 specifically 30 min of restraint stress, an experimental paradigm known to increase circulating 35 corticosterone in vertebrates. We examined all changes in genomic transcription within the HPG 36 axis as compared to both restraint-stressed birds and vehicle-injected controls, as well as between 37 the sexes. We report causal and sex-specific effects of CORT on the HPG stress response at the 38 level of the transcriptome. Restraint stress caused 1567 genes to uniquely differentially express 39 while elevated circulating CORT was responsible for the differential expression of 304 genes. 40 Only 108 genes in females and 8 in males differentially expressed in subjects who underwent 41 restraint stress and those who were given exogenous CORT. In response to CORT elevation 42 characteristic of the stress response, both sexes shared the differential expression of 5 genes, 43 KCNJ5, CISH, PTGER3, CEBPD, and ZBTB16, all located in the pituitary. The known functions 44 of these genes suggest potential influence of elevated CORT on immune function and prolactin 45 synthesis. Gene expression unique to each sex indicated that elevated CORT affected more gene 46 transcription in females than males (78 genes versus 3 genes, respectively). To our knowledge, 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.08.330209; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 47 this is the first study to isolate the role of CORT in HPG genomic transcription during a stress 48 response. These results provide novel targets for new lines of further investigation and therapy 49 development. We present an extensive and openly accessible view of the role corticosterone in 50 the HPG genomic stress response, offering novel gene targets to inspire new lines of 51 investigation of stress-induced reproductive dysfunction. Because the HPG system is well- 52 conserved across vertebrates, these data have the potential to inspire new therapeutic strategies 53 for reproductive dysregulation in multiple vertebrate systems, including our own. 54 55 INTRODUCTION: 56 Unpredictable perturbations or perceived threats in the environment can activate various 57 endocrine cascades to promote behavioral and physiological coping mechanisms associated with 58 survival. The hypothalamic-pituitary-adrenal (HPA) axis plays a central role in mediating a 59 portion of these coping mechanisms (reviewed in Romero and Wingfield, 2015). Parvocellular 60 neurons in the hypothalamus of the brain release hormones such as arginine vasotocin (AVT; in 61 birds and lizards) and corticotropin releasing hormone (CRH) into the portal vasculature, which 62 transports them to the anterior pituitary gland. There, AVT and CRH activate corticotrope cells 63 to induce the release of adrenocorticotropic hormone (ACTH). ACTH travels through the 64 bloodstream to the adrenal glands and binds to melanocortin 2 receptors (MC2R), which 65 stimulates the synthesis of glucocorticoids [including corticosterone (hereafter, CORT) in adult 66 birds and cortisol in many mammals]. CORT travels via the bloodstream to bind to its receptors, 67 either nuclear or membrane bound, including the low affinity glucocorticoid receptor (GR) and 68 high affinity mineralocorticoid (MR) receptor. The binding of CORT to nuclear receptors affects 69 gene transcription throughout the body due to their global distribution (Romero and Wingfield, 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.08.330209; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 70 2015). Receptor activation modifies metabolism, immune function, and induces behavioral and 71 physiological changes (Sapolsky et al., 2000; Wingfield et al., 1998; Wingfield and Kitaysky, 72 2002). 73 When individual survival is favored over other energetically costly activities (Wingfield 74 et al., 1998), the chronic depletion of bodily resources can inhibit other important biological 75 processes (e.g., reproductive function, Sapolsky et al., 2000). The activation and regulation of 76 reproduction and associated behaviors is mediated by a biological system of tissues consisting of 77 the hypothalamus, pituitary, and gonads, referred to as the “HPG” axis. Its most well-studied 78 endocrine cascade involves production and secretion of gonadotropin-releasing hormone 79 (GnRH) from the preoptic area of the hypothalamus, which causes pituitary secretion of 80 gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH and FSH 81 travel through the bloodstream and act upon receptors in the gonads, stimulating gametogenesis 82 and the synthesis of sex steroids, such as testosterone and estradiol. These sex steroids then bind 83 with receptors within the HPG axis to facilitate reproduction and sexual behaviors, and they also 84 act as a negative feedback control mechanism. 85 It has long been known that exposure to certain stressors, and the subsequent 86 physiological response, can negatively impact sexual behavior and reproduction. The activation 87 of the HPA axis affects HPG function at multiple levels because MR and GR are globally 88 distributed and are expressed throughout the body (Angelier and Chastel, 2009; Geraghty and 89 Kaufer, 2015). Elevation of CORT, a significantly influential and well-studied hormone in the 90 stress response, can suppress the release of the reproductive hormone, GnRH, at the level of the 91 brain, by interacting with gonadotropin inhibitory hormone [GnIH; also referred to as RFamide- 92 related peptide (RFRP)], or with kisspeptin neurons (in mammals) (Calisi, 2014). This, in turn, 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.08.330209; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 93 reduces the rate of depolarization of the GnRH-1 neuron and subsequent GnRH-1 release, which 94 causes the reduction of LH, FSH, and steroidogenic capacity and gametogenesis. Thus, 95 activation of the HPA axis in response to environmental perturbations has the ability to affect 96 each endocrine-producing tissue of the HPG axis, and therefore, reproduction. 97 Research on the influential role of CORT on the HPG axis has predominated much of the 98 stress and reproductive biology literature, which has potentially overshadowed other influential 99 mediators of stress. In this study, we used the model of the rock dove (Columba livia) to 100 experimentally test the extent to which changes in gene expression within the HPG axis were 101 explained by an increase in circulating CORT – a key characteristic of a stress response. 102 Previously, our group described the genomic transcriptome community of sexually mature, non- 103 breeding male and female doves (MacManes et al., 2017). Then, we tested how gene 104 transcription within the HPG axis of both sexes was affected by activation of the HPA axis, the 105 latter which we stimulated using a common restraint stress paradigm in which subject mobility is 106 restricted for 30 min (Calisi et al., 2018).