The hypothalamic-pituitary-gonadal (HPG) axis is a key biological system required for reproduction and associated sexual behaviors to occur. In the avian reproductive model of the rock dove (Columba livia), we characterized the transcript community of each tissue of the HPG axis in both sexes, thereby significantly expanding our mechanistic insight into HPG activity. We report greater sex-biased differential expression in the pituitary as compared to the hypothalamus, with multiple genes more highly expressed in the male pituitary being related to secretory function, and multiple genes more highly expressed in the female pituitary being related to reproduction, growth, and development. We report tissue-specific and sex-biased expression in genes commonly investigated when studying reproduction, highlighting the need for sex parity in future studies. In addition, we uncover new targets of investigation in both sexes, which could potentially change our understanding of HPG function.

35The hypothalamic-pituitary-gonadal (HPG) axis is a key biological system required for 36 reproduction and associated sexual behaviors to occur. In the avian reproductive model of the 37 rock dove (Columba livia), we characterized the transcript community of each tissue of the HPG 38 axis in both sexes, thereby significantly expanding our mechanistic insight into HPG activity. We 39 report greater sex-biased differential expression in the pituitary as compared to the 40 hypothalamus, with multiple genes more highly expressed in the male pituitary being related to 41 secretory function, and multiple genes more highly expressed in the female pituitary being 42 related to reproduction, growth, and development. We report tissue-specific and sex-biased 43 expression in genes commonly investigated when studying reproduction, highlighting the need 44 for sex parity in future studies. In addition, we uncover new targets of investigation in both 45 sexes, which could potentially change our understanding of HPG function. 46 47 INTRODUCTION 48

Investigation of the negative impacts of stress on reproduction has largely centered around the effects of the adrenal steroid hormone, corticosterone (CORT), and its influence on a system of tissues vital for reproduction—the hypothalamus of the brain, the pituitary gland, and the gonads (the HPG axis). Research on the action of CORT on the HPG axis has predominated the stress and reproductive biology literature, potentially overshadowing other influential mediators. To gain a more complete understanding of how elevated CORT affects transcriptomic activity of the HPG axis, we experimentally examined its role in male and female rock doves (Columba livia). We exogenously administrated CORT to mimic circulating levels during the stress response, specifically 30 min of restraint stress, an experimental paradigm known to increase circulating CORT in vertebrates. We examined all changes in transcription within each level of the HPG axis as compared to both restraint-stressed birds and vehicle-injected controls. We also investigated the differential transcriptomic response to CORT and restraint-stress in each sex. We report causal and sex-specific effects of CORT on the HPG transcriptomic stress response. Restraint stress caused 1567 genes to uniquely differentially express while elevated circulating CORT was responsible for the differential expression of 304 genes. Only 108 genes in females and 8 in males differentially expressed in subjects that underwent restraint stress and those who were given exogenous CORT. In response to elevated CORT and restraint-stress, both sexes shared the differential expression of 5 genes, KCNJ5, CISH, PTGER3, CEBPD, and ZBTB16, all located in the pituitary. The known functions of these genes suggest potential influence of elevated CORT on immune function and prolactin synthesis. Gene expression unique to each sex indicated that elevated CORT affected more gene transcription in females than males (78 genes versus 3 genes, respectively). To our knowledge, this is the first study to isolate the role of CORT in HPG genomic transcription during a stress response. We present an extensive and openly accessible view of the role corticosterone in the HPG transcriptomic stress response. Because the HPG system is well conserved across vertebrates, these data have the potential to inspire new therapeutic strategies for reproductive dysregulation in multiple vertebrate systems, including our own.

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Hormones mediate physiological and behavioral changes in adults as they transition into reproduction. In this study, we characterize the circulating levels of five key hormones involved in reproduction in rock doves (Columba livia): corticosterone, progesterone, estradiol, testosterone, and prolactin using univariate and multivariate approaches. We show similar patterns as previous studies in the overall patterns in circulating levels of these hormones, i.e., testosterone (males) and estradiol (females) high during nest-building or egg-laying, prolactin increasing at mid-incubation and peaking at hatching (both sexes), and elevated corticosterone levels in later incubation and early nestling development. In our investigation of hormone co-variation, we find a strong correlation between prolactin and corticosterone across sampling stages and similarities in earlier (early to mid-incubation) compared to later (late incubation to nestling d9) sampling stages in males and females. Finally, we utilized experimental manipulations to simulate nest loss or altered caregiving lengths to test whether external cues, internal timing, or a combination of these factors contributed most to hormone variation. Following nest loss, we found that both males and females responded to the external cue. Males generally responded quickly following nest loss by increasing circulating testosterone, but this response was muted when nest loss occurred early in reproduction. Similar treatment type, e.g., removal of eggs, clustered similarly in hormone space. These results suggest internal drivers limited male response early in reproduction to nest loss. In contrast, circulating levels of these hormones in females either did not change or decreased following nest manipulation suggesting responsiveness to external drivers, but unlike males, this result suggests that reproductive processes were decreasing.