Sex-specific behavioral and physiological changes during single parenting in a biparental species, Columba livia.

Many species exhibit biparental care to maximize fitness. When a partner is lost, the surviving partner may alter their behavior to compensate offspring. Whether both sexes use the same physiological mechanisms to manifest their change in behavior remains elusive. We investigated behaviors and mechanisms associated with the alteration of parental care post-partner removal in a biparental avian species, the rock dove (Columba livia). We hypothesized that rock dove single parents experience sex-biased changes in neural genomic transcription and reproductive behaviors, and these changes are related to chick development. We manipulated parental partner presence and measured parental attendance, offspring growth, gene expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in the pituitary, and GR, MR, and estrogen receptor beta (ER-ß) in the hypothalamus. We also measured circulating plasma concentrations of the stress-associated hormone corticosterone and the parental care-associated hormone prolactin. We also quantified prolactin gene (PRL) expression changes in the pituitary, as well as prolactin receptor (PRLR) expression in the hypothalamus and pituitary. We found that single mothers and fathers maintained similar provisioning levels as paired parents, but spent less cumulative time brooding chicks. Chicks of single parents were smaller than paired-parented chicks after three days post-hatch. Mothers in both treatment groups experienced higher expression of hypothalamic GR as compared to fathers. Single parents experienced lower PRL gene expression in the pituitary as compared to paired parents. No significant differences were found for the circulating hormones or other genes listed.

Prolactin and prolactin receptor expression in the HPG axis and crop during parental care in both sexes of a biparental bird (Columba livia).

During breeding, multiple circulating hormones, including prolactin, facilitate reproductive transitions in species that exhibit parental care. Prolactin underlies parental behaviors and related physiological changes across many vertebrates, including birds and mammals. While circulating prolactin levels often fluctuate across breeding, less is known about how relevant target tissues vary in their prolactin responsiveness via prolactin receptor (PRLR) expression. Recent studies have also investigated prolactin (PRL) gene expression outside of the pituitary (i.e., extra-pituitary PRL), but how PRL gene expression varies during parental care in non-pituitary tissue (e.g., hypothalamus, gonads) remains largely unknown. Further, it is unclear if and how tissue-specific PRL and PRLR vary between the sexes during biparental care. To address this, we measured PRL and PRLR gene expression in tissues relevant to parental care, the endocrine reproductive hypothalamic-pituitary- gonadal (HPG) axis and the crop (a tissue with a similar function as the mammalian mammary gland), across various reproductive stages in both sexes of a biparental bird, the rock dove (Columba livia). We also assessed how these genes responded to changes in offspring presence by adding chicks mid-incubation, simulating an early hatch when prolactin levels were still moderately low. We found that pituitary PRL expression showed similar increases as plasma prolactin levels, and detected extra-pituitary PRL in the hypothalamus, gonads and crop. Hypothalamic and gonadal PRLR expression also changed as birds began incubation. Crop PRLR expression correlated with plasma prolactin, peaking when chicks hatched. In response to replacing eggs with a novel chick mid-incubation, hypothalamic and gonadal PRL and PRLR gene expression differed significantly compared to mid-incubation controls, even when plasma prolactin levels did not differ. We also found sex differences in PRL and PRLR that suggest gene expression may allow males to compensate for lower levels in prolactin by upregulating PRLR in all tissues. Overall, this study advances our understanding of how tissue-specific changes in responsiveness to parental hormones may differ across key reproductive transitions, in response to offspring cues, and between the sexes.

Uncovering the Sex-Specific Endocrine Responses to Reproduction and Parental Care.

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.

Isolating the Role of Corticosterone in the Hypothalamic-Pituitary-Gonadal Transcriptomic Stress Response.

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.

Widespread patterns of sexually dimorphic gene expression in an avian hypothalamic-pituitary-gonadal (HPG) axis.

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.