Reproduction is a fundamental stage in the life history of vertebrates. It has high costs: increased susceptibility to disease and resource limitations; but a high reward: the ability to produce successful offspring to carry genes to future generations. In order to minimize costs and maximize reproductive success, many vertebrates birth or hatch young during the times of greatest resource abundance. Breeding must be initiated well in advance of this time to allow for sufficient time to incubate offspring.

Vertebrates use cues from their environment to predict when times of high resource abundance will occur. In temperate zones, day lengths are appreciably different in each season but have low variance on a given day from year to year. As many vertebrates utilize resources that have seasonal cycles of abundance and occur at relatively the same time each year, day length would be an appropriate cue to predict such events. Vertebrates who can detect day lengths and who use changes in day length to appropriately initiate reproductive processes are called photoperiodic seasonal breeders. However, organisms also experience unpredictable environmental events, such as storms, increased predation, food shortage or other environmental stress, that make reproduction unfavorable. Reproductive success requires that they detect and respond to these unpredictable cues as well.

Thus seasonally breeding vertebrates must integrate a primary predictive cue (photoperiod) and supplementary cues to time reproduction appropriately. A system which allows for global changes in physiology and fine points of modulation within the system would be advantageous for such a response.

The hypothalamo-pituitary-gonad (HPG) axis regulates the endocrine control of reproduction. The HPG axis is "switched on" by stimulatory photoperiods in photoperiodic vertebrates. That is, when the organism experiences a stimulatory day length, neurosecretory cells in the hypothalamus of the brain produce and secrete gonadotropin releasing hormone (GnRH) into the external layer of the median eminence. Here, GnRH travels through the hypothalamo-hypophysial portal system into the pituitary gland. Inside the pituitary gland, GnRH binds to its receptor on gonadotroph cells, stimulating the production and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH and FSH travel through the bloodstream to their respective receptors on the testes and ovaries. In the gonads, LH and FSH stimulate the production of sex steroids and the maturation of gametes. Sex steroids, in turn, also stimulate reproductive behaviors in the brain and further stimulate or inhibit the production of GnRH, LH and FSH.

The discovery of gonadotropin inhibitory hormone (GnIH), a second hypothalamic peptide involved in reproduction, indicated that the HPG axis was more finely regulated than previously known. Hypothalamic GnIH inhibits the HPG at two levels: it inhibits the production and secretion of LH and FSH in the pituitary gland and it inhibits the activity of GnRH neurons directly. Furthermore, GnIH secretion is responsive both to endocrine correlates of day length and of stress. These responses indicate GnIH is a candidate for a "fine point modulation" in this system.

Recently, GnIH and GnIHR transcripts were identified in avian gonads. Furthermore, an experiment using systemic administration of GnIH inhibited gonadal function. The work of this dissertation was to identify the functional significance of a GnIH system in the gonads. Experiments using seasonally breeding songbirds and mammals identify the inhibitory function of GnIH on gonadal sex steroid production in males and females and the responsiveness of this system to supplementary cues. Finally, gonadal GnIH is presented in context with the gonadal GnRH system, to foster further research into the implications of gonadal neuropeptide systems. Thus gonadal GnIH is part of a complex system of reproductive regulation in vertebrates.

This dissertation critically investigates research in melatonin across species and disciplines. It offers a broader perspective within the field of reproductive neuroendocrinology by integrating research in pharmacology, genetics, and evolution. The first chapter of this dissertation reviews melatonin administration techniques across fields. The timing, dose, and mode of administration is compared across species. This juxtaposition of previous protocols using melatonin administration illustrates the importance of context to help inform future experimental designs. Furthermore, details of the experiment that affect the findings are emphasized to improve replicability of melatonin administration studies. The second chapter focuses on melatonin synthesizing enzymes in two different songbirds. The zebra finch (Taeniopygia guttata) is an opportunistic breeder in the wild and uses primarily food and water availability to time reproduction (Perfito et al., 2007). The European starling (Sturnus vulgaris) is a photoperiodic breeder whose reproductive state relies on changes in day length (Gwinner, 1977). Melatonin-synthesizing enzymes were expressed in photoreceptive sites of the brain. Key melatonin-synthesizing enzymes were differentially expressed in the hypothalamus of the photoperiodic breeder at certain times of the year, and this expression was not analogously observed in the opportunistic breeder. Melatonin may be a distinguishing physiological factor in reproductive timing between these two breeding types. The last chapter determines the physiological relevance of melatonin binding at the level of the gonad, specifically measuring how melatonin affects testosterone output from cultured testes from Syrian hamsters (Mesocricetus auratus), a photoperiodically-breeding mammal.

Reproduction is a critical component of an animal’s fitness, but it is also energetically intensive. In order to maximize reproductive fitness, organisms modulate their reproductive investment or timing in response to environmental cues that provide information about resource availability or other indicators of reproductive success. Physiologic systems that monitor these cues then influence both reproductive readiness and more scaled measures of investment, including number of offspring or offspring provisioning effort. This kind of physiologic modulation is especially salient to mammals, for whom reproduction (gestation and lactation) is exceptionally intensive, and there is extensive documentation of variation in reproductive effort and success under specific environmental and physiological challenges. Still, the underlying mechanisms that coordinate these shifts in investment remain poorly defined and understood.

This dissertation identifies novel endocrine and paracrine mechanisms that regulate ovarian and uterine function in the context of reproductive success. Chapter 1 summarizes our current understanding of female reproductive function and modulation, ultimately identifying 4 key areas of modulation for which the molecular mechanisms are poorly understood or defined: (1) evaluating autonomic nervous system regulation of reproductive organs in ecophysiological contexts, (2) identifying new signals regulating ovary and uterine function; (3) determining the extent to which reproductive stage (e.g., pregnancy) alters regulatory networks and sensitivity; and (4) scaling function of specific cell types or organs to organism-level reproductive outcomes. Chapters 2-5 address areas 2-4 by identifying new mechanisms of mammalian reproductive system control in the ovary and uterus and by evaluating the function of established signaling networks in other reproductive states.

Chapter 2 interrogates the direct sensitivity of the ovary to physiological cues using mouse ovarian explants. By using pharmacologic inhibition of glucose metabolism, these experiments identify novel glucose sensitivity in the ovary. Chapter 3 examines the effect of neuropeptide gonadotropin inhibitory hormone (GnIH) on ovarian follicle growth, survival, and steroidogenesis in the domestic cat. As has been shown in other mammals to-date, GnIH promotes follicle degradation in vitro. These findings support a conserved role for GnIH in vertebrate ovarian folliculogenesis. Together, these chapters provide new paracrine and endocrine mechanisms of ovarian regulation that adds to our molecular understanding of how reproductive effort and investment are modulated.

Chapter 4 explores the sensitivity of feline uterine endometrial epithelial cells to sex steroids. We demonstrate that sex steroids stimulate morphological changes to endometrial epithelial cell 3D growth in vitro, and that morphological changes are associated with functional changes in gene expression. Though often manipulated as part of normal fertility treatment, we do not have a good understanding of how these hormones facilitate uterine preparedness for implantation and pregnancy in felines. Chapter 4 thus identifies specific mechanisms of reproductive modulation in the feline uterus and also beings to flesh out molecular mechanisms underlying reproductive failure at the organism level. Our results have direct application to endangered felid breeding programs, which utilize in vitro follicle maturation and in vivo hormones treatments to promote breeding success of genetically-important individuals.

Finally, chapter 5 uses a classic stress physiology paradigm to identify the mechanism(s) by which psychological stress inhibits ovarian steroid synthesis during pregnancy in mice. These experiments demonstrate that stress-dependent inhibition of ovarian function does not occur through classical, hypothalamic inhibition, emphasizing the dynamic shifts in endocrine function across mammalian pregnancy.

These studies add to our comparative understanding of reproductive function, which is critical because of its fundamental connection to organismal fitness. Taken together, these studies also highlight the utility of in vitro approaches for experimentally approaching the challenge of connecting molecular variation to organismal function. We summarize the results and their ultimate importance to female reproductive physiology research in Chapter 6.

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide that typically acts in the hypothalamic-pituitary-gonadal (HPG) axis to inhibit reproductive activity and sociosexual behaviors. GnIH is synthesized in the brain and in the gonads, where it can act via its cognate receptor. However, immunohistological evidence in songbirds also shows GnIH projections towards the brainstem1. I propose that GnIH can act within the spinal cord and possibly on a variety of organs to induce rapid behavioral and physiological changes in response to environmental cues. I hypothesize that the hypothalamus directly innervates the gonads via the spinal cord to rapidly regulate steroid synthesis. I first used immunohistochemistry and PCR to provide a description of GnIH and its receptor in the zebra finch (Taeniopygia guttata) spinal cord. I then investigated potential roles and mechanisms of action of GnIH in the spinal cord of European starlings (Sturnus vulgaris) and zebra finches. I found that seasonal changes in daylength correlated with immunoreactive GnIH and its counterpart, gonadotropin-releasing hormone (GnRH), quantity in wild-caught European starling spinal cord. Food restriction and a sickness challenge caused significant behavioral changes but did not correlate with GnIH-immunoreactive quantity nor measured gene expression levels in zebra finches. These results hint at a novel pathway for neuropeptide action in vertebrates. The mechanisms of action of GnIH in the avian spinal cord are still unclear. However, these three chapters provide the first evidence of reproductive neuropeptides in the avian spinal cord and open exciting new paths for research.

Reproduction is a physiologically costly life history stage that varies widely in resource andbehavioral demands depending on the specific stage of courting, mating, and period of parental care. Seasonal shifts in environmental conditions influence animal energy budgets in ways which shape species’ life histories. Animals allocate energy and nutrients among a suite of competing physiological demands across short-, and long-term, timescales and the expression of specific traits or allocation strategy is tightly linked to the environment. Constraints on behavior and/or physiology have important implications for individual fitness and life history evolution, often leading to tradeoffs between processes supporting survival versus those supporting reproduction. In Chapter 1, I provide the first neuroanatomical description of RFRP (precursor peptide of gonadotropin-inhibitory hormone, GnIH) expression and localization in the brain of any bat using a widespread temperate species (Eptesicus fuscus, big brown bat) as a model. We show that RFRP transcripts occur in the hypothalamus, testes, and ovaries of big brown bats. Cellular RFRP immunoreactivity was observed across the hypothalamus, arcuate nucleus, and median eminence (ME), most similar to what is known in naked mole rats. RFRP fiber immunoreactivity was widespread as in other vertebrates. Putative interactions between RFRP-ir fibers and gonadotropinreleasing hormone cell bodies were observed in 16% of GnRH cells, suggesting potential for direct regulation of GnRH via RFRP signaling. This strengthens our fundamental knowledge of how the HPG axis may be regulated in bats and may now guide functional approaches to understanding how reproductive neurophysiology responds, or be well-adapted, to seasonal environmental cues and stressors. Animals face unpredictable challenges that require rapid, facultative physiological reactions to support survival but may compromise reproduction. Bats have a longstanding reputation for being highly sensitive to stressors, with sensitivity and resilience varying both within and among species. yet little is known about how stress affects the signaling that regulates reproductive physiology. Chapter 2 provides the first description of the molecular response of the hypothalamic-pituitary gonadal (HPG) axis of male big brown bats (E. fuscus) in response to short-term stress using a 2 standardized restraint manipulation. This acute stressor was sufficient to upregulate plasma corticosterone and resulted in a rapid decrease in circulating testosterone. While we did not find differences in the mRNA expression of key steroidogenic enzymes (StAR, aromatase, 5-alpha reductase), seminiferous tubule diameter was reduced in stressed bats coupled with a 5-fold increase in glucocorticoid receptor (GR) mRNA expression in the testes. These changes, in part, may be mediated by RFamide-related peptide (RFRP) because fewer immunoreactive cell bodies were detected in the brains of stressed bats compared to controls—suggesting a possible increase in secretion—and increased RFRP expression locally in the gonads. The rapid sensitivity of the bat testes to stress may be connected to deleterious impacts on tissue health and function as supported by significant transcriptional upregulation of key pro-apoptotic signaling molecules (Bax, cytochromec). Experiments like this broadly contribute to our understanding of the stronger ecological predictions regarding physiological responses of bats within the context of stress which may impact decisions surrounding animal handling and conservation approaches. Chapter 3 shifts from examining molecular mechanisms and neuroendocrinology regulating reproduction to a whole organism approach. Using a well-established captive colony of zebra finches, I characterize the behavioral and physiological conflicts between self-maintenance and reproductive investment and the factors that influence responses in line with energy limitation and/or trade-offs. Animals are faced with unpredictable challenges – such as pathogen exposure or infection – requiring quick physiological responses that support survival and may compromise resource allocation to reproduction. Stereotypical “sickness behaviors” may represent an energetic strategy that reduces investment in unnecessary activities to allocate resources toward – and support – the immune system. I use a framework of avian breeding stages to demonstrate the context-dependent behavioral conflict that may/may not be present across a species’ breeding period, and the importance of examining these conflicts through a holistic lens that takes species’ sociality and mating system into account as these variables directly affect routine individual energetic demands.

Stress is a known inhibitor of reproductive function and its associated behaviors. How and why stress acts to influence reproduction have been intensely studied and appear to be extremely varied, though we do not fully understand how these processes operate at the level of the brain. My dissertation work examined the role of the novel neurohormone gonadotropin inhibitory hormone (GnIH) in response to stress and social environment in reproductively active birds. To review the actions of GnIH in brief, GnIH inhibits the activity of gonadotropin releasing hormone (GnRH) neurons in the brain in addition to reducing the release of the gonadotropins luteinizing hormone and follicle-stimulating hormone from the pituitary gland. GnIH also reduces testosterone release from the gonads. Central administration of GnIH can decrease both copulation solicitations in birds and sexual behaviors in rodents. However, how GnIH fluctuates naturally in response to stress and social environment is not well understood. My questions were as follows:

1) Does capture-handling stress affect GnIH, and is there a seasonal effect?

2) Does social environment affect GnIH, and does this vary with breeding stage?

3) Is there a mechanism in place for the stress response to affect reproduction via GnIH? Specifically, are receptors for glucocorticoids expressed in avian GnIH cells? And what are the conservation implications of such neuroendocrine research?

My work is the first to demonstrate the effects of stress (in birds) and social environment (in any organism) on GnIH. My work is also the first to report the co-localization of glucocorticoid receptors in avian GnIH cells, providing a mechanism by which the stress response can influence reproduction at the level of the brain. Because GnIH presence and function are conserved throughout all vertebrates studied, these findings can create new avenues into studies of vertebrate stress and reproductive physiology.

When animals are suffering from an infection, they frequently exhibit symptoms such as reductions in activity, reductions in food and water intake, reductions in libido and in social interactions. Adoption of these "sickness behaviors" is thought to promote immune function by reducing energy expenditure in activities that are not essential for recovery from the infection and investing this energy in mounting an immune response. In other words, during disease, since the body has limited resources, these need to be traded-off between investment in immunity and investment in other activities. My dissertation work was focused on exploiting this trade-off idea by examining how different social contexts affect the expression of sickness behaviors in birds. Social modulation of sickness behaviors should be especially relevant when animals have an opportunity to reproduce. Hence, my work focused as well on how immune challenges affect the reproductive system and how the social environment can determine the extent to which animals invest in reproduction while sick. Finally, it was my purpose to understand whether alterations in sickness behaviors due to social context impact the immune response in ways that are costly for animals.

The work in this dissertation emphasizes the plasticity of the sickness behavior response. Here, I demonstrate that birds are able to adjust the expression of sickness behaviors when subjected to social circumstances that promote other adaptive opportunities. While the reproductive system is extensively shut down during an immune challenge, I demonstrate that this effect is reversed within 30 minutes of presentation of a potential mate. In addition, my work indicates that the social modulation of sickness behaviors comes at the cost of reduced immune defenses. In a world where infectious diseases represent one of the major causes of death, an increased understanding of the way behavior during infection is impacted by the social environment and the costs this might carry might promote better guidelines on how to proceed with infected animals (including humans). As well, a deeper knowledge of the endocrine and immune factors mediating this response has the potential to lead to better tools to treat infections. On the other hand, the results in here alert for the reality that our ability to detect sick animals might be obscured by social context, reducing our chances of controlling the spread of infectious diseases (such as the avian flu). With the added knowledge from this work, I expect that sickness behavior might be used as a new tool for learning about motivation underlying social behaviors.