Evolution of Litter Size: Proximate and Ultimate Mechanisms.

Relative reproductive success and failure are the ultimate determinants of Darwinian fitness. As such, reproductive traits and variation therein have an immediate and considerable impact on the evolutionary trajectory of lineages. Historically, significant attention has been paid to the ecological and evolutionary processes (ultimate factors) that shape the diversity and canalization of reproductive traits within groups to better our understanding of organismal diversity and population or species resilience. In contrast, the physiological systems that mediate variation within and among species (i.e., the proximate factors) in reproductive traits remain a significant black box. To-date, there is comparatively little information about how proximate mechanisms constrain or promote evolutionary potential in reproductive traits. In this mini-review, we focus on litter size in Eutherian mammals as a trait with relatively well-defined diversity (litter sizes are well-described both within and across species) and for which some genetic determinants have been identified. We discuss both the ultimate and potential proximate determinants of litter size with special attention to the breadth of physiological traits that may act as "toggle" switches for evolution of litter size. We close with a brief discussion of the role that physiological plasticity may play in the evolution of litter size and lay out several forward-looking areas for future research.

Adaptive structural and functional evolution of the placenta protects fetal growth in high-elevation deer mice.

Environmental hypoxia challenges female reproductive physiology in placental mammals, increasing rates of gestational complications. Adaptation to high elevation has limited many of these effects in humans and other mammals, offering potential insight into the developmental processes that lead to and protect against hypoxia-related gestational complications. However, our understanding of these adaptations has been hampered by a lack of experimental work linking the functional, regulatory, and genetic underpinnings of gestational development in locally adapted populations. Here, we dissect high-elevation adaptation in the reproductive physiology of deer mice (Peromyscus maniculatus), a rodent species with an exceptionally broad elevational distribution that has emerged as a model for hypoxia adaptation. Using experimental acclimations, we show that lowland mice experience pronounced fetal growth restriction when challenged with gestational hypoxia, while highland mice maintain normal growth by expanding the compartment of the placenta that facilitates nutrient and gas exchange between gestational parent and fetus. We then use compartment-specific transcriptome analyses to show that adaptive structural remodeling of the placenta is coincident with widespread changes in gene expression within this same compartment. Genes associated with fetal growth in deer mice significantly overlap with genes involved in human placental development, pointing to conserved or convergent pathways underlying these processes. Finally, we overlay our results with genetic data from natural populations to identify candidate genes and genomic features that contribute to these placental adaptations. Collectively, these experiments advance our understanding of adaptation to hypoxic environments by revealing physiological and genetic mechanisms that shape fetal growth trajectories under maternal hypoxia.

Evolution in reproductive tempo and investment across the Peromyscus radiation.

Mammals display diverse reproductive strategies, however, the ultimate and proximate mechanisms that underlie this diversity and its composite traits remain poorly understood from both evolutionary and physiological perspectives. The Peromyscus genus of rodents, which is found throughout the north and central Americas, has diversified along life history gradients, varying both within and among species in reproductive strategies. This variation provides a useful model for studying reproductive diversity. Here, we combine a literature review with new analyses of captive colony breeding records from six Peromyscus species to assess our current understanding of how plasticity and local adaptation contribute to diversity in two classes of reproductive traits: phenology and litter investment. There is substantial evidence that many traits underlying phenology and litter investment have diverged among populations in ways that are likely to be locally adaptive, though plasticity in these traits remains common. However, these conclusions are largely based on data collected from the two most widespread Peromyscus species: P. maniculatus and P. leucopus. The majority of Peromyscus species diversity remains understudied regarding reproductive phenology and litter traits. We conclude by discussing key challenges and considerations relevant to using Peromyscus as a mammalian model for reproductive trait diversity and evolution moving forward.

Impact of Chronic Prenatal Stress on Maternal Neuroendocrine Function and Embryo and Placenta Development During Early-to-Mid-Pregnancy in Mice.

Psychological stress, both leading up to and during pregnancy, is associated with increased risk for negative pregnancy outcomes. Although the neuroendocrine circuits that link the stress response to reduced sexual motivation and mating are well-described, the specific pathways by which stress negatively impacts gestational outcomes remain unclear. Using a mouse model of chronic psychological stress during pregnancy, we investigated 1) how chronic exposure to stress during gestation impacts maternal reproductive neuroendocrine circuitry, and 2) whether stress alters developmental outcomes for the fetus or placenta by mid-pregnancy. Focusing on the stress-responsive neuropeptide RFRP-3, we identified novel contacts between RFRP-3-immunoreactive (RFRP-3-ir) cells and tuberoinfundibular dopaminergic neurons in the arcuate nucleus, thus providing a potential pathway linking the neuroendocrine stress response directly to pituitary prolactin production and release. However, neither of these cell populations nor circulating levels of pituitary hormones were affected by chronic stress. Conversely, circulating levels of steroid hormones relevant to gestational outcomes (progesterone and corticosterone) were altered in chronically-stressed dams across gestation, and those dams were qualitatively more likely to experience delays in fetal development. Together, these findings suggest that, up until at least mid-pregnancy, mothers appear to be relatively resilient to the effects of elevated glucocorticoids on reproductive neuroendocrine system function. We conclude that understanding how chronic psychological stress impacts reproductive outcomes will require understanding individual susceptibility and identifying reliable neuroendocrine changes resulting from gestational stress.

Sex-Differences in Phenology: A Tinbergian Perspective.

Shifts in the timing of cyclic seasonal life-history events are among the most commonly reported responses to climate change, with differences in response rates among interacting species leading to phenological mismatches. Within a species, however, males and females can also exhibit differential sensitivity to environmental cues and may, therefore, differ in their responsiveness to climate change, potentially leading to phenological mismatches between the sexes. This occurs because males differ from females in when and how energy is allocated to reproduction, resulting in marked sex-differences in life-history timing across the annual cycle. In this review, we take a Tinbergian perspective and examine sex-differences in timing of vertebrates from adaptive, ontogenetic, mechanistic, and phylogenetic viewpoints with the goal of informing and motivating more integrative research on sexually dimorphic phenologies. We argue that sexual and natural selection lead to sex-differences in life-history timing and that understanding the ecological and evolutionary drivers of these differences is critical for connecting climate-driven phenological shifts to population resilience. Ontogeny may influence how and when sex-differences in life-history timing arise because the early-life environment can profoundly affect developmental trajectory, rates of reproductive maturation, and seasonal timing. The molecular mechanisms underlying these organismal traits are relevant to identifying the diversity and genetic basis of population- and species-level responses to climate change, and promisingly, the molecular basis of phenology is becoming increasingly well-understood. However, because most studies focus on a single sex, the causes of sex-differences in phenology critical to population resilience often remain unclear. New sequencing tools and analyses informed by phylogeny may help generate hypotheses about mechanism as well as insight into the general "evolvability" of sex-differences across phylogenetic scales, especially as trait and genome resources grow. We recommend that greater attention be placed on determining sex-differences in timing mechanisms and monitoring climate change responses in both sexes, and we discuss how new tools may provide key insights into sex-differences in phenology from all four Tinbergian domains.

A push for inclusive data collection in STEM organizations.

Professional societies could better survey, and thus better serve, underrepresented groups.

Fetal growth, high altitude, and evolutionary adaptation: a new perspective.

Residence at high altitude is consistently associated with low birthweight among placental mammals. This reduction in birthweight influences long-term health trajectories for both the offspring and mother. However, the physiological processes that contribute to fetal growth restriction at altitude are still poorly understood, and thus our ability to safely intervene remains limited. One approach to identify the factors that mitigate altitude-dependent fetal growth restriction is to study populations that are protected from fetal growth restriction through evolutionary adaptations (e.g., high altitude-adapted populations). Here, we examine human gestational physiology at high altitude from a novel evolutionary perspective that focuses on patterns of physiological plasticity, allowing us to identify 1) the contribution of specific physiological systems to fetal growth restriction and 2) the mechanisms that confer protection in highland-adapted populations. Using this perspective, our review highlights two general findings: first, that the beneficial value of plasticity in maternal physiology is often dependent on factors more proximate to the fetus; and second, that our ability to understand the contributions of these proximate factors is currently limited by thin data from altitude-adapted populations. Expanding the comparative scope of studies on gestational physiology at high altitude and integrating studies of both maternal and fetal physiology are needed to clarify the mechanisms by which physiological responses to altitude contribute to fetal growth outcomes. The relevance of these questions to clinical, agricultural, and basic research combined with the breadth of the unknown highlight gestational physiology at high altitude as an exciting niche for continued work.

Flexibility in an emergency life-history stage: acute food deprivation prevents sickness behaviour but not the immune response.

The emergency life-history stage (ELHS) can be divided into two subcategories that describe distinct, coordinated responses to disease- or non-disease-related physiological challenges. Whether an individual can simultaneously express aspects of both subcategories when faced with multiple challenges is poorly understood. Emergency life-history theory suggests that disease- and non-disease-related responses are coordinated at the level of the whole organism and therefore cannot be expressed simultaneously. However, the reactive scope and physiological regulatory network models suggest that traits can be independently regulated, allowing for components of both disease- and non-disease-related responses to be simultaneously expressed within a single organism. To test these ideas experimentally, we subjected female zebra finches to food deprivation, an immune challenge, both, or neither, and measured a suite of behavioural and physiological traits involved in the ELHS. We examined whether the trait values expressed by birds experiencing simultaneous challenges resembled trait values of birds experiencing a single challenge or if birds could express a mixture of trait values concurrently. We find that birds can respond to simultaneous challenges by regulating components of the behavioural and immune responses independently of one another. Modularity within these physio-behavioural networks adds additional dimensions to how we evaluate the intensity or quality of an ELHS. Whether modularity provides fitness advantages or costs in nature remains to be determined.

Food access modifies GnIH, but not CRH, cell number in the hypothalamus in a female songbird.

Food deprivation or restriction causes animals to mount a stereotypical behavioral and physiological response that involves overall increases in activity, elevated glucocorticoid production, and (often) inhibition of the reproductive system. Although there is increasing evidence that these responses can differ in their degree or covariation between the sexes, most studies to-date on food restriction/deprivation have focused on male songbirds. We therefore aimed to characterize the behavioral, physiological, and neuroendocrine response to acute food deprivation in a female songbird using a nomadic species, the zebra finch. We quantified behavior during a 6.5 h food deprivation and then measured physiological and neuroendocrine responses of female birds at the 6.5 h timepoint. Within 1 h of acute food deprivation, female zebra finches increased foraging behaviors, and after 6.5 h of food deprivation, females lost 5% of their body mass, on average. Change in body mass was positively associated with elevated corticosterone and (contrary to findings in male zebra finches) negatively related to the number of gonadotropin inhibitory hormone-immunoreactive cells in the hypothalamus. However, there was no effect of food deprivation on corticotropin releasing hormone-immunoreactive cells in the hypothalamus. There was also no relationship between corticotropin releasing hormone-immunoreactive cell number and circulating corticosterone. Our results are consistent with the hypothesis that neuroendocrine responses to food deprivation differ between male and female songbirds. Future studies should work to incorporate sex comparisons to evaluate sex-specific neuroendocrine responses to acute stress.

Developmental and reproductive physiology of small mammals at high altitude: challenges and evolutionary innovations.

High-altitude environments, characterized by low oxygen levels and low ambient temperatures, have been repeatedly colonized by small altricial mammals. These species inhabit mountainous regions year-round, enduring chronic cold and hypoxia. The adaptations that allow small mammals to thrive at altitude have been well studied in non-reproducing adults; however, our knowledge of adaptations specific to earlier life stages and reproductive females is extremely limited. In lowland natives, chronic hypoxia during gestation affects maternal physiology and placental function, ultimately limiting fetal growth. During post-natal development, hypoxia and cold further limit growth both directly by acting on neonatal physiology and indirectly via impacts on maternal milk production and care. Although lowland natives can survive brief sojourns to even extreme high altitude as adults, reproductive success in these environments is very low, and lowland young rarely survive to sexual maturity in chronic cold and hypoxia. Here, we review the limits to maternal and offspring physiology - both pre-natal and post-natal - that highland-adapted species have overcome, with a focus on recent studies on high-altitude populations of the North American deer mouse (Peromyscus maniculatus). We conclude that a combination of maternal and developmental adaptations were likely to have been critical steps in the evolutionary history of high-altitude native mammals.

RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat.

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†.

Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial-blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

Pregnancy stage determines the effect of chronic stress on ovarian progesterone synthesis.

Although stress-induced glucocorticoid release is thought to be a primary driver by which maternal stress negatively impacts pregnancy outcomes, the downstream neuroendocrine targets mediating these adverse outcomes are less well understood. We hypothesized that stress-induced glucocorticoid secretion inhibits pituitary hormone secretion, resulting in decreased ovarian progesterone synthesis. Using a chronic restraint model of stress in mice, we quantified steroid hormone production, pituitary hormones, and expression of ovarian genes that support progesterone production at both early ( day 5) and midpregnancy ( day 10). Females subjected to daily restraint had elevated baseline glucocorticoids during both early and midpregnancy; however, lower circulating progesterone was observed only during early pregnancy. Lower progesterone production was associated with lower expression of steroidogenic enzymes in the ovary of restrained females during early pregnancy. There were no stress-related changes to luteinizing hormone (LH) or prolactin (PRL). By midpregnancy, circulating LH decreased regardless of treatment, and this was associated with downregulation of ovarian steroidogenic gene expression. Our results are consistent with a role for LH in maintaining steroidogenic enzyme expression in the ovary, but neither circulating PRL nor LH were associated with the stress-induced inhibition of ovarian progesterone production during early pregnancy. We conclude that chronic stress impacts endocrine networks differently in pregnant and nonpregnant mammals. These findings underscore the need for further studies exploring dynamic changes in endocrine networks participating in pregnancy initiation and progression to elucidate the physiological mechanisms that connect stress exposure to adverse pregnancy outcomes.

Evidence for a Coupled Oscillator Model of Endocrine Ultradian Rhythms.

Whereas long-period temporal structures in endocrine dynamics have been well studied, endocrine rhythms on the scale of hours are relatively unexplored. The study of these ultradian rhythms (URs) has remained nascent, in part, because a theoretical framework unifying ultradian patterns across systems has not been established. The present overview proposes a conceptual coupled oscillator network model of URs in which oscillating hormonal outputs, or nodes, are connected by edges representing the strength of node-node coupling. We propose that variable-strength coupling exists both within and across classic hormonal axes. Because coupled oscillators synchronize, such a model implies that changes across hormonal systems could be inferred by surveying accessible nodes in the network. This implication would at once simplify the study of URs and open new avenues of exploration into conditions affecting coupling. In support of this proposed framework, we review mammalian evidence for (1) URs of the gut-brain axis and the hypothalamo-pituitary-thyroid, -adrenal, and -gonadal axes, (2) UR coupling within and across these axes; and (3) the relation of these URs to body temperature. URs across these systems exhibit behavior broadly consistent with a coupled oscillator network, maintaining both consistent URs and coupling within and across axes. This model may aid the exploration of mammalian physiology at high temporal resolution and improve the understanding of endocrine system dynamics within individuals.

Low glucose availability stimulates progesterone production by mouse ovaries in vitro.

Steroid production by the ovary is primarily stimulated by gonadotropins but can also be affected by biological cues that provide information about energy status and environmental stress. To further understand which metabolic cues the ovary can respond to, we exposed gonadotropin-stimulated mouse ovaries in vitro to glucose metabolism inhibitors and measured steroid accumulation in media. Gonadotropin-stimulated ovaries exposed to 2-deoxy-d-glucose increased progesterone production and steroidogenic acute regulatory protein mRNA levels. However, oocytes and granulosa cells in antral follicles do not independently mediate this response because targeted treatment of these cell types with a different inhibitor of glucose metabolism (bromopyruvic acid) did not affect progesterone production. Elevated progesterone production is consistent with the homeostatic role of progesterone in glucose regulation in mammals. It also may regulate follicle growth and/or atresia within the ovary. These results suggest that ovaries can regulate glucose homeostasis in addition to their primary role in reproductive activity.

Neural Versus Gonadal GnIH: Are they Independent Systems? A Mini-Review.

Based on research in protochordates and basal vertebrates, we know that communication across the first endocrine axes likely relied on diffusion. Because diffusion is relatively slow, rapid responses to some cues, including stress-related cues, may have required further local control of axis outputs (e.g., steroid hormone production by the gonads). Despite the evolution of much more efficient circulatory systems and complex nervous systems in vertebrates, production of many "neuro"transmitters has been identified outside of the hypothalamus across the vertebrate phylogeny and these neurotransmitters are known to locally regulate endocrine function. Our understanding of tissue-specific neuropeptide expression and their role coordinating physiological/behavioral responses of the whole organism remains limited, in part, due to nomenclature and historic dogma that ignores local regulation of axis output. Here, we review regulation of gonadotropin-inhibitory hormone (GnIH) across the reproductive axis in birds and mammals to bring further attention to context-dependent disparities and similarities in neuropeptide production by the brain and gonads. We find that GnIH responsiveness to cues of stress appears conserved across species, but that the response of specific tissues and the direction of GnIH regulation varies. The implications of differential regulation across tissues remain unclear in most studies, but further work that manipulates and contrasts function in different tissues has the potential to inform us about both organism-specific function and endocrine axis evolution.

The secret life of ground squirrels: accelerometry reveals sex-dependent plasticity in above-ground activity.

The sexes differ in how and when they allocate energy towards reproduction, but how this influences phenotypic plasticity in daily activity patterns is unclear. Here, we use collar-mounted light loggers and triaxial accelerometers to examine factors that affect time spent above ground and overall dynamic body acceleration (ODBA), an index of activity-specific energy expenditure, across the active season of free-living, semi-fossorial arctic ground squirrels (Urocitellus parryii). We found high day-to-day variability in time spent above ground and ODBA with most of the variance explained by environmental conditions known to affect thermal exchange. In both years, females spent more time below ground compared with males during parturition and early lactation; however, this difference was fourfold larger in the second year, possibly, because females were in better body condition. Daily ODBA positively correlated with time spent above ground in both sexes, but females were more active per unit time above ground. Consequently, daily ODBA did not differ between the sexes when females were early in lactation, even though females were above ground three to six fewer hours each day. Further, on top of having the additional burden of milk production, ODBA data indicate females also had fragmented rest patterns and were more active during late lactation. Our results indicate that sex differences in reproductive requirements can have a substantial influence on activity patterns, but the size of this effect may be dependent on capital resources accrued during gestation.

Energy regulation in context: Free-living female arctic ground squirrels modulate the relationship between thyroid hormones and activity among life history stages.

Thyroid hormones (THs), key regulators of lipid and carbohydrate metabolism, are likely modulators of energy allocation within and among animal life history stages. Despite their role in modulating metabolism, few studies have investigated whether THs vary among life history stages in free-living animals or if they exhibit stage-specific relationships to total energy expenditure and activity levels. We measured plasma total triiodothyronine (tT3) and thyroxine (tT4) at four, discrete life history stages of female arctic ground squirrels from two different populations in northern Alaska to test whether plasma THs correlate with life history stage-specific changes in metabolic rate and energy demand. We also tested whether THs explained individual variation in aboveground activity levels within life history stages. T3 peaked during lactation and was lowest during pre-hibernation fattening, consistent with known changes in basal metabolism and core body temperature. In contrast, T4 was elevated shortly after terminating hibernation but remained low and stable across other life-history stages in the active season. THs were consistently higher in the population that spent more time above-ground but the relationship between THs and activity varied among life history stages. T3 was positively correlated with activity only during lactation (r(2)=0.50) whereas T4 was positively correlated with activity immediately following lactation (r(2)=0.48) and during fattening (r(2)=0.53). Our results support the hypothesis that THs are an important modulator of basal metabolism but also suggest that the relationship between THs and activity varies among life history stages.

The timing of embryonic exposure to elevated temperature alters stress endocrinology in domestic chickens (Gallus domesticus).

Patterns of glucocorticoid (GC) release in response to stimuli vary both among individuals and within individuals across their lifetime. While much work has focused on how the prenatal steroid environment can affect GC release, relatively little is known about how environmental parameters, such as incubation temperature affect GCs. We tested the hypothesis that variation and timing of elevated incubation temperature within the thermoneutral zone can alter the pattern of GC release. We incubated domestic chicken eggs (Gallus domesticus) at the optimal incubation temperature (37.5 °C) or at a slightly higher temperature (+1.1 °C) either early, late, or throughout incubation. At three weeks post-hatch, all birds were (i) exposed to a capture-restraint stress to measure stress-induced GC release (naïve). Three days following the naïve stressor, birds were (ii) exposed to a heat challenge, which was followed the next day by a second capture-restraint stress (post-heat challenge). Regardless of treatment, birds had similar patterns of GC release following the naïve stress series. However, during the post-heat challenge stress series, birds incubated at optimal temperatures increased their peak GC release. In contrast, birds exposed to slightly elevated temperatures for any period of development failed to increase peak GC release, and their specific response varied with timing of exposure to the elevated incubation temperature. Our results demonstrate that subtle variation in the embryonic environment, such as elevated incubation temperature within the thermoneutral zone, can impact the pattern of GC release of offspring. Further work is needed to understand the mechanisms underlying these changes and the relationship between fitness and environmentally-altered phenotypes.