Tyler J. Stevenson

Reversible DNA methylation regulates seasonal photoperiodic time measurement

Significance This work examined whether epigenetic mechanisms participate in the regulation of seasonal reproduction. In long-day (summer) breeding hamsters, exposure to inhibitory winter photoperiods, or winter-like patterns of melatonin, altered DNA methyltransferase expression; decreased DNA methylation in the proximal promoter region of deiodinase type III (dio3) in the hypothalamus; and, in turn, increased hypothalamic dio3 expression. Pharmacological blockade of photoperiod-driven demethylation attenuated reproductive responses to winter photoperiods. Winter demethylation was reversed in anticipation of spring: spontaneous reproductive development was accompanied by remethylation of the dio3 promoter and decreases in dio3 mRNA. Methylation dynamics in the adult brain are reversible and may constitute an important component of the mechanism by which seasonal time is represented in the nervous system. In seasonally breeding vertebrates, changes in day length induce categorically distinct behavioral and reproductive phenotypes via thyroid hormone-dependent mechanisms. Winter photoperiods inhibit reproductive neuroendocrine function but cannot sustain this inhibition beyond 6 mo, ensuring vernal reproductive recrudescence. This genomic plasticity suggests a role for epigenetics in the establishment of seasonal reproductive phenotypes. Here, we report that DNA methylation of the proximal promoter for the type III deiodinase (dio3) gene in the hamster hypothalamus is reversible and critical for photoperiodic time measurement. Short photoperiods and winter-like melatonin inhibited hypothalamic DNA methyltransferase expression and reduced dio3 promoter DNA methylation, which up-regulated dio3 expression and induced gonadal regression. Hypermethylation attenuated reproductive responses to short photoperiods. Vernal refractoriness to short photoperiods reestablished summer-like methylation of the dio3 promoter, dio3 expression, and reproductive competence, revealing a dynamic and reversible mechanism of DNA methylation in the mammalian brain that plays a central role in physiological orientation in time.

Disrupted seasonal biology impacts health, food security and ecosystems

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Cloning of Gonadotropin-Releasing Hormone I Complementary DNAs in Songbirds Facilitates Dissection of Mechanisms Mediating Seasonal Changes in Reproduction

Temperate zone animals exhibit seasonal variation in reproductive physiology. In most cases, seasonal changes in reproductive states are regulated by changes in GnRH1 secretion, rather than synthesis, from the preoptic area (POA)/anterior hypothalamus. An important exception occurs in some songbirds that become photorefractory to the stimulatory effects of long days and show profound decreases in brain GnRH1 protein content. Whether this decline reflects changes in gene expression is unknown because of past failures to measure GNRH1 mRNA levels, due in large part to the absence of available GNRH1 gene sequence in this taxon. Here, we report the first cloning of GNRH1 cDNAs in two songbirds: European starlings and zebra finches. Consistent with the size of the prepro-hormone in other avian and non-avian species, the open-reading frames predict proteins of 91 and 92 amino acids, respectively. Whereas the decapeptide in both species is perfectly conserved with chicken GnRH1, the amino acid identity in the signal peptide and GNRH associated peptide subdomains are significantly less well conserved. At the nucleotide level, the starling and zebra finch coding sequences are approximately 88% identical to each other but only approximately 70% identical to chicken GNRH1. In situ hybridization using radiolabeled cRNA probes demonstrated GNRH1 mRNA expression primarily in the POA, consistent with previous studies on the distribution of the GnRH1-immunoreactive cell bodies. Furthermore, we provide evidence for photoperiod-dependent regulation of GNRH1 mRNA in male starlings. Declines in GNRH1 mRNA levels occur in parallel with testicular involution. Thus, photorefractoriness is associated with decreases in GNRH1 gene expression in the medial POA.

Anatomical localization of the effects of reproductive state, castration, and social milieu on cells immunoreactive for gonadotropin-releasing hormone-I in male European starlings (Sturnus vulgaris)

Gonadotropin‐releasing hormone‐I (GnRH‐I) cells are localized primarily to the septopreoptic area (POA) and are responsible for regulating gonadotropin release from the anterior pituitary. Some songbird species exhibit dramatic seasonal variation in the number of detectable GnRH‐I immunoreactive cells, with higher numbers being observed during the breeding season. Here we investigated the anatomical distribution of GnRH‐I‐immunoreactive cells in male starlings that varied in response to manipulations of reproductive state, social context, and gonadal condition. We housed photostimulated, intact and castrated male starlings with a female or alone. Additionally, a fifth treatment group consisted of photorefractory males (i.e., in a nonreproductive state) housed alone. All photostimulated males had significantly greater numbers of GnRH‐I cells compared with photorefractory male starlings. There was a significant main effect of castration and social context. Castrated males had significantly greater numbers of GnRH‐I cells compared with intact males, and males housed in male‐female dyads also had greater numbers of GnRH‐I cells. Furthermore, the significant main effects of castration and social context were the result of an increase in GnRH‐I cell numbers specifically in the rostral and intermediate regions of the POA. These findings indicate that social context and hormonal milieu have profound effects on GnRH‐I immunoreactivity in addition to the previously described effects of reproductive state. These data provide novel insight into the environmental regulation of the hypothalamopituitary axis and suggest that gonadal hormones and female presence independently regulate GnRH‐I cells in specific regions of the POA in male starlings. J. Comp. Neurol. 517:146–155, 2009.

Photoperiodic Condition Is Associated with Region-Specific Expression of GNRH1 mRNA in the Preoptic Area of the Male Starling (Sturnus vulgaris)

Many seasonally breeding avian species exhibit marked changes in hypothalamic content of gonadotropin-releasing vhormone 1 (GNRH1) protein that are reflective of breeding condition. We recently cloned the GNRH1 cDNA in European starlings and demonstrated that changes in GNRH1 mRNA levels occur with a time course similar to what has been observed with GNRH1 protein. However, we did not previously resolve whether these differences were attributable to changes in the number of cells expressing the gene. Herein, we investigated photoperiod-induced changes in the number and distribution of GNRH1 mRNA-expressing cells in the preoptic area of male starlings. GNRH1 mRNA-expressing cell number was significantly greater in breeding birds than in nonbreeding birds. Starlings maintained in short nonstimulatory day length (i.e., prebreeding) showed intermediate cell numbers. Detailed analysis of the rostrocaudal and mediolateral distribution revealed that breeding birds had greater numbers of cells expressing GNRH1 mRNA in the medial intermediate, mediocaudal, and lateral intermediate preoptic area compared with prebreeding and nonbreeding birds. These data demonstrate that photoperiodic changes in reproductive state in starlings are associated with region-specific alterations in the number of cells expressing the GNRH1 gene. It remains to be determined whether these changes reflect quantitative differences in gene expression among an otherwise stable population of cells or a phenotypic switch in which cells gain or lose the ability to make GNRH1 mRNA in response to environmental cues.

Disruption of neuropsin mRNA expression via RNA interference facilitates the photoinduced increase in thyrotropin-stimulating subunit β in birds

It has long been known that the avian brain is capable of light detection independently of the eyes. The photoreceptive molecule neuropsin (OPN5) was identified in mammalian and avian brains, and shown to respond to biologically relevant light wavelengths. Whether OPN5 is functionally involved in light detection is unknown. Daylength plays a critical role in regulating the neuroendocrine control of reproduction in birds. The presence of light during a ‘photoinducible’ phase of the circadian cycle, which occurs 12–16 h after dawn, results in marked changes in hypothalamic gene expression. These changes ultimately control gonadotropin release from the pituitary gland that, in turn, stimulates gonadal development. In this study, we first measured OPN5 expression in the mediobasal hypothalamus (MBH) in border canaries during the photoinducible period in relation to thyrotropin (TSH) β‐subunit mRNA expression, which is implicated in the control of avian reproduction. Second, the knockdown of OPN5 via small interfering RNA antisense in the MBH revealed that there is an inhibitory input in the photoinduced regulation of TSHβ mRNA expression. Our data indicate that a decrease in OPN5 mRNA expression is associated with the facilitation in TSHβ mRNA expression in the MBH, a critical step for the light‐induced increase in gonadal recrudescence. We hypothesise that the removal of an inhibitory input by OPN5 in birds may be a step that occurs during the photoinducible period. Given the distribution of OPN5 in the brain and periphery, this suggests a possible multifunctional role for light information in regulating other physiological processes.

Information theory and the neuropeptidergic regulation of seasonal reproduction in mammals and birds.

Seasonal breeding in the temperate zone is a dramatic example of a naturally occurring change in physiology and behaviour. Cues that predict periods of environmental amelioration favourable for breeding must be processed by the brain so that the appropriate responses in reproductive physiology can be implemented. The neural integration of several environmental cues converges on discrete hypothalamic neurons in order to regulate reproductive physiology. Gonadotrophin-releasing hormone-1 (GnRH1) and Kisspeptin (Kiss1) neurons in avian and mammalian species, respectively, show marked variation in expression that is positively associated with breeding state. We applied the constancy/contingency model of predictability to investigate how GnRH1 and Kiss1 integrate different environmental cues to regulate reproduction. We show that variation in GnRH1 from a highly seasonal avian species exhibits a predictive change that is primarily based on contingency information. Opportunistic species have low measures of predictability and exhibit a greater contribution of constancy information that is sex-dependent. In hamsters, Kiss1 exhibited a predictive change in expression that was predominantly contingency information and is anatomically localized. The model applied here provides a framework for studies geared towards determining the impact of variation in climate patterns to reproductive success in vertebrate species.

Photoperiodic time measurement and seasonal immunological plasticity

Seasonal variations in immunity are common in nature, and changes in day length are sufficient to trigger enhancement and suppression of immune function in many vertebrates. Drawing primarily on data from Siberian hamsters, this review describes formal and physiological aspects of the neuroendocrine regulation of seasonal changes in mammalian immunity. Photoperiod regulates immunity in a trait-specific manner, and seasonal changes in gonadal hormone secretion and thyroid hormone signaling all participate in seasonal immunomodulation. Photoperiod-driven changes in the hamster reproductive and immune systems are associated with changes in iodothyronine deiodinase-mediated thyroid hormone signaling, but photoperiod exerts opposite effects on select aspects of the epigenetic regulation of reproductive neuroendocrine and lymphoid tissues. Photoperiodic changes in immunocompetence may explain a proportion of the annual variance in disease incidence and severity in nature, and provide a useful framework to help understand brain-immune interactions.

Dissociable effects of social context on song and doublecortin immunoreactivity in male canaries

Variation in environmental factors such as day length and social context greatly affects reproductive behavior and the brain areas that regulate these behaviors. One such behavior is song in songbirds, which males use to attract a mate during the breeding season. In these species the absence of a potential mate leads to an increase in the number of songs produced, while the presence of a mate greatly diminishes singing. Interestingly, although long days promote song behavior, producing song itself can promote the incorporation of new neurons in brain regions controlling song output. Social context can also affect such neuroplasticity in these song control nuclei. The goal of the present study was to investigate in canaries (Serinus canaria), a songbird species, how photoperiod and social context affect song and the incorporation of new neurons, as measured by the microtubule‐associated protein doublecortin (DCX) in HVC, a key vocal production brain region of the song control system. We show that long days increased HVC size and singing activity. In addition, male canaries paired with a female for 2 weeks showed enhanced DCX‐immunoreactivity in HVC relative to birds housed alone. Strikingly, however, paired males sang fewer songs that exhibited a reduction in acoustic features such as song complexity and energy, compared with birds housed alone, which sang prolifically. These results show that social presence plays a significant role in the regulation of neural and behavioral plasticity in songbirds and can exert these effects in opposition to what might be expected based on activity‐induced neurogenesis.

Digital epidemiology reveals global childhood disease seasonality and the effects of immunization

Significance Disease surveillance systems largely focus on infectious diseases with high mortality, whereas less severe diseases often go unreported. Using chicken pox as an example, we demonstrate that Internet queries can be used as a proxy for disease incidence when reporting is lacking. We established that Google Trends accurately reflected clinical cases in countries with surveillance, and thus population-level dynamics of chicken pox. Then, we discovered robust seasonal variation in query behavior, with a striking latitudinal gradient on a global scale. Next, we showed that real-time data-mining of queries could forecast the timing and magnitude of outbreaks. Finally, our analyses revealed that countries with government-mandated vaccination programs have significantly reduced seasonality of queries, indicating vaccination efforts mitigated chicken pox outbreaks. Public health surveillance systems are important for tracking disease dynamics. In recent years, social and real-time digital data sources have provided new means of studying disease transmission. Such affordable and accessible data have the potential to offer new insights into disease epidemiology at national and international scales. We used the extensive information repository Google Trends to examine the digital epidemiology of a common childhood disease, chicken pox, caused by varicella zoster virus (VZV), over an 11-y period. We (i) report robust seasonal information-seeking behavior for chicken pox using Google data from 36 countries, (ii) validate Google data using clinical chicken pox cases, (iii) demonstrate that Google data can be used to identify recurrent seasonal outbreaks and forecast their magnitude and seasonal timing, and (iv) reveal that VZV immunization significantly dampened seasonal cycles in information-seeking behavior. Our findings provide strong evidence that VZV transmission is seasonal and that seasonal peaks show remarkable latitudinal variation. We attribute the dampened seasonal cycles in chicken pox information-seeking behavior to VZV vaccine-induced reduction of seasonal transmission. These data and the methodological approaches provide a way to track the global burden of childhood disease and illustrate population-level effects of immunization. The global latitudinal patterns in outbreak seasonality could direct future studies of environmental and physiological drivers of disease transmission.