The mechanisms of amniote photoperiodism: from deep-brain photoreceptors to rhythmic epigenetics

Seasonal reproduction is a strategy conserved across nature. The duration of light (photoperiod) regulates the reproductive molecular control within the Hypothalamic-Pituitary-Gonadal Axis of seasonal species, and supplementary cues fine-tune the exact timing of breeding. In recent years, epigenetic mechanisms have been shown to be involved in an array of circannual rhythms, including reproduction. The aim of this thesis was to explore the molecular reproductive neuroendocrine processes that underlie the onset of reproduction in two summer-breeding animal models, the Japanese quail (Coturnix japonica) and the Siberian hamster (Phodopus sungorus). In birds, light penetrates the skull and is detected by deep-brain photoreceptors (DBPs) within the hypothalamus, stimulating the photoperiodic reproductive response. However, the identity of these DBPs is unclear to this day. In the present thesis, the expression of two photoreceptors, Vertebrate-Ancient Opsin (VA Opsin) and Neuropsin (OPN5), was repressed through adeno-associated viral injection, and the breeding response was monitored in control and treated birds maintained under short-days (SD), or long-days (LD) for 2, 7 or 28 days. The data revealed that both opsins may be involved in seasonal reproduction in the Japanese quail, and that OPN5’s role includes modulating gonadal sensitivity to gonadotropins during breeding. It was also found that hypothalamic OPN5, GNRH and DNAmethyltransferase (Dnmt) expression increases at embryonic day 14 in this species. In addition, higher global methylation levels were found in the pituitary gland of adult LD quail, compared to SD. In the Siberian hamster, two studies were conducted to investigate the effect of triiodothyronine (T3) on the photoperiod-dependent regulation of reproductive physiology and hypothalamic DNA methyltransferase enzyme expression in both males and females. Two weeks of daily T3 injections induced gonadal growth in SD males, but not in females. Female SD hamsters, but not males, were found to express lower levels of de novo Dnmts compared to LD individuals. However, exogenous T3 did not affect hypothalamic Dnmt expression in neither males or females. The data indicated sex differences in the gonadal response to T3, as well as in the regulation of hypothalamic DNA methyltransferase expression. It is likely that female Siberian hamsters require additional cues to initiate reproductive processes. The studies presented allowed for an exploration of reproductive mechanisms in both an avian and a mammalian model, including the role of epigenetic processes in seasonal breeding. Future studies are required to elucidate the precise mechanisms of DBPs, as well as identify downstream targets of maintenance and de novo Dnmts

A comparative perspective on extra-retinal photoreception

Ubiquitous in non-mammalian vertebrates, extra-retinal photoreceptors (ERPs) have been linked to an array of physiological, metabolic, behavioral, and morphological changes. However, the mechanisms and functional roles of ERPs remain one of the enduring questions of modern biology. In this review article, we use a comparative framework to identify conserved roles and distributions of ERPs, highlighting knowledge gaps. We conclude that ERP research can be divided into two largely unconnected categories: (i) identification and localization of photoreceptors and (ii) linkage of non-retinal light reception to behavioral and physiological processes, particularly endocrine systems. However, the emergence of novel gene editing and silencing techniques is enabling the unification of ERP research by allowing the bridging of this divide

Functional inhibition of deep brain non-visual opsins facilitates acute long day induction of reproductive recrudescence in male Japanese quail :Deep brain photoreceptors in birds

For nearly a century, we have known that brain photoreceptors regulate avian seasonal biology. Two photopigments, vertebrate ancient opsin (VA) and neuropsin (OPN5), provide possible molecular substrates for these photoreceptor pathways. VA fulfills many criteria for providing light input to the reproductive response, but a functional link has yet to be demonstrated. This study examined the role of VA and OPN5 in the avian photoperiodic response of Japanese quail (Coturnix japonica). Non-breeding male quail were housed under short days (6L:18D) and received an intracerebroventricular infusion of adeno-associated viral vectors with shRNAi that selectively inhibited either VA or OPN5. An empty viral vector acted as a control. Quail were then photostimulated (16L:8D) to stimulate gonadal growth. Two long days significantly increased pituitary thyrotrophin-stimulating hormone β-subunit (TSHβ) and luteinizing hormone β-subunit (LHβ) mRNA of VA shRNAi treated quail compared to controls. Furthermore, at one week there was a significant increase, compared to controls, in both hypothalamic gonadotrophin releasing hormone-I (GnRH-I) mRNA and paired testicular mass in VA shRNAi birds. Opn5 shRNAi facilitated the photoinduced increase in TSHβ mRNA at 2 days, but no other differences were identified compared to controls. Contrary to our expectations, the silencing of deep brain photoreceptors enhanced the response of the reproductive axis to photostimulation rather than preventing it. In addition, we show that VA opsin plays a dominant role in the light-dependent neuroendocrine control of seasonal reproduction in birds. Together our findings suggest the photoperiodic response involves at least two photoreceptor types and populations working together with VA opsin playing a dominant role

Genome sequencing and transcriptome analyses of the Siberian hamster hypothalamus identify mechanisms for seasonal energy balance

Synthesis of triiodothyronine (T3) in the hypothalamus induces marked seasonal neuromorphology changes across taxa. How species-specific responses to T3 signaling in the CNS drive annual changes in body weight and energy balance remains uncharacterized. These experiments sequenced and annotated the Siberian hamster (Phodopus sungorus) genome, a model organism for seasonal physiology research, to facilitate the dissection of T3-dependent molecular mechanisms that govern predictable, robust, and long-term changes in body weight. Examination of the Phodopus genome, in combination with transcriptome sequencing of the hamster diencephalon under winter and summer conditions, and in vivo-targeted expression analyses confirmed that proopiomelanocortin (pomc) is a primary genomic target for the long-term T3-dependent regulation of body weight. Further in silico analyses of pomc promoter sequences revealed that thyroid hormone receptor 1β-binding motif insertions have evolved in several genera of the Cricetidae family of rodents. Finally, experimental manipulation of food availability confirmed that hypothalamic pomc mRNA expression is dependent on longer-term photoperiod cues and is unresponsive to acute, short-term food availability. These observations suggest that species-specific responses to hypothalamic T3, driven in part by the receptor-binding motif insertions in some cricetid genomes, contribute critically to the long-term regulation of energy balance and the underlying physiological and behavioral adaptations associated with the seasonal organization of behavior. © 2019 National Academy of Sciences. All rights reserved

Genome sequencing and transcriptome analyses of the Siberian hamster hypothalamus identify mechanisms for seasonal energy balance

We thank the manuscript reviewers for constructive feedback; David G. Hazlerigg, Cristina Saenz de Miera, and Valerie Simonneaux for genome sequence contributions; Nicolas Scrutton and Lindsey Duguid for expert technical assistance; and Michael Jarsulic for technical assistance on the high-performance computing clusters. This project was supported by a project research grant from The British Society for Neuroendocrinology (to T.J.S.); Grants BB/M021629/1 and BB/M001555/1 (to F.J.P.E.) from the Biotechnology and Biological Sciences Research Council, and Grants UL1-TR000430 (to T.J.S. and B.J.P.) and R01-AI067406 (to B.J.P.) from the National Institutes of Health. T.J.S. is funded by The Leverhulme Trust. The Center for Research Informatics was supported by the Biological Sciences Division at the University of Chicago with additional support provided by the Institute for Translational Medicine/Clinical and Translational award (NIH 5UL1TR002389-02) and the University of Chicago Comprehensive Cancer Center Support Grant (NIH Grant P30CA014599). The bioinformatics analysis was performed on high-performance computing clusters at the Center for Research Informatics, Biological Sciences Division. P.B. was funded by the Scottish Government Rural and Environment Science and Analytical Services Division grant to the Rowett Institute.Peer reviewedPublisher PD

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification

Sex differences and the neuroendocrine regulation of seasonal reproduction by supplementary environmental cues

Seasonal rhythms in reproduction are conserved across nature and optimize the timing of breeding to environmental conditions favorable for offspring and parent survival. The primary predictive cue for timing seasonal breeding is photoperiod. Supplementary cues, such as food availability, social signals, and temperature, fine-tune the timing of reproduction. Male and female animals show differences in the sensory detection, neural integration, and physiological responses to the same supplementary cue. The neuroendocrine regulation of sex-specific integration of predictive and supplementary cues is not well characterized. Recent findings indicate that epigenetic modifications underlie the organization of sex differences in the brain. It has also become apparent that deoxyribonucleic acid methylation and chromatin modifications play an important role in the regulation and timing of seasonal rhythms. This article will highlight evidence for sex-specific responses to supplementary cues using data collected from birds and mammals. We will then emphasize that supplementary cues are integrated in a sex-dependent manner due to the neuroendocrine differences established and maintained by the organizational and activational effects of reproductive sex hormones. We will then discuss how epigenetic processes involved in reproduction provide a novel link between early-life organizational effects in the brain and sex differences in the response to supplementary cues