Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Recombinant snake antivenoms get closer to the clinic.

Snakebite envenomings kill ~100 000 victims each year and leave many more with permanent sequelae. Antivenoms have been available for more than 125 years but are in need of innovation. A new study by Khalek et al. highlights broadly neutralizing human monoclonal antibodies (mAbs) that might be used to develop recombinant antivenoms with superior therapeutic benefits.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

Alpha-1-B glycoprotein (A1BG) inhibits sterol-binding and export by CRISP2.

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in various processes, including sperm maturation and cancer progression. They are mostly secreted glycoproteins and share a unique conserved CAP domain. The precise mode of action of these proteins, however, has remained elusive. Saccharomyces cerevisiae expresses three members of this protein family, which bind sterols in vitro and promote sterol secretion from cells. This sterol-binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with alpha-1-B glycoprotein (A1BG), a human plasma glycoprotein that is upregulated in different types of cancers. Here we examined whether the interaction between CRISP proteins and A1BG affects the sterol-binding function of CAP family members. Co-expression of A1BG with CAP proteins abolished their sterol export function in yeast and their interaction inhibits sterol-binding in vitro. We map the interaction between A1BG and CRISP2 to the third of five repeated immunoglobulin-like (Ig) domains within A1BG. Interestingly, the interaction between A1BG and CRISP2 requires magnesium, suggesting that coordination of Mg2+ by the highly conserved tetrad residues within the CAP domain is essential for a stable interaction between the two proteins. The observation that A1BG modulates the sterol-binding function of CRISP2, has potential implications for the role of A1BG and related Ig domain containing proteins in cancer progression and the toxicity of reptile venoms containing CRISP proteins.

A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function.

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

From birth to bite: the evolutionary ecology of India's medically most important snake venoms.

BACKGROUND: Snake venoms can exhibit remarkable inter- and intraspecific variation. While diverse ecological and environmental factors are theorised to explain this variation, only a handful of studies have attempted to unravel their precise roles. This knowledge gap not only impedes our understanding of venom evolution but may also have dire consequences on snakebite treatment. To address this shortcoming, we investigated the evolutionary ecology of venoms of Russell's viper (Daboia russelii) and spectacled cobra (Naja naja), India's two clinically most important snakes responsible for an alarming number of human deaths and disabilities. METHODOLOGY: Several individuals (n = 226) of D. russelii and N. naja belonging to multiple clutches (n = 9) and their mothers were maintained in captivity to source ontogenetic stage-specific venoms. Using various in vitro and in vivo assays, we assessed the significance of prey, ontogeny and sex in driving venom composition, function, and potency. RESULTS: Considerable ontogenetic shifts in venom profiles were observed in D. russelii, with the venoms of newborns being many times as potent as juveniles and adults against mammalian (2.3-2.5 ×) and reptilian (2-10 ×) prey. This is the first documentation of the ontogenetic shift in viperine snakes. In stark contrast, N. naja, which shares a biogeographic distribution similar to D. russelii, deployed identical biochemical cocktails across development. Furthermore, the binding kinetics of cobra venom toxins against synthetic target receptors from various prey and predators shed light on the evolutionary arms race. CONCLUSIONS: Our findings, therefore, provide fascinating insights into the roles of ecology and life history traits in shaping snake venoms.

Embryonic development of the neotropical pit viper Bothrops atrox (Serpentes: Viperidae: Crotalinae), with emphasis on pit organ morphogenesis and its evolution in snakes.

BACKGROUND: Bothrops atrox is a pit viper with a loreal pit organ, and its embryological development remains undescribed. Here, we provide a comprehensive description of the embryology of B. atrox, focusing on the loreal pit organ and cephalic scales. RESULTS: We characterized 13 developmental stages of B. atrox based on external features consistent with the embryogenesis of previously described snake species. The loreal pit organ originates from the circumorbital region and migrates to its final position. In Crotalinae, the pit organ first becomes visible at stage 28, whereas in Pythonidae labial, pit organs appear at Stage 35. Pit organs evolved independently three times in Serpentes, encompassing Boidae, Pythonidae, and Crotalinae. Boidae lacks embryological information for pit organs. Furthermore, we observed that head scalation onset occurs at Stage 33 in B. atrox, with fusion of scales surrounding the loreal pit organ. CONCLUSIONS: The embryology of pit organs in Pythonidae and Boidae species remains poorly understood. Our detailed embryological descriptions are critical for proposing developmental scenarios for pit organs and guiding future research on these structures.

Exploring Top-Down Mass Spectrometric Approaches To Probe Forest Cobra (Naja melanoleuca) Venom Proteoforms.

Snake venoms are comprised of bioactive proteins and peptides that facilitate severe snakebite envenomation symptoms. A comprehensive understanding of venom compositions and the subtle heterogeneity therein is important. While bottom-up proteomics has been the well-established approach to catalogue venom compositions, top-down proteomics has emerged as a complementary strategy to characterize venom heterogeneity at the intact protein level. However, top-down proteomics has not been as widely implemented in the snake venom field as bottom-up proteomics, with various emerging top-down methods yet to be developed for venom systems. Here, we have explored three main top-down mass spectrometry methodologies in a proof-of-concept study to characterize selected three-finger toxin and phospholipase A2 proteoforms from the forest cobra (Naja melanoleuca) venom. We demonstrated the utility of a data-independent acquisition mode "MSE" for untargeted fragmentation on a chromatographic time scale and its improvement in protein sequence coverage compared to conventional targeted tandem mass spectrometry analysis. We also showed that protein identification can be further improved using a hybrid fragmentation approach, combining electron-capture dissociation and collision-induced dissociation. Lastly, we reported the promising application of multifunctional cyclic ion mobility separation and post-ion mobility fragmentation on snake venom proteins for the first time.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae).

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Regionalization of the vertebral column and its correlation with heart position in snakes: Implications for evolutionary pathways and morphological diversification.

Spinal regionalization has important implications for the evolution of vertebrate body plans. We determined the variation in the number and morphology of vertebrae across the vertebral column (i.e., vertebral formula) for 63 snake species representing 13 families using intracolumnar variation in vertebral shape. Vertebral counts were used to determine the position of the heart, pylorus, and left kidney for each species. Across all species we observed a conspicuous midthoracic transition in vertebral shape, indicating four developmental domains of the precloacal vertebral column (cervical, anterior thoracic, posterior thoracic, and lumbar). Using phylogenetic analyses, the boundary between the anterior and posterior thoracic vertebrae was correlated with heart position. No associations were found between shifts in morphology of the vertebral column and either the pylorus or left kidney. We observed that among taxa, the number of preapex and postapex vertebrae could change independently from one another and from changes in the total number of precloacal vertebrae. Ancestral state reconstruction of the preapex and postapex vertebrae illustrated several evolutionary pathways by which diversity in the vertebral column and heart position have been attained. In addition, no conspicuous pattern was observed among the heart, pylorus, or kidney indicating that their relative positions to each other evolve independently. We conclude that snakes exhibit four morphologically distinct regions of the vertebral column. We discuss the implications of the forebody and hindbody vertebral formula on the morphological diversification of snakes.

A C-type lectin from Bothrops jararacussu venom reprograms endothelial cell biology.

Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared ß-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Antiangiogenic properties of BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom by VEGF pathway in endothelial cells.

Angiogenesis is a process that is controlled by a delicate combination of proangiogenic and antiangiogenic molecules and can be disrupted in various illnesses, including cancer. Non-cancerous diseases can also have an abnormal or insufficient vascular growth, inflammation and hypoxia, which exacerbate angiogenesis. These conditions include atherosclerosis, psoriasis, endometriosis, asthma, obesity and AIDS. Based on that, the present work assessed the in vitro and ex vivo antiangiogenic properties stemming from BthMP, a P-I metalloproteinase from Bothrops moojeni snake venom, via the VEGF pathway. BthMP at a concentration of 5 and 40 µg/mL showed no toxicity to endothelial cells (HUVEC) in the MTT assay and was not able to induce necrosis and colony proliferation. Interestingly, BthMP inhibited adhesion, migration and invasion of HUVECs in Matrigel and arrested in vitro angiogenesis by reducing the average number of nodules in toxin-treated cells by 9.6 and 17.32 at 5 and 40 µg/mL, respectively, and the number of tubules by 15.9 at 5 µg/mL and 21.6 at 40 µg/mL in a VEGF-dependent way, an essential proangiogenic property. Furthermore, BthMP inhibited the occurrence of the angiogenic process in an ex vivo aortic ring test by decreasing new vessel formation by 52% at 5 µg/mL and by 66% at 40 µg/mL and by increasing the expression of an antiangiogenic gene, SFLT-1, and decreasing the expression of the proangiogenic genes VEGFA and ANGPT-1. Finally, this toxin reduces the production of nitric oxide, a marker that promotes angiogenesis and VEGF modulation, and decreases the protein expression of VEGFA in the supernatant of the HUVEC culture by about 30 %. These results suggest that BthMP has a promising antiangiogenic property and proves to be a biotechnological mechanism for understanding the antiangiogenic responses induced by snake venom metalloproteinases, which could be applied to a variety of diseases that exhibit an imbalance of angiogenesis mechanisms.

Fine mapping of TFL, a major gene regulating fruit length in snake gourd (Trichosanthes anguina L).

Fruit length is a crucial agronomic trait of snake gourd (Trichosanthes anguina L); however, genes associated with fruit length have not been characterised. In this study, F2 snake gourd populations were generated by crossing the inbred lines, S1 and S2 (fruit lengths: 110 and 20 cm, respectively). Subsequently, bulk segregant analysis, sequencing, and fine-mapping were performed on the F2 population to identify target genes. Our findings suggest that the fruit length of snake gourd is regulated by a major-effect regulatory gene. Mining of genes regulating fruit length in snake gourd to provide a basis for subsequent selection and breeding of new varieties. Genotype-phenotype association analysis was performed on the segregating F2 population comprising 6,000 plants; the results indicate that the target gene is located on Chr4 (61,846,126-61,865,087 bp, 18.9-kb interval), which only carries the annotated candidate gene, Tan0010544 (designated TFL). TFL belongs to the MADS-box family, one of the largest transcription factor families. Sequence analysis revealed a non-synonymous mutation of base C to G at position 202 in the coding sequence of TFL, resulting in the substitution of amino acid Gln to Glu at position 68 in the protein sequence. Subsequently, an InDel marker was developed to aid the marker-assisted selection of TFL. The TFL in the expression parents within the same period was analysed using quantitative real-time PCR; the TFL expression was significantly higher in short fruits than long fruits. Therefore, TFL can be a candidate gene for determining the fruit length in snake gourd. Collectively, these findings improve our understanding of the genetic components associated with fruit length in snake gourds, which could aid the development of enhanced breeding strategies for plant species.

Panicolytic-like effects of environment enrichment on male mice threatened by Bothrops jararaca lancehead pit vipers.

Environment enrichment (EE) is a well-known eustress model showing beneficial effects in different psychiatric diseases, but its positive properties in panic disorders are not yet established. The confrontation between prey and predator in complex arenas has been validated as a putative panic attack model. The principal aim of this work was to investigate the role of the EE on panic-like defensive responses elicited by mice threatened by venomous snakes. After 6 weeks of exposure either to an enriched or standard environments, 36 male mice were habituated in a complex polygonal arena for snakes containing an artificial burrow and elevated platforms for escape. The animals were confronted by Bothrops jararaca for 5 min, and the following antipredatory responses were recorded: defensive attention, stretched attend posture, flat back approach, prey versus predator interaction, oriented escape behavior, time spent in a safe place, and number of crossings. Mice threatened by snakes displayed several antipredatory reactions as compared to the exploratory behavior of those animals submitted to a nonthreatening situation (toy snake) in the same environment. Notably, EE causes anxiolytic- and panicolytic-like effects significantly decreasing the defensive attention and time spent in safe places and significantly increasing both prey versus predator interaction and exploratory behavior. In conclusion, our data demonstrate that EE can alter the processing of fear modulation regarding both anxiety- and panic-like responses in a dangerous condition, significantly modifying the decision-making defensive strategy.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Viewpoint: Lupus anticoagulant detection and interpretation in antiphospholipid syndrome.

Lupus anticoagulant (LA) is a well-established risk factor for the clinical manifestations of antiphospholipid syndrome (APS). Accurate LA detection is an essential prerequisite for optimal diagnosis and management of patients with APS or aPL carriers. Variability remains a challenge in LA testing, with reliable detection influenced by multiple factors, including pre-analytical conditions, anticoagulation treatment, choice of tests and procedures performed, as well as interpretation of results, that can lead to false-positives or negatives. A standardised approach to LA testing, following current guidance, based on published data and international consensus, and with attention to detail, is required to underpin accurate detection of LA. Future work should focus on better characterisation of the nature of LA, which may ultimately lead to improved diagnosis and management of patients with APS and aPL carriers. This article reviews current practice and challenges, providing an overview on detection of LA.

Genetic mechanisms and biological processes underlying host response to ophidiomycosis (snake fungal disease) inferred from tissue-specific transcriptome analyses.

Emerging infectious diseases in wildlife species caused by pathogenic fungi are of growing concern, yet crucial knowledge gaps remain for diseases with potentially large impacts. For example, there is detailed knowledge about host pathology and mechanisms underlying response for chytridiomycosis in amphibians and white-nose syndrome in bats, but such information is lacking for other more recently described fungal infections. One such disease is ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, which has been identified in many species of snakes, yet the biological mechanisms and molecular changes occurring during infection are unknown. To gain this information, we performed a controlled experimental infection in captive Prairie rattlesnakes (Crotalus viridis) with O. ophidiicola at two different temperatures: 20 and 26°C. We then compared liver, kidney, and skin transcriptomes to assess tissue-specific genetic responses to O. ophidiicola infection. Given previous histopathological studies and the fact that snakes are ectotherms, we expected highest fungal activity on skin and a significant impact of temperature on host response. Although we found fungal activity to be localized on skin, most of the differential gene expression occurred in internal tissues. Infected snakes at the lower temperature had the highest host mortality whereas two-thirds of the infected snakes at the higher temperature survived. Our results suggest that ophidiomycosis is likely a systemic disease with long-term effects on host response. Our analysis also identified candidate protein coding genes that are potentially involved in host response, providing genetic tools for studies of host response to ophidiomycosis in natural populations.

Neural thyroid hormone metabolism integrates seasonal changes in environmental temperature with the neuroendocrine reproductive axis.

We asked if environmental temperature alters thyroid hormone metabolism within the hypothalamus, thereby providing a neuroendocrine mechanism by which temperature could be integrated with photoperiod to regulate seasonal rhythms. We used immunohistochemistry to assess the effects of low-temperature winter dormancy at 4 °C or 12 °C on thyroid-stimulating hormone (TSH) within the infundibulum of the pituitary as well as deiodinase 2 (Dio2) and 3 (Dio3) within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis). Both the duration and, in males, magnitude of low-temperature dormancy altered deiodinase immunoreactivity within the hypothalamus, increasing the area of Dio2-immunoreactivity in males and females and decreasing the number of Dio3-immunoreactive cells in males after 8-16 weeks. Reciprocal changes in Dio2/3 favor the accumulation of triiodothyronine within the hypothalamus. Whether TSH mediates these effects requires further study, as significant changes in TSH-immunoreactive cell number were not observed. Temporal changes in deiodinase immunoreactivity coincided with an increase in the proportion of males exhibiting courtship behavior as well as changes in the temporal pattern of courtship behavior after emergence. Our findings mirror those of previous studies, in which males require low-temperature exposure for at least 8 weeks before significant changes in gonadotropin-releasing hormone immunoreactivity and sex steroid hormones are observed. Collectively, these data provide evidence that the neuroendocrine pathway regulating the reproductive axis via thyroid hormone metabolism is capable of transducing temperature information. Because all vertebrates can potentially use temperature as a supplementary cue, these results are broadly applicable to understanding how environment-organism interactions mediate seasonally adaptive responses.