Project description:Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used advanced transcriptomic sequencing and high frequency resolution during the initiation, maintenance and recovery of circannual timing for Siberian hamster energy balance to delineate the molecular architecture of a neuroendocrine seasonal clock. Three distinct phases of transcript changes were identified, and deiodinase type-3 (Dio3) expression was activated during the initiation phase. Targeted mutation of Dio3 resulted in a shortening of the circannual interval timer period. Hamsters that exhibit a form of naturally occurring disruption in Dio3 eliminated circannual interval timing. Our work demonstrates that the evolutionary conserved changes in hypothalamic tanycytes expression of Dio3 is critical for timing seasonal life history traits in chordates.
Project description:The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C), or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass-spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass-spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.
Project description:Most temperate animals exhibit seasonal rhythms in reproductive physiology and behaviour. Gonadotropin-releasing hormone (GnRH) cells in the preoptic area (POA) and kisspeptin (Kiss) cells in the anteroventral periventricular (AvPv) nucleus are critical for timing photoperiod-induced changes in seasonal reproduction. In response to prolonged exposure to photoperiod cues, many rodents exhibit endogenously generated programmed changes in physiology that reflect a circannual timing mechanism. Here we used transcriptome sequencing coupled with high tissue sampling across a simulated circannual interval timer to characterise the molecular changes involved in the seasonal control of reproduction and body temperature. Adult male Djungarian hamsters were collected after exposure to short photoperiod on 4-week intervals from 4- to 32-weeks. The POA and AvPv transcriptomes from shot-photoperiod hamsters were compared to a long photoperiod reference group. Our analyses confirmed robust photoperiodic regulation of Kiss1 mRNA expression in the AvPv. Weighted gene co-expression network analyses (WGCNA) identified hundreds (POA: 211, AvPv: 415) of transcript that were associated with testes mass. Gene set enrichment analysis (GSEA) identified ‘gonadotropin secretion’ and ‘glial cell proliferation’ as the primary pathways associated with circannual interval timing. Vimentin had strong negative association with testes mass, indicating AvPv glial morphology increased during gonadal involution. In the POA, pathways associated with ‘regulation at synapse’ and ‘regulation of synaptic plasticity’ were highly enriched and transcripts positively associated with gonadal involution indicated that ribosomal plasticity and intracellular calcium signalling are key mechanisms involved in seasonal reproduction. WGCNA analyses identified 567 in the POA were significantly associated with surface body temperature. GSEA discovered thyrotrophin releasing hormone was significantly negatively associated with surface body temperature. Overall, the POA and AvPv transcriptome datasets provide the foundation to expand our understanding of the molecular representation of seasonal time in the mammalian hypothalamus.
Project description:The post-mortem interval (PMI) is the time that elapses since the death of an individual until the body is found. Different molecules have been analyzed to better estimate the PMI with variable results. The miRNAs draw attention in the forensic field to estimate the PMI, since they can better support degradation. To find potential biomarkers for PMI estimation, we analyzed the miRNome at early PMI in rat skeletal muscle using the Affymetrix GeneChip™ miRNA 4.0 micoarrays. In this dataset, we include the expression of 1218 rat miRNAs at early postmortem interval.
Project description:To identify potential microRNA candidates for the post mortem interval estimation using a transcriptome-based approach, we extracted total RNAs from myocardial tissue samples of BALB/c mice