The loss of antioxidant activities impairs intestinal epithelium homeostasis by altering lipid metabolism.

Reactive oxygens species (ROS) are common byproducts of metabolic reactions and could be at the origin of many diseases of the elderly. Here we investigated the role of ROS in the renewal of the intestinal epithelium in mice lacking catalase (CAT) and/or nicotinamide nucleotide transhydrogenase (NNT) activities. Cat-/- mice have delayed intestinal epithelium renewal and were prone to develop necrotizing enterocolitis upon starvation. Interestingly, crypts lacking CAT showed fewer intestinal stem cells (ISC) and lower stem cell activity than wild-type. In contrast, crypts lacking NNT showed a similar number of ISCs as wild-type but increased stem cell activity, which was also impaired by the loss of CAT. No alteration in the number of Paneth cells (PCs) was observed in crypts of either Cat-/- or Nnt-/- mice, but they showed an evident decline in the amount of lysozyme. Cat deficiency caused fat accumulation in crypts, and a fall in the remarkable high amount of adipose triglyceride lipase (ATGL) in PCs. Notably, the low levels of ATGL in the intestine of Cat -/- mice increased after a treatment with the antioxidant N-acetyl-L-cysteine. Supporting a role of ATGL in the regulation of ISC activity, its inhibition halt intestinal organoid development. These data suggest that the reduction in the renewal capacity of intestine originates from fatty acid metabolic alterations caused by peroxisomal ROS.

Dll1 haploinsufficiency causes brain abnormalities with functional relevance.

Introduction: The Notch pathway is fundamental for the generation of neurons during development. We previously reported that adult mice heterozygous for the null allele of the gene encoding the Delta-like ligand 1 for Notch (Dll1lacZ ) have a reduced neuronal density in the substantia nigra pars compacta. The aim of the present work was to evaluate whether this alteration extends to other brain structures and the behavioral consequences of affected subjects. Methods: Brains of Dll1 +/lacZ embryos and mice at different ages were phenotypically compared against their wild type (WT) counterpart. Afterwards, brain histological analyses were performed followed by determinations of neural cell markers in tissue slices. Neurological deficits were diagnosed by applying different behavioral tests to Dll1 +/lacZ and WT mice. Results: Brain weight and size of Dll1 +/lacZ mice was significantly decreased compared with WT littermates (i.e., microcephaly), a phenotype detected early after birth. Interestingly, enlarged ventricles (i.e., hydrocephalus) was a common characteristic of brains of Dll1 haploinsufficient mice since early ages. At the cell level, general cell density and number of neurons in several brain regions, including the cortex and hippocampus, of Dll1 +/lacZ mice were reduced as compared with those regions of WT mice. Also, fewer neural stem cells were particularly found in the adult dentate gyrus of Dll1 +/lacZ mice but not in the subventricular zone. High myelination levels detected at early postnatal ages (P7-P24) were an additional penetrant phenotype in Dll1 +/lacZ mice, observation that was consistent with premature oligodendrocyte differentiation. After applying a set of behavioral tests, mild neurological alterations were detected that caused changes in motor behaviors and a deficit in object categorization. Discussion: Our observations suggest that Dll1 haploinsufficiency limits Notch signaling during brain development which, on one hand, leads to reduced brain cell density and causes microcephaly and hydrocephalus phenotypes and, on the other, alters the myelination process after birth. The severity of these defects could reach levels that affect normal brain function. Therefore, Dll1 haploinsufficiency is a risk factor that predisposes the brain to develop abnormalities with functional consequences.

Rats exhibit age-related mosaic loss of chromosome Y.

Mosaic loss of the Y chromosome (LOY) is the most frequent chromosomal aberration in aging men and is strongly correlated with mortality and disease. To date, studies of LOY have only been performed in humans, and so it is unclear whether LOY is a natural consequence of our relatively long lifespan or due to exposure to human-specific external stressors. Here, we explored whether LOY could be detected in rats. We applied a locus-specific PCR and target sequencing approach that we used as a proxy to estimate LOY in 339 samples covering eleven tissues from young and old individuals. We detected LOY in four tissues of older rats. To confirm the results from the PCR screening, we re-sequenced 60 full genomes from old rats, which revealed that the Y chromosome is the sole chromosome with low copy numbers. Finally, our results suggest that LOY is associated with other structural aberrations on the Y chromosome and possibly linked to the mosaic loss of the X chromosome. This is the first report, to our knowledge, demonstrating that the patterns of LOY observed in aging men are also present in a rodent, and conclude that LOY may be a natural process in placental mammals.

Sexually dimorphic dynamics of thyroid axis activity during fasting in rats.

Starvation induces tertiary hypothyroidism in adult rodents. Response of the hypothalamus-pituitary-thyroid (HPT) axis to starvation is stronger in adult males than in females. To improve the description of this sexual dimorphism, we analyzed the dynamics of HPT axis response to fasting at multiple levels. In adult rats of the same cohort, 24 and 48 h of starvation inhibited paraventricular nucleus Trh expression and serum concentrations of TSH and T4 earlier in males than in females, with lower intensity in females than in males. In adult females fasted for 36-72 h, serum TSH concentration decreased after 36 h, when the activity of thyrotropin-releasing hormone (TRH)-degrading ectoenzyme was increased in the median eminence. The kinetics of these events were distinct from those previously observed in male rats. We suggest that the sex difference in TSH secretion kinetics is driven not only at the level of paraventricular nucleus TRH neurons, but also by differences in post-secretory catabolism of TRH, with enhancement of TRH-degrading activity more sustained in male than female animals.

Deciphering Ancestral Sex Chromosome Turnovers Based on Analysis of Male Mutation Bias.

The age of sex chromosomes is commonly obtained by comparing the substitution rates of XY gametologs. Coupled with phylogenetic reconstructions, one can refine the origin of a sex chromosome system relative to specific speciation events. However, these approaches are insufficient to determine the presence and duration of ancestral sex chromosome systems that were lost in some species. In this study, we worked with genomic and transcriptomic data from mammals and squamates and analyzed the effect of male mutation bias on X-linked sequences in these groups. We searched for signatures indicating whether monotremes shared the same sex chromosomes with placental mammals or whether pleurodonts and acrodonts had a common ancestral sex chromosome system. Our analyses indicate that platypus did not share the XY chromosomes with placental mammals, in agreement with previous work. In contrast, analyses of agamids showed that this lineage maintained the pleurodont XY chromosomes for several million years. We performed multiple simulations using different strengths of male mutation bias to confirm the results. Overall, our work shows that variations in substitution rates due to male mutation bias could be applied to uncover signatures of ancestral sex chromosome systems.

Corytophanids Replaced the Pleurodont XY System with a New Pair of XY Chromosomes.

Almost all lizard families in the pleurodont clade share the same XY system. This system was meticulously studied in Anolis carolinensis, where it shows a highly degenerated Y chromosome and a male-specific X chromosome dosage compensation mechanism. Corytophanids (casque-headed lizards) have been proposed as the only family in the pleurodont clade to lack the XY system. In this study, we worked with extensive genomic and transcriptomic data from Basiliscus vittatus, a member of the Corytophanidae family that inhabits the tropical rainforests of Mexico. We confirmed that B. vittatus underwent a sex chromosome system turnover, which consisted in the loss of the pleurodont XY system and the gain of a new pair of XY chromosomes that are orthologous to chicken chromosome 17. We estimated the origin of the sex chromosome system to have occurred ∼63 Ma in the ancestor of corytophanids. Moreover, we identified 12 XY gametologues with particular attributes, such as functions related to the membrane and intracellular trafficking, very low expression levels, blood specificity, and incomplete dosage compensation in males.

Voluntary Exercise-Induced Activation of Thyroid Axis and Reduction of White Fat Depots Is Attenuated by Chronic Stress in a Sex Dimorphic Pattern in Adult Rats.

The activity of the hypothalamus-pituitary-thyroid (HPT) axis is inhibited by energy deficit, by acute or chronic stress, but activated by cold exposure or exercise. Because stress curtails acute cold induced activation of HPT, we evaluated the effect of chronic stress on HPT axis response to voluntary exercise, a persistent energy-demanding situation. Adult male and female Wistar rats were exposed to restraint stress, 30 min/day for 2 weeks, or to isolation (Iso) [post-natal day [PND] 30-63]. Exercise was performed (7 p.m.-7 a.m.) in a running wheel, sedentary controls stayed in individual cages (Sed); at 7 a.m. they were housed with their cage mate or individually (Iso); food intake by the exercised group was measured day and night to pair-fed Sed. At sacrifice, hormones, mRNA levels and tissue weights were quantified. Control or restrained adult rats had access to running wheel daily for 2 weeks. Compared to C, exercise decreased white adipose tissue (WAT) mass in females and males, increased hypothalamic paraventricular nucleus (PVN)-Trh expression in males proportionally to exercise performed, and increased TSH and T4 serum concentration in females. These changes were not detected in restrained groups. Starting at PND 63 control (2/cage) and isolated (1/cage) rats either exercised on 10 alternated nights or were sedentary. In control male animals, compared to Sed rats, exercise did not decrease WAT mass, nor changed HPT axis activity, but increased Pomc and deiodinase 2 (Dio2) expression in mediobasal hypothalamus (MBH), adrenergic receptor ß3 and uncoupling protein-1 in brown adipose tissue. In control female animals, exercise decreased WAT mass, increased Pomc, Dio2, and Trhde expression in MBH, and TSH serum concentration. Iso females had lower TSH and T4 serum concentration, Dio2 and Trhde expression in MBH than controls. The stress response was higher in isolated males than females, but in males it did not alter the effects of exercise, in contrast to isolated females that had a blunted response to exercise compared to controls. In conclusion, chronic stress interferes with metabolic effects produced by exercise, such as loss of WAT mass, coincident with dampening of HPT activity.

Neonatal Maternal Separation Alters, in a Sex-Specific Manner, the Expression of TRH, of TRH-Degrading Ectoenzyme in the Rat Hypothalamus, and the Response of the Thyroid Axis to Starvation.

Hypothalamic-pituitary-thyroid (HPT) axis activity is important for energy homeostasis, and is modified by stress. Maternal separation (MS) alters the stress response and predisposes to metabolic disturbances in the adult. We therefore studied the effect of MS on adult HPT axis activity. Wistar male and female pups were separated from their mothers 3 h/d during postnatal day (PND)2-PND21 (MS), or left nonhandled (NH). Open field and elevated plus maze tests revealed increased locomotion in MS males and anxiety-like behavior in MS females. At PND90, MS females had increased body weight gain, Trh expression in the hypothalamic paraventricular nucleus, and white adipose tissue mass. MS males had increased expression of TRH-degrading enzyme in tanycytes, reduced TSH and T3, and enhanced corticosterone serum concentrations. MS stimulated brown adipose tissue deiodinase 2 activity in either sex. Forty-eight hours of fasting (PND60) augmented serum corticosterone levels similarly in MS or NH females but more in MS than in NH male rats. MS reduced the fasting-induced drop in hypothalamic paraventricular nucleus-Trh expression of males but not of females and abolished the fasting-induced increase in Trh expression in both sexes. Fasting reduced serum concentrations of TSH, T4, and T3, less in MS than in NH males, whereas in females, TSH decreased in MS but not in NH rats, but T4 and T3 decreased similarly in NH and MS rats. In conclusion, MS produced long-term changes in the activity of the HPT axis that were sex specific; response to fasting was partially blunted in males, which could affect their adaptive response to negative energy balance.

Acute response of hypophysiotropic thyrotropin releasing hormone neurons and thyrotropin release to behavioral paradigms producing varying intensities of stress and physical activity.

The activity of the hypothalamus-pituitary-thyroid (HPT) axis is essential for energy homeostasis and is differentially modulated by physical and by psychological stress. Contradictory effects of stressful behavioral paradigms on TSH or thyroid hormone release are due to type, length and controllability of the stressor. We hypothesized that an additional determinant of the activity of the HPT axis is the energy demand due to physical activity. We thus evaluated the response of thyrotropin releasing hormone (TRH) neurons of the hypothalamic paraventricular nucleus (PVN) in Wistar male rats submitted to the elevated plus maze (EPM), the open field test (OFT), or restraint, and sacrificed within 1h after test completion; the response to OFT was compared during light (L) or dark (D) phases. Locomotion and anxiety behaviors were similar if animals were tested in L or D phases but their relation to the biochemical parameters differed. All paradigms increased serum corticosterone concentration; the levels of corticotropin releasing hormone receptor 1 and of glucocorticoid receptor (GR) mRNAs in the PVN were enhanced after restraint or OFT-L. Levels of proTRH mRNA increased in the PVN after exposure to EPM-L or OFT-D; serum levels of thyrotropin (TSH) and T(4) only after OFT-D. In contrast, restraint decreased TRH mRNA and serum TSH levels, while it increased TRH content in the mediobasal hypothalamus, implying reduced release. Expression of proTRH in the PVN varied proportionally to the degree of locomotion in OFT-D, while inversely to anxiety in the EPM-L, and to corticosterone in EPM-L and OFT-D. TRH mRNA levels were analyzed by in situ hybridization in the rostral, middle and caudal zones of the PVN in response to OFT-D; they increased in the middle PVN, where most TRH hypophysiotropic neurons reside; levels correlated positively with the velocity attained in the periphery of the OF and negatively, with anxiety. Variations of serum TSH levels correlated positively with locomotor activity in EPM-L and OFT-L or -D, while negatively to serum corticosterone levels in all paradigms. These results support the proposal that the hypophysiotropic PVN TRH neurons are activated by short term physical activity but that this response may be blunted by the inhibitory effect of stress.

The acute response of the amygdalar TRH system to psychogenic stressors varies dependent on the paradigm and circadian condition.

Central administration of thyrotropin releasing hormone (TRH) reduces anxiety; amygdalar TRH expression is inversely proportional to the anxious behavior displayed in the elevated plus maze performed during the dark phase (EPM-D). To better understand the role of TRH in amygdala function, we evaluated the expression of TRH and the elements involved in its transmission in various stressful paradigms and how they associated with behavior. Wistar male rats were exposed to restraint (RES), EPM, or the open field test (OFT) and sacrificed 0-60 min afterwards; OFT, RES and EPM were performed during the light (L), and OFT during the dark phase. Restraint increased amygdalar levels of proCRH mRNA, without change in proTRH. All paradigms augmented corticosterone release, highest after OFT-L that also enhanced proCRH mRNA levels and decreased those of proTRH. OFT-D activated the TRH system. Levels of anxiety or locomotion were similar in animals tested in light or dark phases but their association with biochemical parameters differed. ProTRH expression and TRH release correlated positively with decreased anxiety in EPM-L and in OFT-D. No association with anxiety was detected in OFT-L where proCRH and proTRH expression correlated with locomotion supporting their involvement in arousal. The responses of TRH amygdalar systems appeared modulated by the extent of the stress response and by the circadian conditions. Increased proTRH expression of animals exposed to OFT-D was specifically observed in the cortical nucleus of the amygdala, area involved in processing fear stimuli; these TRH neurons may thus be part of a circuit with anxiolytic properties.

Analysis of the anxiolytic-like effect of TRH and the response of amygdalar TRHergic neurons in anxiety.

Thyrotropin-releasing hormone (TRH) was first described for its neuroendocrine role in controlling the hypothalamus-pituitary-thyroid axis (HPT). Anatomical and pharmacological data evidence its participation as a neuromodulator in the central nervous system. Administration of TRH induces various behavioural effects including arousal, locomotion, analepsy, and in certain paradigms, it reduces fear behaviours. In this work we studied the possible involvement of TRHergic neurons in anxiety tests. We first tested whether an ICV injection of TRH had behavioural effects on anxiety in the defensive burying test (DBT). Corticosterone serum levels were quantified to evaluate the stress response and, the activity of the HPT axis to distinguish the endocrine response of TRH injection. Compared to a saline injection, TRH reduced cumulative burying, and decreased serum corticosterone levels, supporting anxiolytic-like effects of TRH administration. The response of TRH neurons was evaluated in brain regions involved in the stress circuitry of animals submitted to the DBT and to the elevated plus maze (EPM), tests that allow to correlate biochemical parameters with anxiety-like behaviour. In the DBT, the response of Wistar rats was compared with that of the stress-hypersensitive Wistar Kyoto (WKY) strain. Behavioural parameters were analysed in recorded videos. Animals were sacrificed 30 or 60min after test completion. In various limbic areas, the relative mRNA levels of TRH, its receptors TRH-R1 and -R2, and its inactivating ectoenzyme pyroglutamyl peptidase II (PPII), were determined by RT-PCR, TRH tissue content by radioimmunoassay (RIA). The extent of the stress response was evaluated by measuring the expression profile of CRH, CRH-R1 and GR mRNA in the paraventricular nucleus (PVN) of the hypothalamus and in amygdala, corticosterone levels in serum. As these tests demand increased physical activity, the response of the HPT axis was also evaluated. Both tasks increased the levels of serum corticosterone. WKY rats showed higher anxiety-like behaviour in the DBT than Wistar, as well as increased PVN mRNA levels of CRH and GR. TRH mRNA levels increased in the PVN and TSH values remained unchanged in both strains although TRH content decreased in the medial basal hypothalamus of Wistar rats only. TRH content was measured in several limbic regions but only amygdala showed specific task-related changes after DBT exposure of both strains: increased TRH content. Expression of TRH mRNA decreased in the amygdala of Wistar, suggesting inhibition of TRHergic neuronal activity in this region. The participation of amygdalar TRH neurons in anxiety was confirmed in the EPM where TRH expression and release correlated with the number of entries, and the % of time spent in open arms, supporting an anxiolytic role of these TRH-neurons. These results contribute to the understanding of the involvement of TRH during emotionally charged situations and shed light on the participation of particular circuits in related behaviours.