RESUMEN
Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.
Asunto(s)
Pollos , Codorniz , Animales , Humanos , Femenino , Masculino , Codorniz/genética , Patos/genética , MaxilaresRESUMEN
OBJECTIVES: Skeletal malocclusions are common, and severe malocclusions are treated by invasive surgeries. Recently, jaw bone length has been shown to be developmentally controlled by osteoclasts. Our objective was to determine the effect of inhibiting osteoclast-secreted proteolytic enzymes on lower jaw bone length of avian embryos by pharmacologically inhibiting matrix metalloproteinase-9 (MMP9) or cathepsin K (CTSK). METHODS: Quail (Coturnix coturnix japonica) embryos were given a single dose of an inhibitor of MMP9 (iMMP9), an inhibitor CTSK (iCTSK), or vehicle at a developmental stage when bone deposition is beginning to occur. At a developmental stage when the viscerocranium is largely calcified, the heads were scanned via micro-computed tomography and reproducible landmarks were placed on 3D-reconstructed skulls; the landmark coordinates were used to quantify facial bone dimensions. RESULTS: Approximately half of the quail given either iMMP9 or iCTSK demonstrated an overt lower jaw phenotype, characterized by longer lower jaw bones and a greater lower to upper jaw ratio than control embryos. Additionally, iMMP9-treated embryos exhibited a significant change in midface length and iCTSK-treated embryos had significant change in nasal bone length. CONCLUSION: MMP9 and CTSK play a role in osteoclast-mediated determination of lower jaw bone length. Pharmacological inhibition of MMP9 or CTSK may be a promising therapeutic alternative to surgery for treating skeletal jaw malocclusions, but more preclinical research is needed prior to clinical translation.
Asunto(s)
Coturnix , Metaloproteinasa 9 de la Matriz , Animales , Catepsina K/genética , Microtomografía por Rayos X , OsteoclastosRESUMEN
We recently reported that compared with males, female mice have increased hepatic mitochondrial respiratory capacity and are protected against high-fat diet-induced steatosis. Here, we sought to determine the role of estrogen in hepatic mitochondrial function, steatosis, and bile acid metabolism in female mice and investigate potential benefits of exercise in the absence or presence of estrogen via ovariectomy (OVX). Female C57BL mice (n = 6 per group) were randomly assigned to sham surgery (sham), ovariectomy (OVX), or OVX plus estradiol replacement therapy (OVX + Est). Half of the mice in each treatment group were sedentary (SED) or had access to voluntary wheel running (VWR). All mice were fed a high-fat diet (HFD) and were housed at thermoneutral temperatures. We assessed isolated hepatic mitochondrial respiratory capacity using the Oroboros O2k with both pyruvate and palmitoylcarnitine as substrates. As expected, OVX mice presented with greater hepatic steatosis, weight gain, and fat mass gain compared with sham and OVX + Est animals. Hepatic mitochondrial coupling (basal/state 3 respiration) with pyruvate was impaired following OVX, but both VWR and estradiol treatment rescued coupling to levels greater than or equal to sham animals. Estradiol and exercise also had different effects on liver electron transport chain protein expression depending on OVX status. Markers of bile acid metabolism and excretion were also impaired by ovariectomy but rescued with estradiol add-back. Together our data suggest that estrogen depletion impairs hepatic mitochondrial function and liver health, and that estradiol replacement and modest exercise can aid in rescuing this phenotype.NEW & NOTEWORTHY OVX induces hepatic steatosis in sedentary mice which can be prevented by modest physical activity (VWR) and/or estradiol treatment. Estrogen impacts hepatic mitochondrial coupling in a substrate-specific manner. OVX mice have impaired fecal bile acid excretion, which was rescued with estradiol treatment.
Asunto(s)
Estradiol/uso terapéutico , Hígado Graso/prevención & control , Hígado/fisiopatología , Mitocondrias Hepáticas/fisiología , Ovariectomía , Condicionamiento Físico Animal/fisiología , Animales , Terapia Combinada , Estradiol/farmacología , Terapia por Ejercicio , Hígado Graso/etiología , Hígado Graso/patología , Hígado Graso/fisiopatología , Femenino , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/efectos de los fármacos , Ovariectomía/efectos adversosRESUMEN
The impact of sexual dimorphism and mitophagy on hepatic mitochondrial adaptations during the treatment of steatosis with physical activity are largely unknown. Here, we tested if deficiencies in liver-specific peroxisome proliferative activated-receptor-γ coactivator-1α (PGC-1α), a transcriptional coactivator of biogenesis, and BCL-2/ADENOVIRUS EIB 19-kDa interacting protein (BNIP3), a mitophagy regulator, would impact hepatic mitochondrial adaptations (respiratory capacity, H2O2 production, mitophagy) to a high-fat diet (HFD) and HFD plus physical activity via voluntary wheel running (VWR) in both sexes. Male and female wild-type (WT), liver-specific PGC-1α heterozygote (LPGC-1α), and BNIP3 null mice were thermoneutral housed (29-31°C) and divided into three groups: sedentary-low-fat diet (LFD), 16 wk of (HFD), or 16 wk of HFD with VWR for the final 8 wk (HFD + VWR) (n = 5-7/sex/group). HFD did not impair mitochondrial respiratory capacity or coupling in any group; however, HFD + VWR significantly increased maximal respiratory capacity only in WT and PGC-1α females. Males required VWR to elicit mitochondrial adaptations that were inherently present in sedentary females including greater mitochondrial coupling control and reduced H2O2 production. Females had overall reduced markers of mitophagy, steatosis, and liver damage. Steatosis and markers of liver injury were present in sedentary male mice on the HFD and were effectively reduced with VWR despite no resolution of steatosis. Overall, reductions in PGC-1α and loss of BNIP3 only modestly impacted mitochondrial adaptations to HFD and HFD + VWR with the biggest effect seen in BNIP3 females. In conclusion, hepatic mitochondrial adaptations to HFD and treatment of HFD-induced steatosis with VWR are more dependent on sex than PGC-1α or BNIP3.
Asunto(s)
Dieta Alta en Grasa , Mitocondrias Hepáticas/metabolismo , Esfuerzo Físico , Animales , Dieta con Restricción de Grasas , Femenino , Regulación de la Expresión Génica , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Consumo de Oxígeno , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Condicionamiento Físico Animal , Conducta Sedentaria , Caracteres SexualesRESUMEN
Malocclusions are common craniofacial malformations which cause quality of life and health problems if left untreated. Unfortunately, the current treatment for severe skeletal malocclusion is invasive surgery. Developing improved therapeutic options requires a deeper understanding of the cellular mechanisms responsible for determining jaw bone length. We have recently shown that neural crest mesenchyme (NCM) can alter jaw length by controlling recruitment and function of mesoderm-derived osteoclasts. Transforming growth factor beta (TGF-ß) signaling is critical to craniofacial development by directing bone resorption and formation, and heterozygous mutations in TGF-ß type I receptor (TGFBR1) are associated with micrognathia in humans. To identify what role TGF-ß signaling in NCM plays in controlling osteoclasts during mandibular development, mandibles of mouse embryos deficient in the gene encoding Tgfbr1 specifically in NCM were analyzed. Our lab and others have demonstrated that Tgfbr1fl/fl;Wnt1-Cre mice display significantly shorter mandibles with no condylar, coronoid, or angular processes. We hypothesize that TGF-ß signaling in NCM can also direct later bone remodeling and further regulate late embryonic jaw bone length. Interestingly, analysis of mandibular bone through micro-computed tomography and Masson's trichrome revealed no significant difference in bone quality between the Tgfbr1fl/fl;Wnt1-Cre mice and controls, as measured by bone perimeter/bone area, trabecular rod-like diameter, number and separation, and gene expression of Collagen type 1 alpha 1 (Col1α1) and Matrix metalloproteinase 13 (Mmp13). Though there was not a difference in localization of bone resorption within the mandible indicated by TRAP staining, Tgfbr1fl/fl;Wnt1-Cre mice had approximately three-fold less osteoclast number and perimeter than controls. Gene expression of receptor activator of nuclear factor kappa-ß (Rank) and Mmp9, markers of osteoclasts and their activity, also showed a three-fold decrease in Tgfbr1fl/fl;Wnt1-Cre mandibles. Evaluation of osteoblast-to-osteoclast signaling revealed no significant difference between Tgfbr1fl/fl;Wnt1-Cre mandibles and controls, leaving the specific mechanism unresolved. Finally, pharmacological inhibition of Tgfbr1 signaling during the initiation of bone mineralization and resorption significantly shortened jaw length in embryos. We conclude that TGF-ß signaling in NCM decreases mesoderm-derived osteoclast number, that TGF-ß signaling in NCM impacts jaw length late in development, and that this osteoblast-to-osteoclast communication may be occurring through an undescribed mechanism.
RESUMEN
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 adults and contributes to advanced liver injury and cardiometabolic disease. While recent evidence points to involvement of the brain in NAFLD, the downstream neural circuits and neuronal molecular mechanisms involved in this response, remain unclear. Here, we investigated the role of a unique forebrain-hypothalamic circuit in NAFLD. METHODS: Chemogenetic activation and inhibition of circumventricular subfornical organ (SFO) neurons that project to the paraventricular nucleus of the hypothalamus (PVN; SFOâPVN) in mice were used to study the role of SFOâPVN signaling in NAFLD. Novel scanning electron microscopy techniques, histological approaches, molecular biology techniques, and viral methodologies were further used to delineate the role of endoplasmic reticulum (ER) stress within this circuit in driving NAFLD. RESULTS: In lean animals, acute chemogenetic activation of SFOâPVN neurons was sufficient to cause hepatic steatosis in a liver sympathetic nerve dependent manner. Conversely, inhibition of this forebrain-hypothalamic circuit rescued obesity-associated NAFLD. Furthermore, dietary NAFLD is associated with marked ER ultrastructural alterations and ER stress in the PVN, which was blunted following reductions in excitatory signaling from the SFO. Finally, selective inhibition of PVN ER stress reduced hepatic steatosis during obesity. CONCLUSIONS: Collectively, these findings characterize a previously unrecognized forebrain-hypothalamic-ER stress circuit that is involved in hepatic steatosis, which may point to future therapeutic strategies for NAFLD.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Obesidad , Núcleo Hipotalámico Paraventricular/fisiología , Sistema Nervioso SimpáticoRESUMEN
Climate change models often assume similar responses to temperatures across the range of a species, but local adaptation or phenotypic plasticity can lead plants and animals to respond differently to temperature in different parts of their range. To date, there have been few tests of this assumption at the scale of continents, so it is unclear if this is a large-scale problem. Here, we examined the assumption that insect taxa show similar responses to temperature at 96 sites in grassy habitats across North America. We sampled insects with Malaise traps during 2019-2021 (N = 1041 samples) and examined the biomass of insects in relation to temperature and time of season. Our samples mostly contained Diptera (33%), Lepidoptera (19%), Hymenoptera (18%), and Coleoptera (10%). We found strong regional differences in the phenology of insects and their response to temperature, even within the same taxonomic group, habitat type, and time of season. For example, the biomass of nematoceran flies increased across the season in the central part of the continent, but it only showed a small increase in the Northeast and a seasonal decline in the Southeast and West. At a smaller scale, insect biomass at different traps operating on the same days was correlated up to ~75 km apart. Large-scale geographic and phenological variation in insect biomass and abundance has not been studied well, and it is a major source of controversy in previous analyses of insect declines that have aggregated studies from different locations and time periods. Our study illustrates that large-scale predictions about changes in insect populations, and their causes, will need to incorporate regional and taxonomic differences in the response to temperature.