In our previous work, we reported alterations in protamines/histones ratio, in DNA binding of these proteins and their involvement in DNA oxidative damage in 84% of the young men living in the Land ...of Fires. In the present work, we extended our findings, evaluating any alterations in spermatozoa of a family case, a father and son, living in this area, to also give a first look at the possibility of transgenerational inherited effects of environmental contaminants on the molecular alterations of sperm nuclear basic proteins (SNBP), DNA and semen parameters. In the father and son, we found a diverse excess of copper and chromium in the semen, different alterations in SNBP content and low DNA binding affinity of these proteins. In addition, DNA damage, in the presence of CuCl
and H
O
, increased by adding both the father and son SNBP. Interestingly, son SNBP, unlike his father, showed an unstable DNA binding and were able to produce DNA damage even without external addition of CuCl
, in line with a lower seminal antioxidant activity than the father. The peculiarity of some characteristics of son semen could be a basis for possible future studies on transgenerational effects of pollutants on fertility.
The blood–brain barrier (BBB) selectively protects the central nervous system (CNS) from external insults, but its function can represent a limit for the passage of therapeutic molecules. Numerous
in ...vitro
models of the BBB have been realized in order to study the passage of drugs for neurodegenerative diseases, but these
in vitro
models are not very representative of the physiological conditions because of a limited supply of oxygen and nutrients due to static conditions. To avoid this phenomenon, we used a millifluidic bioreactor model that ensures a circulation of the medium and, therefore, of the nutrients, thanks to the continuous laminar flow. This dynamic model consists of a double-culture chamber separated by a membrane on which brain endothelial cells are cultured in order to evaluate the passage of the drug. Furthermore, in the lower chamber, SH-SY5Y were seeded as 3D spheroids to evaluate the drug passage through these cells. As nanodelivery system, we used liposomes functionalized with viral fusion peptide to evaluate the passage of a neuroprotective agent, pituitary adenylate cyclase-activating polypeptide (PACAP), through the dynamic
in vitro
model of the BBB. We showed that our nanodelivery system, made of functionalized liposomes and loaded with specific molecules, efficiently crosses the
in vitro
fluid-dynamic model of the BBB. Our findings represent an important step for further experimental investigations on PACAP administration as a therapeutic agent by an enhanced drug delivery system. Our results can improve the diffusion of good practice in neuroscience laboratories, helping to spread the 3R rules.
SARS-CoV-2 has become one of the most studied viruses of the last century. It was assumed that the only possible host for these types of viruses was mammalian eukaryotic cells. Our recent studies ...show that microorganisms in the human gastrointestinal tract affect the severity of COVID-19 and for the first time provide indications that the virus might replicate in gut bacteria. In order to further support these findings, in the present work, cultures of bacteria from the human microbiome and SARS-CoV-2 were analyzed by electron and fluorescence microscopy. The images presented in this article, in association with the nitrogen (
N) isotope-labeled culture medium experiment, suggest that SARS-CoV-2 could also infect bacteria in the gut microbiota, indicating that SARS-CoV-2 could act as a bacteriophage. Our results add new knowledge to the understanding of the mechanisms of SARS-CoV-2 infection and fill gaps in the study of the interactions between SARS-CoV-2 and non-mammalian cells. These findings could be useful in suggesting specific new pharmacological solutions to support the vaccination campaign.
Objective To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (ΔΨm), and apoptotic status of fresh and ...cryopreserved oocytes. Design Experimental study. Setting University biology research unit and private IVF unit. Patient(s) Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles. Main Outcome Measure(s) Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent ΔΨm reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Result(s) Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 ± 1.94/10 μm vs. fresh 10.30 ± 3.9/10 μm), swelling of smooth endoplasmic reticulum (F/T 0.084 ± 0.03 μm2 vs. fresh 0.040 ± 0.02 μm2 ), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low ΔΨm of pericortical mitochondria, but no signs of apoptosis. Conclusion(s) Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.
Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of
to 1, 10 and 100 pM HgCl
for 24 h on the gonadal morphology and on the expression ...level of three stress genes:
,
and
. In this tissue we also evaluated the level of steroidogenic enzymes 3β-HSD and 17β-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl
and hypo-expression of the three stress genes, particularly for
. Furthermore, decreased labelling with both 3β-HSD and 17β-HSD antibodies was observed following exposure to 1 and 10 pM HgCl
and complete absence at 100 pM HgCl
exposure. Gonads of mussels exposed to all HgCl
doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl
exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.
The objective of this paper is to evaluate adaptations in hepatic mitochondrial protein mass, function and efficiency in a rat model of high‐fat diet‐induced obesity and insulin resistance that ...displays several correlates to human obesity. Adult male rats were fed a high‐fat diet for 7 weeks. Mitochondrial state 3 and state 4 respiratory capacities were measured in liver homogenate and isolated mitochondria by using nicotinamide adenine dinucleotide, flavin adenine dinucleotide and lipid substrates. Mitochondrial efficiency was evaluated by measuring proton leak kinetics. Mitochondrial mass was assessed by ultrastructural observations and citrate synthase (CS) activity measurements. Mitochondrial oxidative damage and antioxidant defence were also considered by measuring lipid peroxidation, aconitase and superoxide dismutase (SOD) specific activity. Whole body metabolic characteristics were obtained by measuring 24‐h oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ) and nonprotein respiratory quotient (NPRQ), using indirect calorimetry with urinary nitrogen analysis. Whole body glucose homeostasis was assessed by measuring plasma insulin and glucose levels after a glucose load. Adult rats fed a high‐fat diet for 7 weeks, exhibit not only obesity, insulin resistance and hepatic steatosis, but also reduced respiratory capacity and increased oxidative stress in liver mitochondria. Our present results indicate that alterations in the mitochondrial compartment induced by a high‐fat diet are associated with the development of insulin resistance and ectopic fat storage in the liver. Our results thus fit in with the emerging idea that mitochondrial dysfunction can led to the development of metabolic diseases, such as obesity, type 2 diabetes mellitus and nonalcoholic steatohepatitis.
The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this ...paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one.
Finally, we demonstrated that in T. marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.
The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, ...i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 17β-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula.
The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard Podarcis sicula, a seasonal breeder, and Rattus rattus, a continuous ...breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both P. sicula and R. rattus. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of P. sicula epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of P. sicula epididymis, whereas in R. rattus it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.
Milk from various species differs in nutrient composition. In particular, human milk (HM) and donkey milk (DM) are characterized by a relative high level of triacylglycerol enriched in palmitic acid ...in sn-2 position. These dietary fats seem to exert beneficial nutritional properties through N-acylethanolamine tissue modulation. The aim of this study is to compare the effects of cow milk (CM), DM, and HM on inflammation and glucose and lipid metabolism, focusing on mitochondrial function, efficiency, and dynamics in skeletal muscle, which is the major determinant of resting metabolic rate. Moreover, we also evaluated the levels of endocannabinoids and N-acylethanolamines in liver and skeletal muscle, since tissue fatty acid profiles can be modulated by nutrient intervention.
To this aim, rats were fed with CM, DM, or HM for 4 weeks. Then, glucose tolerance and insulin resistance were analyzed. Pro-inflammatory and anti-inflammatory cytokines were evaluated in serum and skeletal muscle. Skeletal muscle was also processed to estimate mitochondrial function, efficiency, and dynamics, oxidative stress, and antioxidant/detoxifying enzyme activities. Fatty acid profiles, endocannabinoids, and N-acylethanolamine congeners were determined in liver and skeletal muscle tissue.
We demonstrated that DM or HM administration reducing inflammation status, improves glucose disposal and insulin resistance and reduces lipid accumulation in skeletal muscle. Moreover, HM or DM administration increases redox status, and mitochondrial uncoupling, affecting mitochondrial dynamics in the skeletal muscle. Interestingly, HM and DM supplementation increase liver and muscle levels of the N-oleoylethanolamine (OEA), a key regulator of lipid metabolism and inflammation.
HM and DM have a healthy nutritional effect, acting on inflammatory factors and glucose and lipid metabolism. This beneficial effect is associated to a modulation of mitochondrial function, efficiency, and dynamics and to an increase of OEA levels in skeletal muscle.
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