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Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 μg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erβ, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female.

Anatomia comparata: alla ricerca dell’antenato comune. L’obiettivo primo dello studio anatomico comparativo è la ricostruzione della filogenesi, dei caratteri informativi di un’ascendenza comune, per acquisire le conoscenze sulla morfologia descrittiva e funzionale e per valutare i passaggi evolutivi per i diversi sistemi: in tre parole, forma, funzione ed evoluzione. Il volume presenta peculiari caratteristiche che lo rendono unico nel panorama editoriale: è nuovo e accattivante, per il linguaggio semplice e preciso; è duttile, perché risulta leggero senza essere superficiale, approfondito, ma mai ostico; è coinvolgente, perché la straordinaria eloquenza delle immagini, realizzate appositamente per l'opera, tocca livelli mai raggiunti in un testo di anatomia comparata; è aggiornato all’attualità delle conoscenze scientifiche. Un’altra caratteristica fondamentale dell’opera risiede nei contenuti digitali cui può accedere sia lo studente sia il docente. Sono disponibili filmati, lezioni on line, esperienze di laboratorio e un ampio database di domande a disposizione dei docenti e degli studenti. Inoltre, il docente potrà avere accesso a tutte le immagini in formato elettronico.

Chlorpyrifos (CPF) is one of the most used organophosphorus insecticide (OP) and putative developmental neurotoxicant in different organisms and also in humans.1,2 Maternal exposure to CPF was found to be dose-related to slower brain growth and associated to several structural abnormalities such as thinning of cerebral cortex.3 In mice, prenatal CPF exposure has the potential to affect long-term brain cognitive function, but the mechanisms are still unknown.3 It has been demonstrated that even sub-lethal doses of neurotoxic pesticides affect behaviour by inhibiting acetylcholinesterase (AChE). To elucidate through what mechanism CPF induces alterations in neurogenesis, we used the M u s m u s c u l u s as animal model. F2 offspring (3-8 month) of Mus musculus females, treated with 3 doses of CPF (0.1-1-10 mg/kg/d) during pregnancy, were used to study the effects of chronic exposure to this compound. The analysis was performed on 84 genes associated to Parkinson disease, using RT2 Profiler PCR Arrays. There was a statistically significant dose-dependent correlation between increasing CPF concentration and the down-regulation for many genes studied at 3 and 8 month mice. It was interesting to note that, among the genes significantly down-regulated, there were those more directly related to Parkinson’s disease, such as the Park2, Sv2b, Gabbr2, Sept5, Atxn2, whereas the expression of the Ubiquitin C, Rgs4 and Chgb showed an up-regulation in 8 month mice. Furthermore, we found alteration of the AChE activity, that was inhibited by the exposure to CPF. The results of our study confirm that CPF can elicit alterations in the gene expression profile during neurogenesis and reinforce the view that the critical window of vulnerability of the developing brain to CPF extends from gestational exposure through postnatal period.4

Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen., 2016. © 2016 Wiley Periodicals, Inc.

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

Recently a great interest surrounds the development of nanoparticles (NPs) for potential biomedical applications such as drug delivery, cancer therapy and medical imaging. NP delivery system in vivo promises to overcome the epithelial barriers, key obstacle to the administration of drugs, vaccines, plasmid DNA and RNAi material. However, so far the interplay and hazards between nanoscale materials and biological systems have not been fully understood. Highlight of these data we have evaluated the effect of polystyrene nanoparticles (PS-NPs), widely used as model and reference particles, on primary culture of bovine oviductal epithelial cells (BOEC) that form a epithelial barrier of female reproductive tracts involved in the secretion of products essential for gametes and embryos. BOEC have been cultured with 44 nm fitc-PS-NPs 10ug/ml for 7 days until achievement of cell confluence and treated to study NP influence on the cellular proliferation and chromosomal arrangement, using also confocal microscope, and gene expression by differential display. The confocal analysis has not revealed the NP presence in the nucleus during cell division and the karyotype analysis has not showed stastistically significant chromosomal anomalies compared to control sample. However study of gene expression by differential display has exposed interesting alterations. Particularly, BOEC treated with NPs have showed the activation of expression of a band 100 bp and one of 600 bp. The sequencing has revealed an identity of 99% between the 100 bp band and Bos taurus microsatellite DNA clone RP42-69E7, whereas the 600 bp band shared and identity of 87% with Bos taurus breed Hereford chromosome 5 genomic scaffold (3517 bp at 5' side: GRAM domaincontaining protein 4 6521 bp at 3' side: ceramide kinase (CERK). Microsatellite sequences may occur within promoters and other cisregulatory regions, playing an important role in modulating of gene expression under stress. The phosphorylation of ceramide (C-1-P) via CERK has been shown to stimulate the production of signaling molecules of cell proliferation. Moreover the interaction of C-1-P with phosphatidylinositol 3-kinase/Akt and Mammalian target of rapamycin indicate that disregulated CERK expression may lead to cancer. Our results demonstrate that PS-NPs seem do not affect cell division and chromosomal rearrangement, but may interfere with gene expression and progression in cell cycle. Our data represent a preliminary new contribute to understand the complicated interactions and the effects between NPs and biological systems. However other studies are necessary before NP application in biomedical field.

All eukaryotic genomes contain mobile elements and their role in the shaping of eukaryotic genomes is becoming more and more recognized, although the proportion and activity of the different classes of these elements widely varies between genomes. We used an in silico approach to analyze the presence and distribution of one of this family, the V-SINE sequences, in Danio rerio chromosomes. V-SINEs is a superfamily of vertebrate short interspersed repetitive elements, whose central domain is strongly conserved and is even found in SINEs in the lamprey genome, suggesting that V-SINEs might be ~550 Myr old. The central conserved domain might have been subject to some form of positive selection. A total of 108625 V-SINEs fragments have been identified on the 25 assembled Danio rerio chromosomes (danrer6), accounting for about 0.019% of the entire sequence. The average number of V-SINEs fragments per chromosome is about 4372 (±943), ranging from 2628 to 6571. We estimated that about 20% (±3%) of V-SINES per chromosome were found to be located within genic sequences, the main contribution due to their presence in the introns. In particular, we found a striking bias of V-SINES localization in 5? UTRs with respect to the 3? ones. The 5?UTR/3? ratio was estimate to be about 6.73. Our results seem to confirm that V-SINEs preferentially accumulate within relatively gene-rich regions, as previously seen by their in situ mapping on chromosomes. Besides, since, in mice, evidences have already shown that SINEs located in 5?UTR can contribute to the repression of gene expression, we suggest that, in Danio, the biased distribution of V-SINES in the 5?UTR vs 3? UTR regions, could be a scratch of their potential role in gene regulation.