Reproductive expression dynamics and comparative toxicological perspective of beta estrogen receptor gene in the male wall lizard, Podarcis sicula Rafinesque, 1810 (Chordata: Reptilia)

Over the last few decades, due to its relevant function in male reproduction assessment, important molecular achievements have been made in the molecular characterization of estrogen receptor genes in various species. Our work focuses on a male seasonal breeder, the bioindicator Podarcis sicula, because of its peculiar gonadal anatomy, similar to that of humans. Based on the cloned lizard's gene sequence fragment of estrogen receptor beta, esr2 (GenBank JN705543.1), we found DNA binding domain identity of 99% as well as a homologous sequence with humans. Furthermore, in order to better illustrate how this gene is regulated in the lizard's reproductive system organs, we investigated the transcriptional activity of esr2 in brain and testis tissues during mating and winter stasis phases of the reproductive cycle. Quantitative real time-polymerase chain reaction (qRT-PCR) analyses performed on male gonadal tissues demonstrate a significant increase in esr2 expression during mating compared to the winter stasis period, while in the brain, esr2 shows the opposite trend. Next, we provide morphological evidence of the detrimental effect on spermatogenesis of a pure anti-estrogen treatment (ICI 182,780) and the corresponding effect on esr2 expression in lizard specimens during the mating period which, upon treatment, was found to be no different from the expression levels in winter stasis both in the brain and in the testis. In this study, we explore the potential use of Podarcis sicula as a model for human testis development and maturation, as well as esr2 expression for toxicological screening in one-testis gonadectomy

Fisheries and biodiversity along Mediterranean Sea: Italian and Egyptian coast overview

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The Sarno river and the bio-preservation of species of the genus Pelophylax

The set of living beings, animals and plants that inhabit our planet in their wide variety, as the result of long and complex evolutionary processes, represent the diversity of life on Earth, defined by the term biodiversity. During the twentieth century, the world has experienced an unprecedented technological and industrial explosion, that has had several negative consequences, including an increasing risk due to the effects that some compound and industrial discharges, means of threats to biodiversity (Massa, 2005; Agapow et al., 2004). Environmental degradation, the introduction of non-native species, over-exploitation of species, climate change, the presence of environmental contaminants, and the use of fertilizers, pesticides and often unpleasant herbicides exert toxic effects on the endocrine system and have, each, a prominent place among the risk factors for reproductive health and therefore for the maintenance of a specie (Hoffmann et al., 2010; Stork, 2010). Scientific research, lately, basically it’s oriented at the study of the toxic effects that many compounds have on molecules, cells, tissues, organisms and populations belonging to different classes of vertebrates with the aim to identifying, evaluating the damage and even more to predict the risk of species extinction (Mace et al., 2012). The following study, starting from biosensors potentially at risk of extinction species, such as Amphibians belonging to the genus Pelophylax, present along one of the most polluted river in Europe, develops and validates, with non-invasive biomarkers, skin and testis by gonadectomy, an innovative approach in environmental diagnostics for their preservation and the subsequent evaluation of the state of health of watercourses. In the first part of this research was detected and monitored the state of the health of the Plain of the Sarno river on the base of previous studies, by a synergic analysis of physical, chemical, biological and eco-toxicological components, which permitted to assess the ecological vulnerability of the river ecosystem, evidenced by the actual subletal stress on the organisms present in the Plain, due to the concentration of pollutants deriving from human and industrial activities. The study of the area allowed to define two favorite site, with different pollution degree. In the sites was monitored the presence of different frogs belonging to the genus Pelophylax, which were, in first instance, morphologically identified by classic taxonomic keys; since the difficult to specifically identify some specimens each other, due to the phenomenon of hybridization, only specimens of dubious belonging were analyzed by molecular approach, using the barcoding method. This even permitted to widen the genetic database for the specie Pelophylax bergeri, chosen as biosentinel specie. Furthermore, the evaluation of the oxidative and genotoxic damage on skin biopsy and gonadectomy, was carried out to the assessment of innovative non-invasive techniques. The second part of this research was devoted to develop an effective method for an eco-monitoring based on the analysis of ROS species in frog tissues, by an optimized spin-trapping EPR protocol. Furthermore, the genotoxic damage was detected by mobility shift, by monitoring the fragmentation degree of DNA, extracted by skin biopsy and gonadectomized testis. It’s interesting to note that, while the skin resulted to be damaged in both sites of Sarno River, testis seem to have developed a repair mechanism. This carried out at the study of enzymes involved in repair systems. After characterization and purification of the enzyme PARP-2, which is known to be responsible for the repair of DNA damage caused by heavy metals, the attention was paid to the testis. So, thanks to the examination of repair to DNA during spermatogenesis we realized the PARP, implicated in genome surveillance and protection, might represent in frog spermatogenesis an appealing tool for genotoxic risk assessment, useful to define a warning alarm for its survival

The amphibian Pelophylax bergeri (Günther, 1986) testis poly(ADP-ribose) polymerases: relationship to endocrine disruptors during spermatogenesis

In a growing deterioration of the ecosystem both in terms of biodiversity and ecological features, even more problematic is the conservation of species. The aim of this work was to study seasonal variations of biochemical and molecular DNA damage markers in Pelophylax bergeri exposed to potential endocrine disrupting chemicals. Frogs were collected in the main phase of the reproductive cycle: in April and May, during active spermatogenesis and the breeding season, and in October and November, in spermatogenetic regression, from a sampling site in the polluted Sarno River and from a clean reference site (Matese Lake). DNA profile and poly(ADPribose) polymerase (PARP) activity were evaluated in the testis. In the main phases of the reproductive cycle, all specimens from Sarno River showed a PARP activity higher than that measured in the same frog’s testis from Matese Lake. In addition, the PARP activity in active spermatogenesis was always higher compared with the activity in the spermatogenetic regression in Sarno River frogs. PARP2, usually activated in response to exposure to heavy metals, was expressed in all testes. In the species examined from Sarno River, no evident correlation between testis DNA damage and PARP activation was found. The working hypothesis is that PARP, implicated in genome surveillance and protection, might represent in frog spermatogenesis an appealing tool for genotoxic risk assessment useful to define a warning alarm for its survival

Molecular characterization of some species in the Sarno river

LOCUS HQ199217 238 bp DNA linear VRT 13-SEP-2010 DEFINITION Salmo trutta fario cytochrome b gene, partial cds; mitochondrial. ACCESSION HQ19921

Lizard neuroendocrine disruptor assessment by Phospholipid Hydroperoxide Glutathione Peroxidase (GPx4/PHGPx) expression

Phospholipid hydroperoxide glutathione peroxidase (GPx4/PHGPx) is a most abundant selenoprotein in the brain and GPx4 null mice die in utero at midgestation or knockdown of GPx4 during mammalian embryogenesis disturbs brain development. The current experiments were designed to investigate, by semiquantitative RT-PCR, the cerebral function of GPx4 of the lizards collected in both polluted (Sarno River, Italy) and unpolluted (Lago Matese, Italy) areas in order to provide useful molecular information on this widely distributed seleno-enzyme in adult as well through the development of the lizard, Podarcis sicula. Our research demonstrates the lizard GPx4 hormonal control. Treatments with HCG increases immunoreactivity while the anti-estrogen ICI 182-780 induces its reduction. The partial region of the cloned cDNA is of 340 bp; conserved protein domains reports its membership to selenoprotein and analysis amino acid residues the value as an antioxidant. The semiquantitative expression of GPx 4 of adult lizard brain in polluted station resulted generally very high and let sustain its importance as lizard neuroendocrine disruptor alarm

Roles of reactive oxygen species in the spermatogenesis regulation

Spermatogenesis is a complex process of male germ cells proliferation and maturation from diploid spermatogonia, through meiosis, to mature haploid spermatozoa.The process involves dynamic interactions between the developing germ cells and their supporting Sertoli cells. The gonadal tissue, with abundance of highly unsaturated fatty acids, high rates of cell division, and variety of testis enzymes results very vulnerable to the overexpression of reactive oxygen species (ROS). In order to address this risk, testis has developed a sophisticated array of antioxidant systems comprising both enzymes and free radical scavengers.This chapter sets out the major pathways of testis generation, the metabolism of ROS, and highlights the transcriptional regulation by steroid receptors of antioxidant stress enzymes and their functional implications. It also deals with of the advantages of the system biology for an antioxidant under steroid control, the major selenoprotein expressed by germ cells in the testis, the phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) having multiple functions and representing the pivotal link between selenium, sperm quality, and species preservatio