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 preservation.

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Model of the our research was the adult male amphibian anura, Pelophylax bergeri, poikilotherm species not considered threatened by the IUCN, sampled in representative sites at different degree. In the first phase, a biochemical characterization of the ADP-ribosylation on the skin of barcoded amphibian anura collected from Matese Lake (clean reference site in CE, Italy) was carried out. Two PARP isoforms were evidence: the first of 66 kDa is localized into nucleus and activated by DNA damage; the second of 150 kDa is in cytoplasm, as demonstrated by biochemical and immunohistochemical analysis. Subsequently, the PARP activity, the quantitative expression of androgen receptor gene, and the levels of arsenic and chromium in skin and testis of frog and soil, water, and sediment collected from sites at different degrees of pollution were measured. A significant variation of PARP activity and androgen receptor expression levels was detected in both tissues of barcoded frogs from Sarno and Scafati, along Sarno River (SA, Italy), suggesting that a PARP activation is correlated to pollution and to steroid-regulated physiology disruption.

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.

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.