Fertility preservation for prepubertal male patients undergoing gonadotoxic therapies, potentially depleting spermatogonial cells, is an expanding necessity, yet most of the feasible options are still in the experimental phase. We present our experience and a summary of current and novel possibilities regarding the different strategies to protect or restore fertility in young male patients, before proceeding with chemotherapy or radiotherapy for malignances or other diseases. Adult oncological patients should always be counselled to cryopreserve the semen before starting treatment, however this approach is not suitable for prepubertal boys, who aren’t capable to produce sperm yet. Fortunately, since the survival rate of pediatric cancer patients has skyrocketed in the last decade and it’s over 84%, safeguarding their future fertility is becoming a major concern for reproductive medicine. Surgical and medical approaches to personalize treatment or protect the gonads could be a valid first step to take. Testicular tissue autologous grafting or xenografting, and spermatogonial stem cells (SSCs) transplantation, are the main experimental options available, but spermatogenesis in vitro is becoming an intriguing alternative. All of these methods feature both strong and weak prospects. There is also relevant controversy regarding the type of testicular material to preserve and the cryopreservation methods. Since transplanted cells are bound to survive based on SSCs number, many ways to enrich their population in cultures have been proposed, as well as different sites of injection inside the testis. Testicular tissue graft has been experimented on mice, rabbits, rhesus macaques and porcine, allowing the birth of live offspring after performing intracytoplasmic sperm injection (ICSI), however it has never been performed on human males yet. In vitro spermatogenesis remains a mirage, although many steps in the right direction have been performed. The manufacturing of 3D scaffolds and artificial spermatogenetic niche, providing support to stem cells in cultures, seems like the best way to further advance in this field.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 μg/ml and 5 μg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn’t sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 μg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

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IntroductionAmong substances released into the environment by anthropogenic activities, the heavy metal cadmium (Cd) is known to induce severe testicular injury causing male subfertility/infertility. Zinc (Zn) is another heavy metal that, unlike Cd, is physiologically present in the testis, being essential for spermatogenesis. We aimed to examine the possibility that 50 µM ZnCl2 could counteract the toxic effects induced by Cd in an in vitro model of porcine prepubertal Sertoli cells (SCs) exposed to both subtoxic (5 μM) and toxic (10 μM) concentrations of CdCl2 for 48 h.Materials and MethodsApoptosis, cell cycle, and cell functionality were assessed. The gene expression of Nrf2 and its downstream antioxidant enzymes, ERK1/2, and AKT kinase signaling pathways were evaluated.Materials and ResultsWe found that Zn, in co-treatment with subtoxic and toxic Cd concentration, increased the number of metabolically active SCs compared to Cd exposure alone but restored SC functionality only in co-treatment with subtoxic Cd concentration with respect to subtoxic Cd alone. Exposure of Cd disrupted cell cycle in SCs, and Zn co-treatment was not able to counteract this effect. Cd alone induced SC death through apoptosis and necrosis in a dose-dependent manner, and co-treatment with Zn increased the pro-apoptotic effect of Cd. Subtoxic and toxic Cd exposures activated the Nrf2 signaling pathway by increasing gene expression of Nrf2 and its downstream genes (SOD, HO-1, and GSHPx). Zn co-treatment with subtoxic Cd attenuated upregulation on the Nrf2 system, while with toxic Cd, the effect was more erratic. Studying ERK1/2 and AKT pathways as a target, we found that the phosphorylation ratio of p-ERK1/2 and p-AKT was upregulated by both subtoxic and toxic Cd exposure alone and in co-treatment with Zn.DiscussionOur results suggest that Zn could counteract Cd effects by increasing the number of metabolically active SCs, fully or partially restoring their functionality by modulating Nrf2, ERK1/2, and AKT pathways. Our SC model could be useful to study the effects of early Cd exposure on immature testis, evaluating the possible protective effects of Zn.