A new model to study the effects of gonadotropins on an “in vitro” prepubertal artificial porcine mini-testis

At present, there is no reliable experimental model “in vitro” to analyze the complex interactions between gonadotropins on the pre-pubertal Sertoli cells (SC) and Leydig cells (LD). Considering that, in the pre-pubertal period, only the anti-mullerian hormone (AMH) is upregulated by FSH and down-regulated by androgens [1-2], AMH could be considered a potential marker of pre-pubertal testis function. The aim of our work was to study the effects of FSH, LH and HCG on an in-vitro model of “mini-testis”. SC and LD, obtained from 15-20 days old neonatal pigs, were isolated and evaluated in terms of purity by AMH (unique pre-pubertal SCs marker), INSL3 (LD marker), ASMI (peritubular cells marker) and PGP9.5 (gonocytes and spermatogonial cells marker). Finally, purified SC and LD were co-cultured to obtain the “mini-testis” and were stimulated with gonadotropins. We have then evaluated: a) AMH, inhibin B and testosterone levels released in the culture medium (by ELISA), both in basal conditions and after stimulations; b) analysis of the follicle-stimulating hormone receptor (FSHR), MAPkinasi (Erk1/2, AKT) by Real Time PCR. We show an increase in inhibin B levels after FSH and FSH/LH stimulation and a selectively increase in testosterone production after LH treatment. AMH secretion was downregulated by FSH treatment. These data seem to preliminarily suggest that ERK1/ ERK2 expression was up-regulated by FSH and FSH/LH stimulation while FSHreceptor expression was down-regulated by FSH and increased by FSH/LH treatment; AKT was up-regulated in all conditions. The proposed model, by creating an artificial mini- testis, could help better understanding the complex and still partially unknown interactions between human gonadotropins, SC and LD possibly creating a novel background to shed light inside a future therapy of male infertilit

Effects of cadmium on viability and function of porcine pre-pubertal Sertoli cells

Cadmium, an ubiquitous environmental pollutant mainly used for industrial purposes, is highly associated with reproductive toxicity. Sertoli cells (SC), by providing an appropriate microenvironment for the development of germ cells, play a pivotal role on spermatogenesis regulation (Geoffroy-Siraudin et al. 2012). Aim of our investigation was to assess the effects of cadmium on high mammalian SC viability and function. Porcine pre-pubertal SC were isolated, according to previously established methods (Fallarino et al. 2009) and treated with 3 different concentrations (5-10-15 μM) of cadmium chloride. The evaluation of SC function in terms of inhibin B and anti-Müllerian hormone (AMH) secretion showed a significant decrease in all SC treated conditions respect as compared to SC control. On the contrary, evaluation of the FSH-R integrity on SC surface, in terms of 17-b-estradiol production under FSH stimulation, showed no difference between SC control and 5 μM cadmium treated SC monolayers in comparison to 10 and 15μM cadmium treated SC monolayers, where FSH-R was impaired. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all cadmium treated SC conditions in comparison with SC control. In conclusion, our data demonstrate that cadmium, even at low dose, exerts toxic effects on Sertoli cells function possibly adversely affecting the spermatogenesis

'In vitro' Effect of Different Follicle¿Stimulating Hormone Preparations on Sertoli Cells: Toward a Personalized Treatment for Male Infertility

Follicle-stimulating hormone (FSH), a major regulator of spermatogenesis, has a crucial function in the development and function of the testis and it is extensively given as a fertility treatment to stimulate spermatogenesis. We analyzed the effects of different FSH preparations (α-follitropin, β-follitropin, and urofollitropin) in combination with testosterone on porcine pre-pubertal Sertoli cells. To study the effect of the different FSH treatments in the Sertoli cell function we performed Real Time PCR analysis of AMH, inhibin B, and FSH-r, an ELISA assay for AMH and inhibin B, and a high-throughput comparative proteomic analysis. We verified that all three preparations induced a reduction of AMH in terms of mRNA and secreted proteins, and an increase of inhibin B in terms of mRNA in all the FSH formulations, while solely α-follitropin produced an increase of secreted inhibin B in the culture medium. Comparative proteomic analysis of the three FSH preparations identified 46 proteins, 11 up-regulated and 2 down-regulated. Surprisingly, the combination of testosterone with β-follitropin specifically induced an up-regulation of eight specific secreted proteins. Our study, showing that the three different FSH preparations induce different effects, could offer the opportunity to shed light inside new applications to a personalized reproductive medicine

Pb effects on an experimental model of porcine prepubertal Sertoli cells

The environmental pollution is one of the main factors implicated in the world’s fertility decline. Lead (Pb) is one of the major heavy metal contaminants that impairs several organs but preferentially accumulates in male reproductive organs and alters in vivo and in vitro sperm quality [1]. Nowadays, the underlying mechanisms remain unclear. Sertoli cells (SC) provides structural and metabolic support to the spermatogenic cells within the seminiferous tubules, therefore, metabolic and structural changes in SC affect the developing germ cells and consequently alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of Pb would adversely affect superior mammalian SC function. Highly purified and functional porcine pre-pubertal SC were isolated [2] and treated with three different Pb acetate concentrations. Parameters of SC functionality, such as inhibin B and anti-Müllerian hormone (AMH) mRNAs and proteins were decreased by Pb exposure respect to the control, such as the FSH-r integrity in terms of 17-β-estradiol production, under FSH stimulation. In addition, we observed an increase of AKT and mTOR mRNAs, p38 phosphorylation ratio and Akt phosphorylation ratio in all experimental conditions, respect to the control. In conclusion, the Pb-related toxicity on SC, even at low concentrations, is expected to alter spermatogenesis

Nickel oxide nanoparticles exposure as a risk factor for male infertility: “In vitro” effects on porcine pre-pubertal Sertoli cells

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

Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: A potential product for cell transplant therapy

Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGβ-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation