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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.

Follicle-stimulating hormone (FSH) is the main regulator of spermatogenesis and plays a key role in the development and function of the reproductive system. To assess the effects of different FSH preparations in combination with testosterone on porcine pre-pubertal Sertoli cells, we performed Real Time PCR analysis of AMH, inhibin B and FSH-r, Western blotting analysis of AKT-posphoAKT, ERK1/2-posphoERK1/2, ELISA assay for AMH and inhibin B and a high-throughput proteomic analysis.We observed that all three preparations induced a reduction of AMH in terms of mRNA and secreted protein and, an increase of inhibin B in terms of mRNA in all the formulations while, only α-follitropin induced an increase of inhibin B secreted in the culture medium. Proteomic analysis permitted us to identify 46 secreted proteins.Of those, the SPARC protein was down-regulated after the treatment with testosterone associated with α-follitropin, β-follitropin and urofollitropin (vs group stimulated with T alone). 11 proteins were up-regulated by the different FSH preparations. In detail, Hemoglobin subunit beta, TPA and TPI have been observed to be up-regulated by stimulation with testosterone in addition with α-follitropin or with β-follitropin and or with urofollitropin. All preparations induced an increase in the secreted inhibin beta A chain, but in the medium after stimulation with urofollitropin we observed an higher increase in the levels of this protein. β-follitropin, associated with testosterone, specifically induces an up-regulation of 8 specific secreted proteins.Our study, showing that the three FSH preparations were associated with different effects, could offer the opportunity to shed light inside applications to personalized reproductive medicine.

Recent studies have shown that extracellular vesicles (Ev) are an important mechanism of intercellular communication. In fact, Ev may contain proteins, DNA and mRNA. In particular, the latter play an important role in various biological processes including regulation and cell differentiation [1]. Sertoli cells (SC), previously considered as a mere “sustentacular cell”, were re-evalued in their functions and elevated to the rank of a “sentinel” in spermatogenesis due to production of trophic, differentiation and immune-modulators factors. Porcine pre-pubertal SC, isolated by our method [2], upon 48 hours culture, SC were stimulated with recombinant a-follitropin (rFSH) or FSH + testosterone (T) to evaluate both the presence in the medium of SC-derived Ev (SC-Ev) and SC-Ev content, in terms of mRNA for Anti-Müllerian hormone (AMH), inhibin B, Androgen Binding Protein (ABP) and FSH-receptor (FSH-r), by RT-PCR. SEM analysis highlighted the presence of SC-Nv in culture medium with mean diameters < 149 nm. We have also demonstrated, within the SC-Ev, significant increase in mRNA for AMH, ABP and FSH-r after both FSH and FSH+T stimulation, as compared to unstimulated SC-Ev. Differently from unstimulated SC-Ev, mRNA inhibin B levels were unchanged in FSH-stimulated SC-Ev, and increased after FSH+T stimulation. Interestingly, an opposite trend was shown in mRNA secretion, in control SC monolayer where, we demonstrated a decrease of AMH and FSH-r mRNA (after both stimulations with FSH or FSH + T) and an increase of inhibin B mRNA. On the contrary, mRNA ABP levels, in SC monolayer, decreased after stimulation with FSH but were unchanged in the presence of FSH+T. For the first time in the Literature, our work has shown the presence of SC-Nv containing AMH, inhibin B, ABP and FSH-r mRNA regulated by FSH with or without T. This result may suggest that other testicular cells could produce factors that, until now, were considered an exclusive SC secretory product.This work was supported by Mr.Gary Harlem (Altucell Inc. 3 Astor Court, Dix Hills, New York, NY) and Merck-Serono (London, UK)., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

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., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

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 infertility, Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

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., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Introduction and Aim. Type 2 Diabetes Mellitus (T2DM), one of the world’s most important, common and costly diseases associated with devastating complications, is caused by insulin resistance mainly due to a chronic inflammation of the visceral adipose tissue with local and systemic increases in proinflammatory cytokines and adipokines such as tumor necrosis factor-α (TNF-α) and IL-6. Sertoli cells (SC), considered mere mechanical cell aids, have been recently revisited with respect to their functional competence showing that these cells may provide immunomodulatory and trophic factors that are able to ameliorate survival and development of different cell types and constitute an immuno-protective shield for transplantation in many diseases such as Type 1 Diabetes Mellitus. Aim of this work was to verify if the injection of free (subcutaneously) or microencapsulated (intraperitoneally) SC would reverse hyperglycaemia in db/db mice spontaneous T2DM Materials and Methods. Porcine pre-pubertal SC were isolated, according to previously established methods, after finely chopping the retrieval testicles, with double enzymatic digestion with Collagenase P and a mixed solution of trypsin and DNase I. SC enveloped in Barium alginate-based microcapsules (Ba-SCMCs) were prepared according to our method, by a mono air-jet device system. Free SC and Ba-SCMCs were examined as far as: (a) SC morphology by light microscopy; (b) SC viability, by fluorescence microscopy after staining with ethidium bromide and fluorescein diacetate; (c) SC in vitro function (α-aromatase activity and IGF-I secretion); (d) reversal of T2DM in spontaneous diabetes db/db mice, were concerned. Results. Ba-SCMCs showed excellent features in terms of size, morphology, sphericity and coalescence. SC viability, both in free and microencapsulated SC, was very high (over 90%). Very good α-aromatase activity and IGF-I secretion were associated with the examined SC preparations. Both subcutaneous free SC injection and intraperitoneal transplantation of Ba-SCMCs demonstrated a significant reduction, in 60% of the treated mice, of HbA1c (6.6 % vs 8.8 %) with a normalization of intraperitoneal glucose tolerance test. Conclusions. SC may be enveloped in Ba-SCMCs with no loss of their functional properties and morphology. Xenograft of SC, both free and enveloped in barium alginate microcapsules, induced an important reduction of HbA1c blood levels with a normalization of glucose tolerance test (IPGTT). This result might open new perspectives for the future therapy of human T2DM., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011