Isolation and characterization of extracellular vesicles secreted by pre-pubertal Sertoli cells

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)

'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

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

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

Xenograft of free or microencapsulated Sertoli cells as a potential therapy for experimental Type 2 Diabetes Mellitus

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

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

Comparative in vitro studies on the fibrogenic effects of two samples of silica on epithelial bronchial cells

Abstract The small dimension and particle shape of silica in gypsum used to prepare moulds for lost wax casting might be responsible for the high prevalence of silicosis in gold jewellery workers. To test this hypothesis, human pulmonary epithelial cell (BEAS 2B) cultures were exposed to two samples of silica with different crystal micro-morphologies: Silica Powder dust (Silica P) which is used in casting gold jewellery, and no powder Silica (Silica F). Extracellular matrix (ECM) production was evaluated using radio labelled precursors and quantified by RT PCR analysis. Expression of basic fibroblast growth factor (FGF 2) and its receptor (FG FR2) was also evaluated. The results demonstrated Silica P particles had a very fine lamellar crystalline structure, while Silica F particles was characterized by larger rounded crystals. Silica P stimulated collagen production significantly more than Silica F and downregulated laminin and metalloprotease expression. Both silica samples down-regulated FGF 2 but only Silica F enhanced FGF2 receptor expression. In conclusion each Silica sample promoted a profibrotic lung microenvironment in a different manner and also elicited different FGF 2 signalling pathways. The data confirm that different micromorphology of Silica particles affects the fibrogenic potential and the molecular mechanisms of silica dust pathogenicity

Silica particle size and shape: in vitro effects on extracellular matrix metabolism and viability of human bronchial epithelial cells

Crystal micro-morphology and dimension of silica particles could be responsible for the high prevalence of silicosis as recently found among goldsmiths. In the present study we investigated two samples of silica particles with different surface sizes and shapes for their capacity to induce changes in ECM component production. In addition we investigated if their different effects could be related to cytotoxicity and apoptotic effects. Human bronchial epithelial cells were cultured with or without a sample of Silica used for casting gold jewellery, named in our experiments Silica P or a commercial sample of Silica with different physical and chemical properties, named in our experiments Silica F. After 48 h of exposure PCR analysis determined levels of several matrix components. As induction of the apoptosis cascade, annexin assay, caspase 3 activity and cellular cytoxicity by MTT assay were assayed. Silica F promoted fibronectin, MMP12, tenascin C and Integrins b5 gene expressions more than Silica P. Silica P stimulated more TGFß1 and its TGFßR1 receptor than Silica F. Cytotoxic effects were induced by the two samples of Silica. On the contrary, no alteration in classic apoptotic marker protein expression was observed in presence of either Silica F or Silica P, suggesting silica particles affect ECM production and metalloproteases through a mechanism that does not involve apoptotic activation. Different Silica micromorphology and TGFß signal pathway are linked to lung fibrotic effects but the potential role Silica in apoptotic and toxic reaction remains to be ascertained