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.

Effects of Follicle-Stimulating Hormone on Human Sperm Motility In Vitro.

To evaluate whether the follicle-stimulating hormone (FSH) receptor (FSHR) is expressed in human spermatozoa and the effects of FSH incubation on sperm function. Twenty-four Caucasian men were recruited. Thirteen patients had asthenozoospermia, and the remaining 11 had normal sperm parameters (controls). After confirming FSHR expression, spermatozoa from patients and controls were incubated with increasing concentrations of human purified FSH (hpFSH) to reassess FSHR expression and localization and to evaluate progressive and total sperm motility, the mitochondrial membrane potential, and protein kinase B (AKT) 473 and 308 phosphorylation. FSHR is expressed in the post-acrosomal segment, neck, midpiece, and tail of human spermatozoa. Its localization does not differ between patients and controls. Incubation with hpFSH at a concentration of 30 mIU/mL appeared to increase FSHR expression mainly in patients. Incubation of human spermatozoa with hpFSH overall resulted in an overall deterioration of both progressive and total motility in patients and controls and worse mitochondrial function only in controls. Finally, incubation with FSH increased AKT473/tubulin phosphorylation to a greater extent than AKT308. FSHR is expressed in the post-acrosomal region, neck, midpiece, and tail of human spermatozoa. Contrary to a previous study, we report a negative effect of FSH on sperm motility and mitochondrial function. FSH also activates the AKT473 signaling pathway.

Zinc restores functionality in porcine prepubertal Sertoli cells exposed to subtoxic cadmium concentration via regulating the Nrf2 signaling pathway.

Introduction: Among 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 Methods: Apoptosis, 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 Results: We 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. Discussion: Our 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.

Sperm-carried IGF2 downregulated the expression of mitogens produced by Sertoli cells: A paracrine mechanism for regulating spermatogenesis?

Introduction: Insulin-like growth factor 2 (IGF2) mRNA has been found in human and mouse spermatozoa. It is currently unknown whether the IGF2 protein is expressed in human spermatozoa and, if so, its possible role in the cross-talk between germ and Sertoli cells (SCs) during spermatogenesis. Methods: To accomplish this, we analyzed sperm samples from four consecutive Caucasian men. Furthermore, to understand its role during the spermatogenetic process, porcine SCs were incubated with increasing concentrations (0.33, 3.33, and 10 ng/mL) of recombinant human IGF2 (rhIGF2) for 48 hours. Subsequently, the experiments were repeated by pre-incubating SCs with the non-competitive insulin-like growth factor 1 receptor (IGF1R) inhibitor NVP-AEW541. The following outcomes were evaluated: 1) Gene expression of the glial cell-line derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), and stem cell factor (SCF) mitogens; 2) gene and protein expression of follicle-stimulating hormone receptor (FSHR), anti-Müllerian hormone (AMH), and inhibin B; 3) SC proliferation. Results: We found that the IGF2 protein was present in each of the sperm samples. IGF2 appeared as a cytoplasmic protein localized in the equatorial and post-acrosomal segment and with a varying degree of expression in each cell. In SCs, IGF2 significantly downregulated GDNF gene expression in a concentration-dependent manner. FGF2 and SCF were downregulated only by the highest concentration of IGF2. Similarly, IGF2 downregulated the FSHR gene and FSHR, AMH, and inhibin B protein expression. Finally, IGF2 significantly suppressed the SC proliferation rate. All these findings were reversed by pre-incubation with NVP-AEW541, suggesting an effect mediated by the interaction of IGF2 with the IGFR. Conclusion: In conclusion, sperm IGF2 seems to downregulate the expression of mitogens, which are known to be physiologically released by the SCs to promote gonocyte proliferation and spermatogonial fate adoption. These findings suggest the presence of paracrine regulatory mechanisms acting on the seminiferous epithelium during spermatogenesis, by which germ cells can influence the amount of mitogens released by the SCs, their sensitivity to FSH, and their rate of proliferation.

Effect of EPA on Neonatal Pig Sertoli Cells "In Vitro": A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies.

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 µM vs SCs and ***p < 0.001 at 3.33µM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 µM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 µM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Effects of Titanium Dioxide Nanoparticles on Porcine Prepubertal Sertoli Cells: An "In Vitro" Study.

The increasing use of nanomaterials in a variety of industrial, commercial, medical products, and their environmental spreading has raised concerns regarding their potential toxicity on human health. Titanium dioxide nanoparticles (TiO2 NPs) represent one of the most commonly used nanoparticles. Emerging evidence suggested that exposure to TiO2 NPs induced reproductive toxicity in male animals. In this in vitro study, porcine prepubertal Sertoli cells (SCs) have undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposures at both subtoxic (5 µg/ml) and toxic (100 µg/ml) doses of TiO2 NPs. After performing synthesis and characterization of nanoparticles, we focused on SCs morphological/ultrastructural analysis, apoptosis, and functionality (AMH, inhibin B), ROS production and oxidative DNA damage, gene expression of antioxidant enzymes, proinflammatory/immunomodulatory cytokines, and MAPK kinase signaling pathway. We found that 5 µg/ml TiO2 NPs did not induce substantial morphological changes overtime, but ultrastructural alterations appeared at the third week. Conversely, SCs exposed to 100 µg/ml TiO2 NPs throughout the whole experiment showed morphological and ultrastructural modifications. TiO2 NPs exposure, at each concentration, induced the activation of caspase-3 at the first and second week. AMH and inhibin B gene expression significantly decreased up to the third week at both concentrations of nanoparticles. The toxic dose of TiO2 NPs induced a marked increase of intracellular ROS and DNA damage at all exposure times. At both concentrations, the increased gene expression of antioxidant enzymes such as SOD and HO-1 was observed whereas, at the toxic dose, a clear proinflammatory stress was evaluated along with the steady increase in the gene expression of IL-1α and IL-6. At both concentrations, an increased phosphorylation ratio of p-ERK1/2 was observed up to the second week followed by the increased phosphorylation ratio of p-NF-kB in the chronic exposure. Although in vitro, this pilot study highlights the adverse effects even of subtoxic dose of TiO2 NPs on porcine prepubertal SCs functionality and viability and, more importantly, set the basis for further in vivo studies, especially in chronic exposure at subtoxic dose of TiO2 NPs, a condition closer to the human exposure to this nanoagent.

"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, ß-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 ß-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.

Effects of nicotine on porcine pre-pupertal sertoli cells: An in vitro study.

Smoke components, such as nicotine and its major metabolites, cross the blood-testis barrier and are detectable in the seminal plasma of both active smokers and individuals exposed to cigarette smoke. In vivo studies in a rat model have further demonstrated that nicotine exposure reduces the weight of the testis, as well as the number of spermatocytes and spermatids, and affects the ultrastructure of Sertoli cells (SC) - which serve as sentinels of spermatogenesis - causing intense germ cell sloughing in the tubular lumen that compromises offspring fertility. This study sought to determine the effects of nicotine on the viability and function of purified pig pre-pubertal SC. Nicotine exposure reduced the mRNA expression and protein levels of anti-Mullerian hormone (AMH) and inhibin B and impaired FSH-r sensitivity via the downregulation of FSH-r and aromatase gene expression compared to untreated SC. Overall, our study suggests that nicotine can significantly alter extracellular matrix and tight junction protein gene expression (e.g., laminin, integrin, and occludin), thus compromising cross-talk between the interstitial and tubular compartments and enhancing blood-testis barrier (BTB) permeability via downregulation of the mitogen-activated protein kinase (MAPK) pathway. These findings further elucidate a potential mechanism of action underlying nicotine exposure's detrimental effects on SC function in vivo.

In "Vitro" Lps-Stimulated Sertoli Cells Pre-Loaded With Microparticles: Intracellular Activation Pathways.

Sertoli cells (SC) are immune privileged cells with the capacity of modulating the immune response by expressing several immune-regulatory factors. SC have the capacity to respond to external stimuli through innate phagocytic and antibacterial activities. This evidence evoked a potential role of SC as drug carriers and therapeutic agents. Such stimuli drive SC towards a still unknown evolution, the clinical relevance of which as yet remains undisclosed. This study sought to investigate the effects of external stimuli in the form of polymeric microparticles (MP) and bacteria derived endotoxins, such as lipopolysaccharides (LPS), in order to identify the pathways potentially involved in cell phenotype modifications. Compared to single stimulation, when combined, MP and LPS provoked a significant increase in the gene expression of IDO, PD-L1, FAS-L, TLR-3, TLR-4, MHC-II, ICAM-1, TFGß1, BDF123, BDF129, BDF3 and pEP2C. Western Blotting analysis demonstrated up-regulation of the ERK 1-2 and NF-kB p65 phosphorylation ratios. Our study, showing the exponential increase of these mediators upon combined MP and LPS stimulation, suggests a "switch" of SC function from typical cells of the blood-testicular barrier to nonprofessional tolerogenic antigen-presenting cells. Further studies should target the clinical and technological implications of such stimuli-induced SC transformation.

Effects of Insulin on Porcine Neonatal Sertoli Cell Responsiveness to FSH In Vitro.

There is ongoing debate as to whether the decline of sperm production in recent times may be related to a parallel increase in the rate of obesity and diabetes. Lower anti-Müllerian hormone (AMH) and inhibin B secretion have been observed in young hyperinsulinemic patients compared to healthy controls, suggesting a Sertoli cell (SC) dysfunction. The pathophysiological mechanisms underlying SC dysfunction in these patients are poorly understood. To the best of our knowledge, no evidence is available on the effects of insulin on SC function. Therefore, this study was undertaken to assess the effects of insulin on basal and follicle-stimulating hormone (FSH)-stimulated SC function in vitro. To accomplish this, we evaluated the expression of AMH, inhibin B and FSHR genes, the secretion of AMH and inhibin B and the phosphorylation of AKT473 and SC proliferation on neonatal porcine SC after incubation with FSH and/or insulin. We found that similar to FSH, the expression and secretion of AMH is suppressed by insulin. Co-incubation with FSH and insulin decreased AMH secretion significantly more than with FSH alone. Insulin had no effect on the expression and secretion of the inhibin B gene, but co-incubation with FSH and insulin had a lower effect on inhibin B secretion than that found with FSH alone. FSH and/or insulin increased AKT473 phosphorylation and SC proliferation. In conclusion, the results of this study showed that insulin modulates SC function. We hypothesize that hyperinsulinemia may therefore influence testicular function even before puberty begins. Therefore, particular care should be taken to avoid the onset of hyperinsulinemia in children to prevent a future deleterious effect on fertility.

Effects of GH and IGF1 on Basal and FSH-Modulated Porcine Sertoli Cells In-Vitro.

Several lines of evidence suggest that insulin-like growth factor 1 (IGF1) is involved in Sertoli cell (SC) proliferation and that its receptor (IGF1R) could mediate follicle-stimulating hormone (FSH) effects. To examine the role of the growth hormone (GH)-IGF1 axis on SC function, we evaluated the effects of GH and IGF1 on basal and FSH-modulated SC proliferation, as well as on anti-Müllerian hormone (AMH) and inhibin B expression and secretion in-vitro. SCs from neonatal pigs were incubated with (1) placebo, (2) 100 nM highly purified urofollitropin (hpFSH), (3) 100 nM recombinant GH (rGH), (4) 100 nM recombinant IGF1 (rIGF1), (5) 100 nM hpFSH plus 100 nM rGH, (6) 100 nM hpFSH plus 100 nM rIGF1, for 48 h. We found that IGF1, but not FSH nor GH, stimulated SC proliferation. Furthermore, an inhibitory effect of FSH, GH and IGF1 on AMH secretion, and a stimulatory role of FSH and IGF1, but not GH, on inhibin B secretion were found. These results suggest that the GH-IGF1 axis influences basal and FSH-modulated SC proliferation and function. We speculate that SC proliferation occurring in childhood might be supported by the increased serum IGF1 levels observed during this period of life.

The IGF1 Receptor Is Involved in Follicle-Stimulating Hormone Signaling in Porcine Neonatal Sertoli Cells.

Experimental evidence has shown that the IGF1 receptor (IGF1R) is involved in testicular development during embryogenesis. More recently, data gathered from mice granulosa cells and zebrafish spermatogonia suggest that IGF1R has a role in Follicle-stimulating hormone (FSH) signaling. No evidence has been reported on this matter in Sertoli cells (SCs) so far. The aim of the study was to evaluate the role, if any, of the IGF1R in FSH signaling in SCs. The effects of FSH exposure on myosin-phosphatase 1 (MYPT1), ERK 1/2, AKT308, AKT473, c-Jun N-terminal kinase (JNK) phosphorylation and on anti-Müllerian hormone (AMH), inhibin B and FSH receptor (FSHR) mRNA levels were assessed with and without the IGF1R inhibitor NVP-AEW541 in purified and functional porcine neonatal SCs. Pre-treatment with NVP-AEW541 inhibited the FSH-induced MYPT1 and ERK 1/2 phosphorylation, decreased the FSH-dependent Protein kinase B (AKT)308 phosphorylation, but did not affect the FSH-induced AKT473 and JNK phosphorylation rate. It also interfered with the FSH-induced AMH and FSHR down-regulation. No influence was observed on the FSH-stimulated Inhibin B gene expression. Conclusion. These findings support the role of theIGF1R in FSH signaling in porcine SCs. The possible influence of IGF1 stimulation on the FSH-mediated effects on SCs should be further explored.

Acute effects of lead on porcine neonatal Sertoli cells in vitro.

Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-ß-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.

An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3ß-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

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

BACKGROUND: 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 (TFGß-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.

In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFß/TGFß receptor (TGFßR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, ß1 and ß5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and ß3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFß and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFß/TGFßR signaling.

Reversal of experimental Laron Syndrome by xenotransplantation of microencapsulated porcine Sertoli cells.

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

Toremifene decreases type I, type II and increases type III receptors in desmoid and fibroma and inhibits TGFbeta1 binding in desmoid fibroblasts.

Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.

Differences in extracellular matrix production and basic fibroblast growth factor response in skin fibroblasts from sporadic and familial Alzheimer's disease.

Extracellular matrix (ECM) molecules and growth factors, such as fibroblast growth factor (FGF), play a crucial role in Alzheimer's disease (AD). The purpose of this investigation was to determine whether phenotypic alterations in ECM production are present in non-neuronal AD cells associated with different FGF expression and response. Synthesis of glycosaminoglycans (GAG) and collagen were measured in skin fibroblasts from patients with familial, sporadic AD (FAD and SAD respectively), and from age-matched controls by radiolabeled precursors. Proteoglycans (PG), metalloprotease (MMP)-1, and FGF gene expressions were measured by reverse transcription-polymerase chain reaction. The results showed different ECM neosynthesis and mRNA levels in the two AD fibroblast populations. FAD accumulated more collagen and secreted less GAG than SAD. Biglycan PG was upregulated in FAD while betaglycan, syndecan, and decorin were markedly downregulated in SAD fibroblasts. We found a significant decrease of MMP1, more marked in FAD than in SAD fibroblasts. Constitutive FGF expression was greatly reduced in both pathological conditions (SAD>FAD). Moreover, an inverse high affinity/low affinity FGF receptor ratio between SAD and FAD fibroblasts was observed. FGF treatment differently modulated ECM molecule production and gene expression in the two cell populations. These observations in association with the changes in FGF gene expression and in the FGF receptor number, suggest that cellular mechanisms downstream from FGF receptor binding are involved in the two different forms of AD.