M. Galdieri

Prevention of spontaneous autoimmune diabetes in NOD mice by transferring in vitro antigen-pulsed syngeneic dendritic cells.

To evaluate the effect of antigen-pulsed dendritic cell (DC) transfer on the development of diabetes, 5-week-old female NOD mice received a single iv injection of splenic syngeneic DC from euglycemic NOD mice pulsed in vitro with human γ globulin (HGG). Eleven of 12 mice were protected from the development of diabetes up to the age of 25 weeks, and the insulitis score was significantly reduced. In contrast, NOD mice receiving unpulsed splenic DCs showed histological signs of insulitis and course of type 1 diabetes similar to untreated NOD mice. Treatment with HGG-pulsed DC was associated with profound modifications of cytokine secretory capacities within the islets. Thus, supernatants of islets from these mice contained increased levels of interleukin (IL)-4, IL-10, and, to a lesser extent, interferon-γ and diminished levels of tumor necrosis factor-α compared with controls. Because exogenous IL-4 and IL-10 exert antidiabetogenic effect in NOD mice and early blockade of endogenous tumor necrosis factor-α ...

Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide.

Streptozotocin (STZ) is a widely used diabetogenic agent that damages pancreatic islet β cells by activating immune mechanisms, when given in multiple low doses, and by alkylating DNA, when given at a single high dose. Actually, STZ contains a nitroso moiety. Incubation of rat islets with this compound has been found to generate nitrite; moreover, photoinduced NO production from STZ has been demonstrated. These reports have suggested that direct NO generation may be a mechanism for STZ toxicity in diabetogenesis. Several other studies have denied such a mechanism of action. This study has shown that (1) the multiple low‐dose (MLDS) treatment does not stimulate NO production at the islet level; in fact, nitrite + nitrate levels and aconitase activity (also in the presence of an NO‐synthase inhibitor, namely NAME) remain unmodified; RT‐PCR analysis demonstrates that this treatment does not stimulate iNOS activity; (2) the high‐dose (HDS) treatment does not stimulate NO production; in fact nitrite + nitrate levels remain unmodified and iNOS mRNA levels are not altered, although aconitase activity is significantly decreased. Moreover, we have confirmed that the MLDS treatment is able to decrease SOD activity by day 11 and that STZ, given in a single high dose, transiently increases superoxide dismutase (SOD) values (24 h from the administration), then dramatically lowers SOD levels. On the basis of our results, we conclude that STZ, “in vivo” is unable to generate NO, both as a MLDS or HDS treatment, thus excluding that NO exerts a role in streptozotocin‐dependent diabetes mellitus. J. Cell. Biochem. 77:82–91, 2000.

Diabetic rat testes: morphological and functional alterations

Reproductive dysfunction is a consequence of diabetes, but the underlying mechanisms are poorly understood. This study investigated the histological and molecular alterations in the testes of rats injected with streptozotocin at prepuperal (SPI rats) and adult age (SAI rats) to understand whether diabetes affects testicular tissue with different severity depending on the age in which this pathological condition starts. The testes of diabetic animals showed frequent abnormal histology, and seminiferous epithelium cytoarchitecture appeared altered as well as the occludin distribution pattern. The early occurrence of diabetes increased the percentage of animals with high number of damaged tubules. The interstitial compartment of the testes was clearly hypertrophic in several portions of the organs both in SPI and SAI rats. Interestingly, fully developed Leydig cells were present in all the treated animals although abnormally distributed. Besides the above‐described damages, we found a similar decrease in plasma testosterone levels both in SPI and SAI rats. Oxidative stress (OS) is involved in the pathogenesis of various diabetic complications, and in our experimental models we found that manganese superoxide dismutase was reduced in diabetic animals. We conclude that in STZ‐induced diabetes, the altered spermatogenesis, more severe in SPI animals, is possibly due to the effect of OS on Leydig cell function which could cause the testosterone decrease responsible for the alterations found in the seminiferous epithelium of diabetic animals.

Embryonic mouse testis development: Role of platelet derived growth factor (PDGF-BB)

Platelet‐derived growth factors (PDGFs) are paracrine growth factors mediating epithelial–mesenchymal interactions and exerting multiple biological activities which include cell proliferation, motility, and differentiation. As previously demonstrated, PDGFs act during embryonic development and recently, by culturing male genital ridges, we have demonstrated that PDGF‐BB is able to support in vitro testicular cord formation. In the present paper, we report that PDGF‐BB is present during embryonic testis development and, in organ culture, induces cord formation although with reduced diameters compared with the cords formed in the genital ridges cultured in the presence of HGF. Moreover we have analyzed the roles exerted by this growth factor during the morphogenesis of the testis. We demonstrate by immunohistochemical experiments that PDGF‐BB and its receptors are synthesized by the male UGRs isolated from 11.5 and 13.5 dpc embryos and by Western blot that the factor is secreted in a biologically active form by testicular cells isolated from 13.5 dpc embryos. The biological roles of the factor have also been studied and we demonstrate that PDGF‐BB acts as a migratory factor for male mesonephric cells whose migration is a male specific event necessary for a normal testicular morphogenesis. In addition we demonstrate that during testicular development, PDGF‐BB induces testicular cell proliferation being in this way responsible for the increase in size of the testis. Finally we demonstrate that PDGF‐BB is able to reorganize dissociated testicular cells inducing the formation of large cellular aggregates. However the structures formed in vitro under PDGF‐BB stimulation never had a cord‐like morphology similar to the cord‐like structures formed in the presence of HGF (Ricci et al., 2002, Mech Dev 118:19–28), suggesting that this factor does not act as a morphogenetic factor during testicular development. All together the data presented in this paper demonstrate that PDGF‐BB and its receptors (α‐ and β‐subunits) are present during the crucial ages of embryonic mouse testis morphogenesis and indicate the multiple roles exerted by this factor during the development of the male gonad.

Hepatocyte growth factor modulates Sertoli–Sertoli tight junction dynamics

In mammalian testes Sertoli cells form tight junctions whose function is fundamental for the maintenance of a normal spermatogenesis. Hepatocyte growth factor (HGF) is a cytokine influencing the cellular tight junctions either in normal or in tumor cells. We have previously demonstrated that HGF is expressed in the rat testis and influences many functional activities of somatic and germ cells. We now report that HGF decreases the levels of testicular occludin and influences the position of the molecule in the tight junctions as demonstrated by confocal microscopy analysis. In fact in the presence of the factor occludin was mainly localized in the suprabasal region of the tubules whereas in its absence occludin was prevalently localized in the basal region. Occludin production is known to be regulated by different cytokines including TGFβ. We have investigated the role of HGF in the regulation of the levels of TGFβ and we report that HGF significantly increases the amount of the active fraction of the factor without affecting the amount of the total TGFβ. Urokinase type plasminogen activator (uPA) is closely related with the tight junctions and is one of the molecules able to activate the inactive TGF‐β. We found that HGF significantly increases the amount of uPA present in the testis suggesting that HGF regulates the amount of active TGFβ via uPA levels. In conclusion we report that in the testis HGF regulates Sertoli–Sertoli tight junctions inducing a reduction and redistribution of occludin possibly modulating the levels of uPA and active TGFβ. J. Cell. Physiol. 216: 253–260, 2008.

Hepatocyte growth factor (HGF) regulates blood-testis barrier (BTB) in adult rats.

We have studied the effects of HGF on BTB dynamics in adult rats. We demonstrate that, at stages VII-VIII of the epithelium wave when germ cells traverse the BTB, HGF reduces the levels of occludin and influences its distribution pattern and assembling. Moreover, we report that, at stages VII-VIII, HGF significantly increases the amount of active TGF-β and the amount of uPA present in the tubules. For the first time we report that, in the same stages, HGF reduces the amount of actin present in the BTB region, in which occludin levels are highest, and modifies the morphology of the actin cytoskeleton network. At the level of maximal intensity of occludin fluorescence, we report that HGF also modifies the colocalization of occludin and actin. Lastly, we demonstrate that HGF is maximally expressed at stages VII-VIII, whereas its levels fall in the subsequent stages.

The effect of hepatocyte growth factor on the initial stages of mouse follicle development.

Interactions between theca and granulosa cells of the follicle are critical for the coordination of ovarian follicle development. The cell–cell interactions are mediated through the local production and actions of a variety of factors. The current study is designed to investigate the expression of Hgf and its receptor, c‐Met, in the mouse ovary during in vivo folliculogenesis. We found that Hgf and c‐Met mRNAs were already expressed in 2‐day‐old ovaries, and that, while c‐Met levels remained constant until 22‐day‐old, Hgf levels slightly but not significantly increased with age. The expression of Hgf mRNA in theca/interstitial cells was higher than in granulosa cells in 22‐day‐old ovaries. Immunohistochemistry analysis confirmed the expression pattern demonstrated by RT‐PCR. We investigated the role of hepatocyte growth factor (HGF) at the beginning of mouse folliculogenesis and its possible interaction with kit ligand (KL). Interestingly, both KL and HGF were able to increase the expression of each other, creating a positive feedback loop. In the presence of HGF, we observed an increase of granulosa cell proliferation and an increase in the number of pre‐antral and early antral follicles in ovary organ cultures. We also observed a significant increase in the diameters of follicles in individual follicle cultures. Moreover, HGF stimulated the expression of the FSH receptors, both in the whole ovary and in isolated pre‐antral follicle cultures. Based on the data presented, we concluded that HGF exerts multiple levels of control over follicular cell functions, which collectively enable the progression of follicular development. J. Cell. Physiol. 226: 520–529, 2011.

Hepatocyte Growth Factor (HGF) Modulates Leydig Cell Extracellular Matrix Components

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays multiple roles during mammalian development. We previously demonstrated that in the postnatal testes, the HGF receptor, c-met, is expressed by Leydig cells and HGF increases the steroidogenetic activity of the cells. In the present article, we report that HGF modifies the composition of the extracellular matrix of cultured Leydig cells. We show that HGF increases the metabolic activity of isolated Leydig cells; in particular, the factor increases urokinase plasminogen activator and matrix metalloproteinase 2 secretion. We have also shown that the levels of active transforming growth factor beta are increased by HGF. On the contrary, using the Western blotting technique, a strong reduction in the amount of fibronectin present in the culture medium of cells cultured in the presence of HGF has been detected. The presented data demonstrate that HGF modulates several functional activities of Leydig cells, further supporting the hypothesis that this factor has a relevant role in the regulation of mammalian spermatogenesis.

Direct effects of microgravity on testicular function: Analysis of hystological, molecular and physiologic parameters

Spaceflight experiments carried out in microgravity environments have revealed that exposure to altered gravity condition results in alteration of several cellular functions and, consequently, of several apparatuses. There is some evidence in the literature indicating that space-flight affects the physiology of the testis. The data on effects of spaceflight or simulated microgravity on testicular function, however, sometimes appear contradictory. In the present study we used an in vitro experimental model in order to investigate the direct effects of microgravity on testicular tissue. We generated a microgravity environment using the Rotating Wall Vessel and performed experiments on testicular fragments isolated from pre-pubertal rats. In this model we then analyzed several parameters such as histological integrity, cell proliferation, cell apoptosis, occludin distribution pattern, and hormonal secretions. The emerging picture shows some alterations of testicular tissue physiology. Interestingly, we also demonstrate for the first time that, in organ culture, Leydig cell survival is severely affected by simulated microgravity.

Hepatocyte Growth Factor Is a Mouse Fetal Leydig Cell Terminal Differentiation Factor

ABSTRACT The hepatocyte growth factor (HGF) is a pleiotropic cytokine and a well-known regulator of mouse embryonic organogenesis. In previous papers, we have shown the expression pattern of HGF and its receptor, C-MET, during the different stages of testis prenatal development. We demonstrated that C-MET is expressed in fetal Leydig cells (FLCs) and that HGF stimulates testosterone secretion in organ culture of late fetal testes. In the present study, we analyzed the proliferation rate, apoptotic index, and differentiation of FLCs in testicular organ culture of 17.5 days postcoitum (17.5 dpc) embryos to clarify the physiological role of HGF in late testis organogenesis. Based on our data, we conclude the following: 1) HGF acts as an antiapoptotic factor that is able to reduce the number of apoptotic FLCs and testicular caspase-3 active fragment; 2) HGF does not affect FLC proliferation; 3) HGF significantly increases expression of insulin-like 3 (INSL3), a marker of Leydig cell terminal differentiation, without affecting 3beta-hydroxysteroid dehydrogenase (3betaHSD) expression; 4) HGF significantly decreases the expression of nestin, a marker of Leydig cell progenitors; and 5) HGF significantly increases the number of fully developed FLCs. Taken together, these observations demonstrate that HGF is able to act in vitro as a survival and differentiation factor in FLC population.