Roberto Ponzio

Expression of Aromatase, Estrogen Receptor α and β, Androgen Receptor, and Cytochrome P-450scc in the Human Early Prepubertal Testis

The expression of aromatase, estrogen receptor α (ERα) and β (ERβ), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERα by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERβ and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERβ, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERβ were immunoexpressed strongly whereas no expression of ERα, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.

Effects of di(2-ethylhexyl) phthalate on gap and tight junction protein expression in the testis of prepubertal rats.

The aim of this study was to analyze whether di(2‐ethylhexyl) phthalate (DEHP), a Sertoli and Leydig cell toxicant, is able to induce alterations in the expression of testicular gap and tight junction proteins. DEHP was administered by gavage (1 g/5 mL corn oil/kg body weight/day) to 25‐day‐old male Sprague–Dawley rats for 2 days (DEHP‐27d) and control rats were treated with corn‐oil vehicle for 2 days (C‐27d); animals were killed 24 h after the last treatment. Testes of DEHP‐27d rats showed different degrees of germ cell sloughing of seminiferous tubules (ST). No alterations of the blood testis barrier (BTB) by lanthanum tracer study were observed. ST of DEHP‐27d rats showed a milder immunofluorescence and more restricted expression of connexin‐43 (Cx43) in the adluminal and basal compartment compared to C‐27d. In DEHP‐27d rats, we found a discontinuous immunofluorescent (IF) pattern for zonula occludens (ZO‐1), contrasting with the continuous IF profile observed in C‐27d, and a delocalization of claudin‐11. A decrease in Cx43 and ZO‐1 and no changes in occludin expression were detected by Western blot in the testes of DEHP‐27d rats. Results from 57‐day‐old rats treated with DEHP for 2 days and held for 30 days without treatment showed that the alterations in protein expression induced by DEHP are reversible. However, a delay of spermatogenesis compared to C‐57d rats, occurred. Data demonstrated that DEHP does not impair BTB permeability but induces germ cell sloughing that might respond to a down regulation of Cx43 and ZO‐1 that alters cell junction proteins. Microsc. Res. Tech. 2009.

Role of IGFs and Insulin in the Human Testis During Postnatal Activation: Differentiation of Steroidogenic Cells

Immunoexpression of IGF-I, IGF-II, type 1 IGF receptor (IGFR), insulin receptor (IR), and GH receptor (GHR) was analyzed in human testis, in three age groups (Gr): Gr1 (neonates), Gr2 (postnatal testicular activation), and Gr3 (early prepuberty). In interstitial cells, low IGF-I and GHR, but moderate IR immunoexpression was observed in all Grs. However, high expression of IGF-II in Gr1, and moderate expression of IGFR in Gr1 and Gr2 were found. In Leydig cell (LC), high expression of IGF-II, moderate expression of IGFR and GHR, and undetectable IGF-I was found. Moreover, IR was highly expressed in Gr2. The effect of IGF-I on cell proliferation (PI) and apoptosis (AI), induction of cytochrome P450 side chain cleavage (cP450scc) immunoexpression, 3β-hydroxysteroid dehydrogenase mRNA and testosterone (T) secretion was evaluated in human testis cell cultures. IGF-I increased P450scc immunoexpression, 3β-hydroxysteroid dehydrogenase mRNA, T secretion, and PI, but decreased AI. We propose that IGF-II, mainly through IR, is involved in functional LC differentiation. In some interstitial cells, probably in LC precursors, IGF-II/IR could be involved, among other factors, in the stimulation of PI and/or inhibition of AI, and in LC differentiation.

Cyclooxygenase-2 in testes of infertile men: evidence for the induction of prostaglandin synthesis by interleukin-1β.

As we previously reported, testes of men suffering from hypospermatogenesis and germ cell arrest or Sertoli cell-only syndrome show a major increase in the number of macrophages expressing interleukin-1β (IL-1β) and abundant expression of cyclooxygenase-2 (COX-2), the inducible isoform of the key enzyme in the biosynthesis of prostaglandins (PGs), in Leydig cells. In the present study we report [1] a positive correlation between IL-1β levels and COX-2 expression in testes of infertile patients, [2] the induction of COX-2 by IL-1β in mouse Leydig cells (TM3) and human macrophages (THP-1), and therefore [3] evidence for an IL-1β-dependent induction of testicular inflammatory states.

New insights into melatonin/CRH signaling in hamster Leydig cells

We have previously described that melatonin inhibits androgen production in hamster testes via melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone (CRH) system. This study attempted to determine the initial events of the melatonin/CRH signaling pathway. In Leydig cells from reproductively active Syrian hamsters, Western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and a colorimetric assay demonstrated that melatonin and CRH activate tyrosine phosphatases and subsequently reduce the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk), down-regulate the expression of c-jun, c-fos and steroidogenic acute regulatory (StAR), and inhibit the production of testosterone. These effects were prevented by a highly selective CRH antagonist, thus indicating that melatonin does not exert a direct role. Specific mitogen-activated protein kinase kinase (MEK) and jnk blockers inhibited expression of c-jun, c-fos, StAR and the production of testosterone, confirming that these are events triggered downstream of erk and jnk. In Leydig cells from photoperiodically regressed adult hamsters, CRH inhibited the production of androstane-3α,17β-diol (3α-diol), the main androgen produced, through the same signaling pathway. Testicular melatonin concentration was 3-4-fold higher in reproductively inactive hamsters than that detected in active animals. Since melatonin, CRH, and their receptors are present not only in hamster testes but also in testicular biopsies of infertile men, we can conjecture about the relevance of this previously uncharacterized pathway in human fertility disorders. In summary, our study identifies crucial intracellular events triggered by melatonin/CRH in the testis that lead to a down-regulation of the steroidogenic process.

Passive Immunization with Anti-Laminin Immunoglobulin G Modifies the Integrity of the Seminiferous Epithelium and Induces Arrest of Spermatogenesis in the Guinea Pig

Abstract In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.

Expression of the TGF-beta1 system in human testicular pathologies

BackgroundIn non-obstructive azoospermia, histological patterns of Sertoli cell-only Syndrome (SCO) and hypospermatogenesis (H) are commonly found. In these pathologies, Leydig cell hyperplasia (LCH) is detected in some patients. Since TGF-β1 is involved in cellular proliferation/development, the aim of this work was to analyze the expression of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), and the co-receptor endoglin in human biopsies from patients with idiopathic infertility.MethodsSpecific immunostaining of TGF-β1, its receptors TGFBRII, TGFBRI (ALK-1 and ALK-5), co-receptor endoglin and Smads proteins, were carried out in testicular biopsies from normal and infertile men with SCO or H. Gene expression of TGF-β1 system were made in biopsies from infertile patients with semi-quantitative and quantitative PCR.ResultsImmunohistochemical studies revealed that TGF-β1 and its specific receptors are present in Leydig cells in biopsies from normal tissue or patients with SCO or H with or without LCH. Smad proteins, which are involved in TGF-β1 signaling, are also detected in both their phosphorylated (activated) and dephosphorylated form in all samples TGF-β1, ALK-1 and endoglin gene expression are stronger in human biopsies with LCH than in those with SCO or H. Neither TGFBRII nor ALK-5 gene expression showed significant differences between pathologies. A significant correlation between ALK-1 and endoglin expression was observed.ConclusionsIn conclusion, the high levels of mRNA and protein expression of the TGF-β1 system in patients with LCH, particularly ALK1 and its correlation with endoglin, suggest that these proteins acting in concert might be, at least in part, committed actors in the Leydig cell hyperplasia.

Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells

Reactive oxygen species (ROS) regulate testicular function in health and disease. We previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli cells, and also induces antioxidant enzymes expression suggesting that defense systems are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and specially H2O2 may act as second messengers regulating signal transduction pathways and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase (LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and CREB/ATF-1 phosphorylation. Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant status in Sertoli cells and, more importantly, in the modulation of LDH expression which takes place through ROS generation and the Akt-CREB/ATF-1 pathway.

Melatonin in testes of infertile men: evidence for anti-proliferative and anti-oxidant effects on local macrophage and mast cell populations.

Melatonin acting through the hypothalamus and pituitary regulates testicular function. In addition, direct actions of melatonin at the testicular level have been recently suggested. We have described that melatonin inhibits androgen production in hamster Leydig cells via melatonin subtype 1a (mel1a) receptors and the local corticotrophin‐releasing hormone (CRH) system. The initial events of the melatonin/CRH signalling pathway have also been established. Melatonin and all components of the melatonergic/CRH system were also detected in Leydig cells of infertile men. This study attempted to search for additional targets of melatonin in the human testis, and to investigate the effects of melatonin on proliferation and the oxidative state in these novel target cells. To this aim, evaluation of human testicular biopsies of patients suffering from hypospermatogenesis or Sertoli cell only syndrome and cell culture studies were performed. Melatonergic receptors were found in macrophages (MACs) and mast cells (MCs) of the human testis. In biopsies of patients suffering idiopathic infertility, melatonin testicular concentrations were negatively correlated with MAC number per mm2 and TNFα, IL1β and COX2 expression, but positively correlated with the expression of the anti‐oxidant enzymes SOD1, peroxiredoxin 1 and catalase. Melatonin inhibited proliferation and the expression of pro‐inflammatory cytokines and cyclooxygenase 2 (COX2) in both the human non‐testicular THP‐1 MAC cell line and primary cell cultures of hamster testicular MACs. In the human HMC‐1 MC line, melatonin increased the expression of anti‐oxidant enzymes and decreased reactive oxygen species (ROS) generation. The results reveal new testicular targets of melatonin and describe anti‐proliferative and anti‐inflammatory effects of this hormone on testicular MACs. Furthermore, melatonin might provide protective effects against oxidative stress in testicular MCs.

Evidence for an adaptation in ROS scavenging systems in human testicular peritubular cells from infertility patients

Fibrosis, increased amounts of immune cells and expression of COX-2 in the testes of infertility patients provide circumstantial evidence for a specific testicular milieu, in which reactive oxygen species (ROS) could be increased. If ROS level increase and/or ROS scavengers decrease, the resulting testicular oxidative stress may contribute to human male infertility. Primary peritubular cells of the human testis, from men with normal spermatogenesis (HTPCs) and infertile patients (HTPC-Fs), previously allowed us to identify an end product of COX-2 action, a prostaglandin derivative (15dPGJ2), which acts via ROS to alter the phenotype of peritubular cells, at least in vitro. Using testicular biopsies we now found 15dPGJ2 in patients and hence we started exploring the ROS scavenger systems of the human testis. This system includes catalase, DJ-1, peroxiredoxin 1, SOD 1 and 2, glutathione-S-transferase and HMOX-1, which were identified by RT-PCR/sequencing in HTPCs and HTPC-Fs and whole testes. Catalase, DJ-1, peroxiredoxin 1 and SOD 2 were also detected by Western blots and in part by immunohistochemistry in testicular samples. Western blots of cultured cells further revealed that catalase levels, but not peroxiredoxin 1, SOD 2 or DJ-1 levels, are significantly higher in HTPC-Fs than in HTPCs. This particular difference is correlated with the improved ability of HTPC-Fs to handle ROS, which became evident when cells were exposed to 100 μm H(2)O(2). H(2)O(2) induced stronger responses in HTPCs than in HTPC-Fs, which correlates with the lower level of the H(2)O(2)-degrading defence enzyme catalase in HTPCs. The results provide evidence for an adaptation to elevated ROS levels, which must have occurred in vivo and which persist in vitro in HTPC-Fs. Thus, in infertile men with impaired spermatogenesis elevated ROS levels likely exist, at least in the tubular wall.