Mario Arizzi

Expression and Cellular Localization of Follicle-Stimulating Hormone Receptor in Normal Human Prostate, Benign Prostatic Hyperplasia and Prostate Cancer

PURPOSE FSH, identified as an endogenous product of the prostate, is a glycoprotein with proliferative activity. Increasing evidence of autocrine/paracrine activities of gonadotropins at extragonadal sites led us to investigate the gene expression and cellular localization of FSH-R in normal and diseased human prostates. MATERIALS AND METHODS Prostate specimens, including normal gland, BPH, PCa and human androgen refractory (PC3) and androgen dependent (LNCaP) prostate cancer cell lines (European Collection of Cell Cultures, Salisbury, United Kingdom), were analyzed for FSH-R expression by semiquantitative and real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated cyclic adenosine monophosphate production by cultured PC3 and LNCaP stimulated with human FSH. RESULTS Little FSH-R expression was seen in 9 of 13 normal and 8 of 15 BPH specimens. Of 30 PCa samples 21 were FSH-R positive with generally higher expression compared to normal prostate and BPH samples. Real-time reverse transcriptase-polymerase chain reaction of matched normal/tumor pairs confirmed higher FSH-R mRNA expression in PCa. PC3 cells expressed FSH-R, while LNCaP cells were FSH-R negative. FSH-R protein was mainly localized in the glandular epithelium and in some stromal cells in normal prostate, BPH and PCa specimens. PC3 cells expressed FSH-R protein and their treatment with FSH induced a significant increase in cyclic adenosine monophosphate production. CONCLUSIONS These results indicate that a subset of PCa expresses FSH-R mRNA and protein at levels higher than those of normal and hyperplastic tissues that express FSH-R. This suggests that FSH might contribute to some cases of PCa via a receptor mediated mechanism.

Ontogenesis of Leptin Receptor in Rat Leydig Cells

Abstract There are still many controversies about the role of leptin in reproductive function and sexual development. We recently demonstrated that leptin receptors are expressed in rodent Leydig cells and that leptin has inhibitory effects on hCG-stimulated testosterone production by adult rat Leydig cells in culture. In this study, we evaluated the expression of leptin receptor (Ob-R) in rat testes from gestational to adult age in comparison with the pattern of expression of relaxin-like factor (RLF), a specific marker of Leydig cell differentiation status. Immunohistochemical analysis showed that, in prenatal life, Ob-R immunoreactivity was absent at early embryonic ages (E14.5) and appeared at a late embryonic age (E19.5); in postnatal life, immunoreactivity was evident only after sexual maturation (35-, 60-, and 90-days old), whereas it was absent in testes from sexually immature rats (7-, 14-, and 21-days old). Immunoreaction was always confined to Leydig cells and no signal of Ob-R was detected within the tubules. The pattern of expression of Ob-R during testicular development was similar with that of RLF immunoreactivity, which was present in mature fetal as well as adult-type Leydig cells. In contrast with the findings in the testis, in the hypothalamus, the immunohistochemical pattern of Ob-R was very similar between pre- and postpubertal life. Reverse transcription-polymerase chain reaction studies showed that Ob-R expression was present in embryonic, prepubertal, and adult rat testes; semiquantitative analysis showed that mRNA levels were much higher in late versus early embryonic testes, as well as in mature adults versus sexually immature testes, with a gradual increase from younger to older ages. Functional studies showed that, while leptin (150 ng/ml) significantly inhibited hCG-stimulated testosterone production in adult rat Leydig cells (46% reduction; P > 0.01), it did not modify prepubertal rat Leydig cells steroidogenic function in vitro. In conclusion, we showed that, in rat testis, Ob-R expression is characteristic of mature Leydig cells (fetal and adult type) and it is functional in adult but not prepubertal life.

Platelet-Derived Growth Factor Receptor β-Subtype Regulates Proliferation and Migration of Gonocytes

Proliferation and migration of gonocytes, the precursors of spermatogonial stem cells, to the germline niche in the basal membrane of the seminiferous tubules, are two crucial events that take place between postnatal d 0.5 (P0.5) and P5.0 in the mouse and involve a selection of the cells that are committed to the germline stem cells lineage. Here we show that from embryonic d 18.0 (E18) and up to P5, the gonocytes express platelet-derived growth factor (PDGF) receptor beta-subtype (PDGFR-beta) and that during the same time period, the Sertoli cells express PDGF-B and PDGF-D, both ligands for PDGFR-beta. Inhibition of the PDGFR-beta tyrosine kinase activity during the first five postnatal days provokes a profound reduction of gonocyte number through inhibition of their proliferation and induction of apoptosis. Moreover, we found that PDGFR-beta ligands are chemotactic for gonocytes. These data suggest that PDGFR-beta activation has the remarkable capability to drive the selection, survival, and migration of the gonocytes from the center of the seminiferous tubules to the testicular germline niche on the basal membrane.

PDGF and the testis

Testicular development is controlled by a complex hierarchy of gene regulatory proteins, growth factors, cell adhesion molecules, signaling molecules and hormones that interact, often acting within short time windows, via reciprocal control relationships. The identification in the testis of platelet-derived growth factor (PDGF), a key regulator of connective tissue cells in embryogenesis and pathogenesis, has focused attention on the role of this growth factor in testicular pathophysiology. This review summarizes recent advances in the study of the actions of PDGF in the male gonad, and attempts to incorporate complex in vitro and in vivo experimental data into a model that might clarify the role played by PDGF in the mammalian testis.

Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

Immunohistochemical localization of growth hormone-releasing hormone in human gonads

Human gonads were examined for the presence of ir growth hormone-releasing-hormone. We demonstrated the presence of immu-nostainable cells both in ovarian and in testicular tissue using a non-crossreactive antiGRH antiserum and the immunoperoxidase detection technique. In ovaries from ovulating women, GRH immunoreactivity was localized in the corpórea lutea; granulosa cells, theca cells and cells of primary follicles did not stain. In premenopausal ovaries, staining was detectable in scattered luteinized stromal cells. In testes from post-puberal men GRH immunoreactivity was localized in the Leyding cells; cells of the germinal epithelium did not stain. These results demonstrate the presence of GRH in human gonads and suggest that this peptide may exert regulatory function at the testicular and ovarian levels.

Expression of Platelet-Derived Growth Factor (PDGF) in the Epididymis and Analysis of the Epididymal Development in PDGF-A, PDGF-B, and PDGF Receptor β Deficient Mice

Abstract The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) α- and β-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-β loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-α immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-β were absent. The cellular localization of PDGF-A within the epithelium and the α-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-α system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR α- and β-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-α mRNAs were seen by reverse transcription–polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-β were almost not detectable. During the first days of life, PDGF-B and PDGFR-β genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-β, and PDGFR-α were constant with the only exception of a progressive decrease of PDGFR-α in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-β −/− mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

Iodine supplementation restores fertility of sheep exposed to iodine deficiency

The aims of the study were to monitor sheep iodine intake in different sheep breeding farms in Abruzzo and to evaluate the effects of iodine supplementation on ovine fertility. The urinary iodine concentrations (UIC) in animals of 8 out of the 11 breeding farms analyzed were borderline (UIC 100–150 μg/l) or very low (UICФ50 μg/l). Only animals bred in 3 farms showed an adequate iodine intake with a mean UICГ300 μg/l. Animals with very low iodine intake had lower T4 and T3 (plt;0.01) serum levels, compared to those with adequate iodine intake. To investigate the effects of iodine supplementation on ovine fertility, 32 ewes and 20 rams, characterized by low UIC, were randomly divided into 2 groups. One group (16 ewes and 10 rams) received a sc injection of 1 ml of Lipiodol, containing 480 mg of iodine, while the remaining animals were employed as control. This treatment was able to maintain UIC above 300 μg/l for 3 months and to increase T4 and T3 serum levels (plt;0.01). After 9 months, the fertility of control and treated animals was assessed by monitoring the rate of successful matings by ultrasonography. The results showed that 100% of treated ewes mated with treated rams were pregnant vs 37% of the control ewes mated with control rams (p=0.007). The iodine content was 4-fold higher in milk from treated ewes (2393±453 μg/l), compared to controls (675±154 μg/l).The results demonstrated that iodine supplementation restores fertility of sheep living in iodine deficient areas and may represent a means to achieve a silent iodine prophylaxis of local populations.

PACAP and Type I PACAP Receptors in Human Prostate Cancer Tissue

Abstract:  We characterized the expression and localization of pituitary adenylate cyclase‐activating polypeptide (PACAP) and its specific type I receptor variants in prostatic, hyperplastic, and carcinomatous tissue collected from patients undergoing prostate biopsy and surgery for benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The immunohistochemical studies using an indirect immunoperoxidase technique evidenced positive immunostaining for PACAP in the cytoplasm of epithelial cells of hyperplastic and carcinomatous prostate specimens and in some scattered cells of the stroma. Type I PACAP receptors (PAC1 R) in healthy and BPH tissues were localized in all epithelial cells lining the lumen of the acini and in some stromal cells, while in specimens from PCa the anti‐PAC1 R antibody stained the apical portion of a large percentage of cells. Furthermore, our molecular studies provide evidence that several PAC1 R isoforms (null, SV1/SV2) are present in normal, hyperplastic, and neoplastic tissue, the null variant being the most intensely expressed in PCa. These observations provide additional evidence for a role of PACAP and PAC1 R in the events determining the outcome of PCa.