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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.

Inhibition of platelet-derived growth factor receptor (PDGFR) signaling restricts the growth of human breast cancer in the bone of nude mice. We hypothesized that osteoblast-secreted substances may alter the response capacity of breast cancer cells to the PDGFRs tyrosine kinase inhibitor imatinib mesylate. We found that osteoblast-conditioned medium (OCM) increases the proliferation rate of the estrogen receptor negative (ER-) MDA-MB-231 and of the ER+ MCF-7 human breast cancer cell lines and the growth-promoting effect on ER+ cells is independent from estrogen. OCM significantly improved the dose- and the time-dependent sensitivity of the tumor cells to the anti-proliferative effect of imatinib. We also found that MDA-MB-231 and MCF-7 cells express the two PDGFRs subtypes, PDGFR-alpha and PDGFR-beta, and OCM treatment increases the expression of the PDGFRs. Furthermore, imatinib inhibited the phosphorylation rate of its target tyrosine kinase receptors. We conclude that bone microenvironment, through osteoblast-secreted substances may cause estrogen-independent proliferation of breast cancer cells by a mechanism mediated by the induction of PDGFRs expression. The enhanced sensitivity of OCM-treated breast cancer cells to imatinib would justify investigation on the efficacy of imatinib in bone breast cancer metastasis.

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.

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.

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.

Objective: To evaluate whether defective cyclic adenosine monophosphate responsive element modulator (CREM) expression is the causative factor of spermatid maturation arrest (SMA)., Design: Comparative evaluation of the testicular histology in patients with SMA or normal spermatogenesis., Setting: University clinic of andrology., Patient(s): Azoospermic patients undergoing testicular biopsy., Intervention(s): None., Main Outcome Measure(s): Expression of CREMtau in quantitative immunohistochemistry analysis of testicular biopsy samples., Result(s): Regular CREM expression was observed in the tubules with round, but not elongated, spermatids of patients with SMA (n = 9). Quantitative analysis showed that round spermatids of patients with SMA had a staining intensity similar to that observed in controls (n = 7)., Conclusion(s): Lack of spermatid elongation was not due to defective CREM expression. Therefore, CREM did not play a pathogenetic role in the onset of SMA in humans.

Biochemical investigations have identified putative enzymatic pathways for the synthesis and metabolism of endogenous cannabinoids. Anandamide amidase is an enzyme that metabolizes anandamide into arachadonic acid and ethanolamine. Using in vitro methods, various inhibitors of amidase have been identified. The present studies were undertaken to determine if the amidase inhibitor AM 374 could enhance the effects of intraperitoneal (IP) injections of anandamide. Three studies were conducted to investigate the effects of various drug treatments on fixed ratio 5 operant lever pressing for food reinforcement. In the first study, the effects of different doses of anandamide were assessed, and it was demonstrated that 5.0 and 10.0 mg/kg anandamide IP significantly suppressed lever pressing, while 2.5 mg/kg produced very little effect. The second study tested the effects of intraventricular (ICV) injections of AM 374, and it was observed that doses up to 10.0, 20.0 and 40 microg AM 374 had no significant effect upon lever pressing. The third study investigated the combined effect of AM374 with a low dose of anandamide. Rats received two drug injections: one ICV and one IP. Four different drug treatments were assessed: 1) ICV vehicle + IP vehicle, 2) ICV vehicle + 2.5 mg/kg anandamide IP, 3) ICV 20.0 microg AM 374 + IP vehicle, and 4) ICV 20 microg AM 374 + 2.5 mg/kg anandamide IP. Combined administration of AM 374 plus anandamide led to a significant decrease in lever pressing compared to either AM374 or anandamide administered alone. Observations of the animals treated with the combination of AM374 plus anandamide indicated that the drug combination resulted in motor slowing, which is consistent with the notion that stimulation of cannabinoid receptors produced a motor deficit that interfered with lever pressing. Although AM374 produced no effect on its own, this amidase inhibitor did enhance the behavioral effect of a low dose of anandamide. These results are consistent with the notion that AM 374 inhibited the enzymatic breakdown of exogenously injected anandamide. This type of procedure can be used to assess a variety of different compounds for their ability to inhibit cannabinoid metabolism.

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) alpha- and beta-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-beta loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-alpha immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-beta were absent. The cellular localization of PDGF-A within the epithelium and the alpha-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-alpha system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR alpha- and beta-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-alpha mRNAs were seen by reverse transcription-polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-beta were almost not detectable. During the first days of life, PDGF-B and PDGFR-beta 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-beta, and PDGFR-alpha were constant with the only exception of a progressive decrease of PDGFR-alpha in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-beta -/- 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.

The typical response to acute peripheral administration of low to high doses of ethanol in rats is a dose-dependent depression of motor activity. Nevertheless, recent studies indicate that intraventricular (ICV) injections of ethanol can produce signs of behavioral activation. In addition, considerable evidence indicates that brain metabolism of ethanol is involved in modulating some of the behavioral effects of this drug, which suggests that ethanol may have active metabolites with central actions. The present study was undertaken to investigate the effects of ICV ethanol, and its two major metabolites acetaldehyde and acetate, on open field locomotor activity in rats. Male Sprague-Dawley rats received different doses of ethanol, acetaldehyde or acetate ICV and immediately were placed in an open field chamber in which locomotion was measured. Rats injected with ICV ethanol or acetaldehyde showed an inverted U-shaped dose-response curve, with moderate doses increasing motor activity. In contrast, acetate produced a dose-dependent decrease in motor activity. These results demonstrate that central administration of low doses of ethanol can increase locomotor activity in rats, and suggest that acetaldehyde may be an active metabolite of ethanol that also can facilitate locomotor activity. Moreover, it is possible that some of the motor suppression or sedation produced by ethanol is due to the central actions of acetate.

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 (PDGF) plays a critical role in regulating the development and functional control of various tissues and has been implicated in the pathogenesis of serious diseases, including cancer and atherosclerosis. Given the emerging role of PDGF in the development and function of male gonads, we compared the expression profiles of the mRNAs of the PDGF A- and B-chains and of the PDGF receptor (PDGFR) alpha- and beta-subunits in fetal and adult human testis. The immunohistochemical localization of the corresponding proteins in fetal, adult, and diseased human testicular tissues was also analyzed. PDGFs and PDGFRs mRNAs were readily detected by both Northern analysis and RT-PCR. The transcript levels were higher during 16-20 wk gestation, significantly lower at 24-28 wk, and increased in the adult. An identical pattern of protein expression was confirmed by immunohistochemistry, although the cellular localization of the PDGF system changes during postnatal development, concomitantly with the progression of spermatogenesis. In the testicular samples from patients affected by either complete aplasia of germ cells or various grades of spermatogenic arrest, the immunohistochemical localization of PDGFs and PDGFRs was different from normal, confirming a close connection between germ cells and PDGF system distribution. These results indicate that PDGF, through complex interactions, could play a leading role in ontogenesis and testicular pathophysiology in humans. Finally, the expression of PDGF ligands and receptor proteins in Leydig cell tumors suggests a relationship of the PDGF system to tumorigenesis or tumor progression in this testicular neoplasm.

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.

Rationale: Previous research with both humans and animals suggests that there are sex differences in cocaine self-administration; in rodents, ovarian hormones may underlie these differences., Objectives: A two-lever drug self-administration procedure was used to compare regulation of intravenously self-administered cocaine in male and female rats and among females in different phases of the estrous cycle., Methods: Eleven female and seven male age-matched Wistar rats were trained to self-administer nine doses of cocaine (0.0-2.4 mg/kg) during daily 5-h sessions. Experimental test chambers were equipped with two levers and associated stimulus lights. A response on the lever with stimuli signaling an increase in cocaine dose increased the infusion duration by 3 s, and a response on the other lever decreased the infusion duration by 3 s., Results: After responding for cocaine stabilized, regulation was disrupted more in females than in males (r2=78.9, r2=92.6, respectively) with the greatest disruption observed in females during the estrus phase (r2=48.5). Mean dose size varied considerably for males and for females in the metestrus/diestrus and proestrus phases; however, estrus females responded almost exclusively on the lever associated with an increase in cocaine dose., Conclusions: These findings indicate sex differences in the regulation of cocaine self-administration, and they suggest that ovarian hormones may be responsible for the observed sex differences.

In mice, the Fas/Fas ligand (FasL) system has been shown to be involved in germ cell apoptosis. In the present study we evaluated the expression of Fas and Fas ligand (FasL) in fetal and adult human testis. Semiquantitative RT-PCR demonstrated the expression of Fas and FasL messenger ribonucleic acids in adult testis, but not in fetal testis (20-22 weeks gestation). In situ RT-PCR and immunohistochemistry experiments on adult human testis demonstrated the expression of FasL messenger ribonucleic acid and protein in Sertoli and Leydig cells, whereas the expression of Fas was confined to the Leydig cells and sporadic degenerating spermatocytes. The number of Fas-positive germ cells per 100 Sertoli cell nuclei was increased in 10 biopsies with postmeiotic germ cell arrest compared to 10 normal testis biopsies (mean, 3.82 +/- 0.45 vs. 2.02 +/- 0.29; P = 0.0001), but not in 10 biopsies with meiotic germ cell arrest (mean, 1.56 +/- 1.07). Fas and FasL proteins were not expressed in cases of idiopathic hypogonadotropic hypogonadism. Together, these findings may suggest that Fas/FasL expression in the human testis is developmentally regulated and under gonadotropin control. The increased germ cell expression of Fas in patients with postmeiotic germ cell arrest suggests that the Fas/FasL system may be involved in the quality control mechanism of the produced gametes.

Platelet-derived growth factor (PDGF)- A-deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Ralpha expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A-/- mice, combined with the normal developmental expression of PDGF-A and PDGF-Ralpha, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.

The aims of the study were to evaluate the association between male infertility and risk of developing testis cancer and to establish guidelines for the early diagnosis of testis neoplasia in subfertile men. 32-year-old infertile man. The patient underwent random testicular biopsy to establish the exact cause of infertility. An incidental diagnosis of seminoma was made and the patient then underwent right testis excision. Anatomopathologic macroscopic examination revealed two nodules, the sizes of which were 0.8 x 0.4 and 0.3 x 0.2 cm, respectively. Histologic examination confirmed the diagnosis of typical seminoma, pT1, with copious lymphocytic struma infiltration. There appears to be a correlation between male infertility and occurrence of seminoma. Diagnosis of testis cancer is often incidental and sometimes occurs in men undergoing testicular biopsy to investigate infertility. Since the biopsy was not specifically targeted in our case, the diagnosis of seminoma was casual. This suggests the need for a careful follow-up, including testicular ultrasonography as a screening test to achieve an early diagnosis of testis cancer in all infertile men, because of their higher risk of developing testis cancer than the normal population.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Acid phosphatase activity was found to have a similar distribution in the suprarenal glands of Discoglossus pictus, Xenopus laevis and Bufo Bufo (Anurans, Amphibia) as determined by light and electron histochemical localization. The enzymatic activity is localized in the lysosomes of both the interrenal cells and the chromaffin cells. It is, moreover, positive on the granule membranes of the adrenaline cells whereas it appears only occasionally on the granule membranes of the noradrenaline cells. Some precipitates can also be seen occasionally at the level of the Golgi membranes.

Protein synthetic activity has been studied during spermiogenesis of Paracentrotus lividus by high-resolution autoradiography using 3 H-leucine as a labeled precursor. Under the adopted experimental conditions 3 H-leucine is incorporated during the whole spermiogenesis period. The early spermatid is the most active stage and it shows labeling over the nucleus, the cytosol and the mitochondria. Nuclear 3 H-leucine incorporation progressively decreases as spermiogenesis proceeds. Cytosol labeling shows similar values at early and intermediate spermatid and it undergoes a considerable decreases at late spermatid. Mitochondrial grain density increases from early to intermediate spermatid and it remains almost constant at late spermatid. Our results are compared with the data reported for other animal groups and possible functions of the observed protein synthesis are discussed.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterases (PsChE) in chick adrenal gland during the first phases of organogenesis was studied. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for the two enzymes, respectively, whereas BW284c51 (1,5 bis (4-allyldimethylammonium-phenyl)pentan-3-one-dibromide) and ISO-OMPA (tetraisopropylpyrophosphoramide) were used as respective inhibitors of AchE and PsChE. AchE was present on the plasma membrane, in the perinuclear cisterna and in some cisternae of the rough endoplasmic reticulum of both interrenal and chromaffin cells; moreover enzymatic activity was found in the same sites of ganglion cells and mesenchymatic undifferentiated cells, i.e. on the inside and in the proximity of the glandular anlage. PsChE activity was localized in the perinuclear space and in the rough endoplasmic reticulum of all types of cells in the anlage. It is suggested that these enzymatic activities may be implicated in morphogenetic mechanisms.

Sea urchin eggs are known to display a significant level of monoamine oxidase B activity. Deprenyl, a highly selective inhibitor of monoamine oxidase B, was found to prolong the length of the first two mitotic cycles of the sea urchin egg. The prophase seems to be specifically prolonged by the drug, while the length of the other phases of the cycle is unaffected. These results suggest that the monoamine oxidase B may be a component of the monoaminergic system acting during the divisions of the sea urchin eggs.