Expression of Fas and Fas ligand in human testicular germ cell tumours

In the present study, we analysed the expression of Fas ligand (FasL) and its cognate receptor Fas in 14 seminomatous testicular germ cell tumours (TGCT) and six normal testicular tissues obtained following orchiectomy. Tissue samples have been processed to prepare either total RNA or protein extracts or fixed and embedded in paraffin for immunohistochemistry (IHC) experiments. Quantitative RT-PCR experiments demonstrated in TGCT a significant ( p  < 0.01) increase of the FasL mRNA expression of 21.1 ± 5.4 fold, with respect to normal tissues. On the contrary, in the same cancer tissues, the levels of Fas mRNA were significantly ( p  < 0.01) reduced to 0.27 ± 0.06 fold. These observations were confirmed in western blot experiments showing a significant increase of FasL and a concomitant decrease of Fas proteins in testicular cancer tissues, with respect to normal testis. Moreover, IHC experiments showed a strong FasL immuno-reactivity in six out of eight TGCT samples analysed, while Fas immuno-positivity was found in cancer cells of only two TGCT tissues. In addition, in all tumour samples, infiltrating lymphocytes were Fas positive. However, no correlation could be observed between Fas or FasL mRNA variations and clinical parameters such as patient's age, TNM stage or tumour size. We also compared the serum levels of soluble FasL (sFasL) of 15 patients affected by seminomatous TGCT, of four patients with non-seminomatous TGCT and six age-matched healthy males. No significant differences in sFasL serum level could be identified. In conclusion, our data demonstrated that the majority of seminomas are characterized by an increased expression of FasL and a concomitant reduction of Fas, with respect to human normal testis, and that sFasL serum level is not a tumour marker for patients affected by TGCT.     

Experimental testicular germ cell tumorogenesis in mouse strains with and without spontaneous tumours differs from development of germ cell tumours of the adult human testis

The aim of this study was to undertake a morphological analysis of the earliest stages of experimentally induced (by genital ridge grafting) germ cell tumours in mouse strains with (129/Sv-ter) and without (MA) spontaneous tumorogenesis. Genital ridges from fetuses aged 12 or 13 days from 129/Sv-ter and MA were transplanted into the testes of adult 129/Sv-ter . The results show clearly that experimentally induced carcinoma-in-situ in mouse testes differs considerably from its human counterpart, found in patients with and without testicular germ cell tumours, and considered to be the precursor for all kinds of germ cell tumours of the adult testis apart from spermatocytic seminoma. The results indicate that development of testicular germ cell tumours is different in man and the mouse.     

Bilateral testicular microlithiasis predicts the presence of the precursor of testicular germ cell tumors in subfertile men

PURPOSE: A high prevalence of testicular microlithiasis has been described in adolescent and adult clinical cases of invasive testicular germ cell tumor (TGCT), that is seminomas and nonseminomas. However, to our knowledge it remains to be established whether testicular microlithiasis also indicates the presence of the pre-invasive lesion of this cancer, known as carcinoma in situ (CIS). We determined the predictive value of unilateral and bilateral testicular microlithiasis for CIS in subfertile men, a known risk population for TGCTs (approximately 1%). MATERIALS AND METHODS: In a retrospective cross-sectional study the association between testicular microlithiasis and CIS was studied in a group of 263 men referred for subfertility. Testicular microlithiasis and CIS were diagnosed in all men by scrotal ultrasound and in testicular histology specimens as part of the routine evaluation of all patients. RESULTS: Of the 263 subfertile men 53 (20%) had testicular microlithiasis. No CIS or TGCT was identified in the 23 men with unilateral testicular microlithiasis. In contrast, 6 of the 30 men (20%) with bilateral testicular microlithiasis were diagnosed with CIS. Therefore, the prevalence of CIS in subfertile men with bilateral testicular microlithiasis is significantly higher than in patients without testicular microlithiasis (1 of 210, 0.5%) and with unilateral testicular microlithiasis (0 of 23, 0%) (p <0.0001). CONCLUSIONS: Bilateral testicular microlithiasis is indicative for CIS in subfertile men. Since these men are at particular risk for invasive TGCT, an assessment of testicular microlithiasis is a valuable tool for the early diagnosis of this disease.     

Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats

Pituitary adenylate cyclase activating polypeptide (PACAP) has several different actions in the nervous system. Numerous studies have shown its neuroprotective effects both in vitro and in vivo. Previously, it has been demonstrated that PACAP reduces brain damage in rat models of global and focal cerebral ischemia. Based on the protective effects of PACAP in cerebral ischemia and the presence of common pathogenic mechanisms in cerebral ischemia and traumatic brain injury (TBI), the aim of the present study was to investigate the possible protective effect of PACAP administered 30 min or 1 h postinjury in a rat model of diffuse axonal injury. Adult Wistar male rats were subjected to impact acceleration, and PACAP was administered intracerebroventricularly 30 min (n = 4), and 1 h after the injury (n = 5). Control animals received the same volume of vehicle at both time-points (n = 5). Two hours after the injury, brains were processed for immunohistochemical localization of damaged axonal profiles displaying either beta-amyloid precursor protein (beta-APP) or RMO-14 immunoreactivity, both considered markers of specific features of traumatic axonal injury. Our results show that treatment with PACAP (100 microg) 30 min or 1 h after the induction of TBI resulted in a significant reduction of the density of beta-APP-immunopositive axon profiles in the corticospinal tract (CSpT). There was no significant difference between the density of beta-APP-immunopositive axons in the medial longitudinal fascicle (MLF). PACAP treatment did not result in significantly different number of RMO-14-immunopositive axonal profiles in either brain areas 2 hours post-injury compared to normal animals. While the results of this study highlighted the complexity of the pathogenesis and manifestation of diffuse axonal injury, they also indicate that PACAP should be considered a potential therapeutic agent in TBI.     

Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma

Based on evidence from morphological and histochemical studies and from clinical experience, the following hypotheses are proposed: carcinoma-in-situ (CIS) germ cells are malignant gonocytes; these CIS gonocytes have some capacity to regress into more primitive, totipotent embryonic cells which can give rise to all types of nonseminomatous germ cell tumours; the tumour germ cells of classical seminomas are malignant gonocytes derived from CIS gonocytes which have lost their ability to regress into totipotent embryonic cells; the ability of CIS gonocytes to regress into totipotent embryonic cells decreases with age, whereas the capacity to form classical seminoma cells is preserved; the transformation of CIS gonocytes into invasive tumours is dependent on factors such as gonadotrophins and/or testicular steroids; the pathogenesis of classical and spermatocytic seminoma are unrelated. As a consequence of these hypotheses an alternative nomenclature for carcinoma-in-situ, seminoma and dysgerminoma is suggested.     

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin‐43, Zona Occluding‐1 and N‐cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood–testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed.     

Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumours

Immunohistochemical localization of placental alkaline phosphatase (PlAP) has been performed on eighty-two samples of normal (embryonic, fetal, infantile or adult), cryptorchid or tumorous testicular tissue. The isoenzyme could be demonstrated at the cell membrane of primitive, embryonic germ cells but not in other normal tissues. In-situ carcinomas and seminomas were positively stained in 93% and 94% of cases respectively. The percentage of positivity decreased with tumour differentiation. It is suggested that PlAP is a marker of primitive germ cells and that it reappears in germ cell neoplasms by gene derepression.     

Possible mechanism responsible for the acquisition of resistance to cis-diamminedichloroplatinum (II) by cultured human testicular seminoma cells

PURPOSE: We established a cis-diamminedichloroplatinum (II) (CDDP) resistant cell line and determined the mechanisms of CDDP resistance. MATERIALS AND METHODS: The CDDP resistant cell line JKT/DDP was established from the highly metastatic human testicular seminoma cell line JKT-HM by long-term intermittent administration of low dose CDDP. Growth curves, CDDP sensitivity and intracellular CDDP concentrations were compared in JKT/DDP, the seminoma cell line JKT-1 and JKT-HM cells. Expression of the mRNA of MDR1, MRP, LRP and GST-pi was also compared. RESULTS: The growth curve and doubling time of JKT/DDP were similar to those of JKT-1 and JKT-HM in culture without CDDP but different with CDDP added to the culture. Morphologically cytoplasm segmentation and chromatin aggregation in JKT-1 and JKT-HM were observed when CDDP was present, while JKT/DDP cells were spindle-shaped and showed cobblestone growth.The IC50 for CDDP determined by the collagen gel drop embedded drug sensitivity test showed that JKT/DDP was more resistant to CDDP than the other 2 cell lines. The expression of MDR1, MRP and GST-pi was higher than in JKT-1 or JKT-HM cells and the intracellular CDDP concentration was lower in JKT/DDP. CONCLUSIONS: The JKT/DDP cell line acquired CDDP resistance by increased cytoplasmic CDDP metabolism.     

Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasms

The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs. The LPL protein, however, was restricted to the cell membranes of pachytene spermatocytes and spermatids in normal tubules, absent from CIS cells and scarcely represented in tumours. The distribution of LPL protein in non-seminomas resembled the distribution of OCT3/4, a marker of embryonal carcinoma. The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases in normal testis suggests the existence of distinct biological roles, perhaps developmentally regulated, as indicated by the LPL expression in GCTs with embryonic features. A high expression of EL and abundance of lipid in tubules with CIS may have a diagnostic value.     

Evaluation of the testis function of mice exposed in utero and during lactation to Pfaffia glomerata (Brazilian ginseng)

Pfaffia glomerata (Spreng.) Pedersen, popularly known as “Brazilian ginseng,” is used as medicinal plant in Brazil to treat inflammatory diseases in general. Previous studies showed that its extract increases the nitric oxide (NO) levels. Knowing that NO downregulates steroidogenesis and that alterations in the action/production of androgens during perinatal life could alter testis development, the present studies sought to investigate the reproductive toxicity of Pfaffia glomerata on male mice exposed to hydroalcoholic extract in utero and during lactation. The present study shows that P. glomerata extract does not alter body weight, tubular diameter and testis function in male mice. Although a reduction in the testis weight was observed in the animals that received the highest dose directly in early post‐natal life, our findings show clearly that P. glomerata may not act as an endocrine disruptor, and it is not an “antiandrogenic” compound that could lead to testicular dysgenesis syndrome.     

Protective efficiency of Ashrasi date palm hydroalcoholic extract against diabetes‐induced testicular toxicity: A biochemical and stereological study

The present study was designed to investigate the protective effects of hydroalcoholic extract of Ashrasi date palm (ADP) on diabetes‐induced testicular injuries. Adult male rats were randomly allocated into five groups (n = 8 in each group): 1: control; 2: diabetic; 3: diabetic + 30 mg/kg of ADP extract; 4: diabetic + 90 mg/kg of ADP extract; and 5: diabetic + 270 mg/kg of ADP extract. Diabetes was induced by a single dose of streptozotocin (55 mg/kg) intraperitoneally. Testicular changes were assessed quantitatively using stereological method followed by measuring antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase and serum testosterone level. Malondialdehyde (MDA) and Bcl‐2 expression were also evaluated in tissue samples. Diabetes resulted in significant deleterious alterations in the architecture of testicular tissue, suppressed antioxidant enzymes and testosterone levels and increased lipid peroxidation. The expression of Bcl‐2 was downregulated in diabetic testis and resulted in enhanced apoptosis. Eight weeks of ADP extract treatment especially at higher doses could markedly improve structural changes of testis and restore the antioxidant defence and testosterone levels in testicular tissue. In conclusion, this findings showed that ADP extract can play as a potent antioxidant and can attenuate the adverse effects of diabetes on male reproduction.     

Nasturtium Officinale L. hydroalcoholic extract improved oxymetholone‐induced oxidative injury in mouse testis and sperm parameters

Testicular tissue and sex hormones are sensitive to the anabolic steroids (oxymetholone/OM) due to increased free radical damage and hormonal changes. The Nasturtium officinale L. have various antioxidant compounds. The aim of the present study was to investigate N. officinale effect on OM‐induced oxidative injury in mouse testis and sperm parameters. Thirty BALB/c mice were divided into five groups, including control, OM (5 ml/kg) and three N. officinale doses (25, 50 and 100 mg/kg) + OM. At the end of the study (40 days), serum luteinising hormone (LH), follicle‐stimulating hormone (FSH), testosterone, nitric oxide (NO) levels, ferric reducing ability of power (FRAP) and testis stereological factors were measured. The sperm parameters were evaluated. Liquid chromatography‐electrospray ionisation‐tandem mass spectrometry (LC‐ESI/MS) analysis was yielded a fingerprint of N. officinale phenolic constituents. 100 mg/kg of N. officinale extract significantly reduced the serum level of testosterone and a significant increase in LH and FSH in comparison with the control group. This dose also significantly improved the stereological factors and sperm parameters. 50 and 100 mg/kg of N. officinale extract significantly increased the testis tissue FRAP levels, and 100 doses reduced the serum levels of NO. Fourteen compounds and 34 peaks were identified in the extract with LC‐ESI/MS. Nasturtium officinale extract has protective effects against testicular toxicity caused by OM.