Aurora B expression in post-puberal testicular germ cell tumours

Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target. J. Cell. Physiol. 221: 435–439, 2009. © 2009 Wiley-Liss, Inc.     

Rat synapsin 1 promoter mediated transgene expression in testicular cell types

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.     

Bilateral germ cell testicular tumors

PURPOSE: To study the clinical characteristics of bilateral testicular tumors in the cisplatin era. PATIENTS AND METHODS: Between November 1988 and November 1998 2386 testicular cancer patients were treated in our Department and 72 bilateral germ cell testicular cancer patients were retrospectively explored (3%). The incidence, the clinical and histological characteristics and, in the case of asynchronous tumor, the interval between the two tumors were analyzed. RESULTS: During the 10 years 19 synchronous (26.4%) and 53 asynchronous bilateral germ cell testicular cancers (73.6%) were treated. The incidence of bilateral synchronous seminoma was 68.4%. Among the asynchronous tumors 9 concordant seminomas and 9 concordant nonseminomas were detected. In the first, second and third 5-year follow-up period 39.6, 30.2, and 28.2% of asynchronous tumors were diagnosed. The incidence of seminoma after the first castration in the 5, 10 and 15 years was 19, 37.5, and 60%, respectively. The overall survival rates of synchronous and asynchronous testicular cancer were 84 and 93%. In cases of asynchronous tumor the prevalence of stage I cancer was significantly greater in a regularly controlled population (p=0.014) than in the not regularly followed population, but the survival rate was good in both groups. Nonseminoma showed up earlier as first and second tumor than seminoma (p=0.05, p=0.045). The interval between the two asynchronous tumors was shorter in the case of nonseminoma than in the case of seminoma (p=0.002). CONCLUSION: The prognosis of bilateral germ cell testicular cancer is good because of the high incidence rate of seminoma and the effective treatment. With regular follow-up the early diagnosis of second testicular tumors is probable. The interval between the tumors depends on the patients' age and the histology of the second tumor, in the case of seminoma it is longer. The effect of the previous treatment on the incidence of seminoma and the interval between the two asynchronous tumors requires further investigations.     

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.     

Spermatogenesis and testicular tumours in ageing dogs

Spermatogenesis was examined in testes from 74 dogs of various breeds without clinically detected testicular disease. A modified Johnsen score system was used to determine whether spermatogenesis deteriorates with ageing. The diameter of seminiferous tubules was measured in dogs without testicular disease to examine other possible effects of ageing on tubular performance. There appeared to be no relation between age and these variables. The influence of testicular tumours on spermatogenesis was also investigated in both affected and unaffected testes. The testes of 28 dogs with clinically palpable tumours and 21 dogs with clinically non-palpable tumours were investigated. In cases of unilateral occurrence of a tumour, impairment of spermatogenesis was observed only in the affected testis of dogs with clinically detected tumours. Bilateral occurrence of tumours, whether detected clinically or non-clinically, was associated with severe impairment of spermatogenesis. The prevalence of tumours increased during ageing. Eighty-six per cent of the clinically detected and 57% of the non-clinically detected tumours were found in old dogs. Multiple types of tumour and bilateral occurrence were very common. Seminomas and Leydig cell tumours were more frequent than Sertoli cell tumours. It was concluded that spermatogenesis per se did not decrease during ageing in dogs but the occurrence of testicular tumours increased with ageing and affected spermatogenesis significantly, as reflected by a lower Johnsen score.     

Cytoplasmic β-actin promoter produces germ cell and preimplantation embryonic transgene expression

The cytoplasmic β-actin promoter, commonly used as strong promoter in many gene regulation studies, produces a pattern of male germ cell and preimplantation, embryonic gene expression in transgenic mice. In seven of ten expressing transgenic lines, a chicken β-actin-lacZ fusion gene was expressed in adult testes. In addition, five of the ten lines demonstrated transgene expression in the preimplantation mouse embryo. This is the first example of transgene expression at the stages of both gamete and early embryo. Overall, the site or transgene integration appeared to influence transgene expression in adult tissues. © 1993 Wiley-Liss, Inc.     

Genetic aspects of testicular germ cell tumors

Testicular Germ Cell Tumor (TGCT) is the most common malignant tumor in young Caucasian men with an annual increase of 3-6% in the past 50 years. Data in the literature indicate that both environmental and genetic factors acting on the primordial gonocyte/gonocyte are implicated in the etiopathogenesis of this tumour. Genetic linkage and genome-wide analyses did not reveal a major gene effect so far, implying that multiple loci must contribute to the development of TGCTs. Only one significant genetic risk factor has been reported, the so called gr/gr deletion of the Y chromosome which still request further confirmation by independent studies. On the other side, the analysis of somatic genetic changes through mutation and genome imbalance analyses and expression profiling has just began to unravel the complex interaction of multiple pathways involved in TGCTs. This review focuses on genetic factors (both genomic and somatic) involved in the etiology, progression and treatment sensitivity of TGCTs.     

Pathobiology of testicular germ cell tumors: views and news

Human germ cell tumors (GCTs) are a heterogeneous group of neoplasms. They can occur in different anatomic locations, predominantly in the gonads (both ovary and testis) and in the midline of the body, including the retroperitoneal, mediastinal and hypothalamus/pineal gland regions. This distribution has been related to the migration routefollowed by primordial germ cells from the yolk sac to the genital ridge. The clinical behavior of these tumors depends on the sex of the patient, the age at clinical presentation and the histology of the tumor, Within the testis, three groups of GCTs can be distinguished; (I) yolk sac tumors and teratomas of neonates and infants; (II) seminomas and nonseminomas of adolescents and adults, the so-called testicular germ cell tumors; and (III) spermatocytic seminomas. This review discusses the histology, epidemiology and chromosomal constitution of GCT, in particular of the seminomas and nonseminomas of the adult testis, including their precursor, carcinoma in situ. In addition, the available data on the molecular basis of treatment sensitivity and resistance of GCT are reviewed.     

Functional role of caspases in heat-induced testicular germ cell apoptosis

In the present study, we determined whether a pan caspase inhibitor could prevent or attenuate heat-induced germ cell apoptosis. Groups of five adult (8 wk old) C57BL/6 mice pretreated with vehicle (DMSO) or Quinoline-Val-Asp (Ome)-CH2-O-Ph (Q-VD-OPH), a new generation broad-spectrum caspase inhibitor, were exposed once to local testicular heating (43 degrees C for 15 min) and killed 6 h later. The inhibitor (40 mg/kg body weight) or vehicle was administered intraperitoneally (i.p.) 1 h before local testicular heating. Germ cell apoptosis was detected by TUNEL assay and quantitated as number of apoptotic germ cells per 100 Sertoli cells at stages XI-XII. Compared with controls (16.8 +/- 3.1), mild testicular hyperthermia within 6 h resulted in a marked activation (277.3 +/- 21.6) of germ cell apoptosis, as previously reported by us. Q-VD-OPH at this dose markedly inhibited caspase 3 activation and significantly prevented (by 67.0%) heat-induced germ cell apoptosis. Q-VD-OPH-mediated rescue of germ cells was independent of cytosolic translocation of mitochondrial cytochrome c and DIABLO. Electron microscopy further revealed normal appearance of these rescued cells. Similar protection from heat-induced germ cell apoptosis was also noted after pretreatment with minocycline, a second-generation tetracycline that effectively inhibits cytochrome c release and, in turn, caspase activation. Collectively, the present study emphasizes the role of caspases in heat-induced germ cell apoptosis.     

Role of Sertoli cells in injury-associated testicular germ cell apoptosis

This review examines experimental models of Sertoli cell injury resulting in germ cell apoptosis. Since germ cells exist in an environment created by Sertoli cells, paracrine signaling between these intimately associated cells must regulate the process of germ cell death. Germ cell apoptosis may be signaled by a decrease in Sertoli cell pro-survival factors, an increase in Sertoli cell pro-apoptotic factors, or both. The different models of Sertoli cell injury indicate that spermatogenesis is susceptible to disruption, and that targeting critical Sertoli cell functions can lead to rapid and massive germ cell death.     

Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue

Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P < 0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P < 0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P < 0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P < 0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P > 0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue.     

Estradiol treatment induces testicular oxidative stress and germ cell apoptosis in rats

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.     

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.     

Ultrastructural localization of hCG in the placenta and in testicular germ cell tumors

Human chorionic gonadotrophin(hCG)-producing cells in the human placenta and in testicular germ cell tumors have been investigated by ultrastructural immunohistochemistry using labeled Fab'fragments of anti-hCG-alpha and anti-hCG-beta. Most reaction products were demonstrated in the rough endoplasmic reticulum (rER) and perinuclear spaces of syncytiotrophoblastic giant cells ( STGC ) occurring in embryonal carcinomas and in syncytial cells observed in choriocarcinomas and in normal placenta. The rER of placental and choriocarcinomatous syncytial cells was highly developed and invariably showed a positive reaction. However, the rER of STGCs varied both with regard to its degree of development and its immunoreactivity with hCG. Localization of hCG in the Golgi apparatus and in cytoplasmic granules was indefinite. Mononuclear cells reacting positively with anti-hCG-alpha or -beta were also demonstrated. Cytotrophoblast-like cells which failed to react with anti-hCG-alpha or -beta were noted adjacent to sTGCs and the significance of these cells is discussed.