BACKGROUND:Human testicular germ cell tumours (TGCT) arise from germ cell neoplasia in situ (GCNIS) cells that originate from foetal germ cell precursors. Activin A is central to normal foetal testis development, and its dysregulation may contribute to TGCT aetiology.OBJECTIVE:(i) To test whether the expression profiles of activin A targets in normal and neoplastic human testes indicates functional links with TGCT progression. (ii) To investigate whether activin A levels influence MMP activity in a neoplastic germ cell line.MATERIALS AND METHODS:(1) Bouin's fixed, paraffin-embedded human testes were utilized for PCR-based transcript analysis and immunohistochemistry. Samples (n = 5 per group) contained the following: (i) normal spermatogenesis, (ii) GCNIS or (iii) seminoma. CXCL12, CCL17, MMP2 and MMP9 were investigated. (2) The human seminoma-derived TCam-2 cell line was exposed to activin A (24 h), and target transcripts were measured by qRT-PCR (n = 4). ELISA (n = 4) and gelatin zymography (n = 3) showed changes in protein level and enzyme activity, respectively.RESULTS:(i) Cytoplasmic CXCL12 was detected in Sertoli and other somatic cells, including those surrounding seminoma cells. Anti-CCL17 labelled only the cytoplasm of Sertoli cells surrounding GCNIS, while anti-MMP2 and anti-MMP9 labelled germline and epithelial-like cells in normal and neoplastic testes. (ii) Exposing TCam-2 cells to activin A (50 ng/mL) elevated MMP2 and MMP9 transcripts (fourfold and 30-fold), while only MMP2 protein levels were significantly higher after activin A (5 ng/mL and 50 ng/mL) exposure. Importantly, gelatin zymography revealed activin A increased production of activated MMP2.DISCUSSION:Detection of CCL17 only in GCNIS tumours may reflect a change in Sertoli cell phenotype to a less mature state. Stimulation of MMP2 activity by activin A in TCam-2 cells suggests activin influences TGCT by modulating the tumour niche.CONCLUSION:This knowledge provides a basis for understanding how physiological changes that influence activin/TGF-β superfamily signalling may alter germ cell fate.

Endometriose wird durch das Vorhandensein von endometrialen Drüsen und Stroma außerhalb der Cavitas uteri definiert, wobei diese Herde histologisch weitgehend dem eutopen Endometrium entsprechen. In letzter Zeit wird zunehmend die Epithelial-Mesenchymale-Transition (EMT) bei der Endometriose diskutiert. Die EMT ist eine Veränderung des Zell-Phänotyps, die durch den Verlust von epithelialen und den Zugewinn mesenchymaler Eigenschaften gekennzeichnet ist. Wir analysierten die EMT im humanen Endometrium sowie endometriotischen Läsionen, um eine Beteiligung in der Pathogenese der Endometriose zu untersuchen.

BACKGROUND:Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR.RESULTS:Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.CONCLUSION:We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.

In mammals, the oocyte and preimplantation embryo are protected by the zona pellucida (ZP) consisting mainly of ZP glycoproteins, which are responsible for sperm binding, induction of the acrosome reaction and zona pellucida hardening to prevent polyspermia. The ZP proteins become increasingly important as possible predictors for in vitro cultured oocytes competence. As little is known about the stage-dependent expression of ZP1, ZP2 and ZP3 in marmoset monkey (Callithrix jacchus) oocytes, mRNA expression was investigated with real-time RT-PCR. Total-RNA was isolated from three different classes of marmoset oocytes; Class 1 oocytes from periantral follicles (<600 μm, n = 10), Class 2 oocytes from small antral follicles (600–1000 μm, n = 10) and Class 3 oocytes from large antral follicles (>1000 μm, n = 9). Compared with Class 1 oocytes mRNA expression of ZP1, ZP2 and ZP3 in Class 2 oocytes was significantly decreased. In Class 3 oocytes, the transcription of ZP1, ZP2 and ZP3 genes showed also a significant decrease compared with Class 1 oocytes. In this study a differently regulated expression of the ZP genes during late folliculogenesis with an obvious downregulation of ZP1, ZP2 and ZP3 could be demonstrated for the first time in the marmoset monkey.

Objectives: In clinical trials, transforming growth factor betas (TGF-betas) and plasminogen activator inhibitor-1 (PAI-1) were observed to be highly expressed at endometriotic sites as well as in the peritoneal fluid or serum of patients who suffer from endometriosis. PAI-1 is one of the most important genes to test for upregulation of mRNA expression after treatment of cells with TGF-betas. Furthermore, PAI-1 might contribute to activation of latent TGF-betas via plasmin or is misregulated in cancer tissue, e.g. breast cancer. Thus, we have analyzed PAI-1 secretion in endometrial and endometriotic cells after treatment with TGF-betas and have measured PAI-1 serum levels in patients suffering from endometriosis.

You have accessJournal of Urology1 Apr 2008PROTAMINE AND Bcl2 mRNA IN TESTICULAR SPERMATIDS AND EJACULATED SPERMATOZOA AS PROGNOSTIC BIOMARKER FOR MALE FERTILITY Klaus Steger, Jochen Wilhelm, Lutz Konrad, Thomas Stalf, Robert Greb, Thorsten Diemer, Sabine Kliesch, Martin Bergmann, and Wolfgang Weidner Klaus StegerKlaus Steger More articles by this author , Jochen WilhelmJochen Wilhelm More articles by this author , Lutz KonradLutz Konrad More articles by this author , Thomas StalfThomas Stalf More articles by this author , Robert GrebRobert Greb More articles by this author , Thorsten DiemerThorsten Diemer More articles by this author , Sabine KlieschSabine Kliesch More articles by this author , Martin BergmannMartin Bergmann More articles by this author , and Wolfgang WeidnerWolfgang Weidner More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(08)61851-0AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "PROTAMINE AND Bcl2 mRNA IN TESTICULAR SPERMATIDS AND EJACULATED SPERMATOZOA AS PROGNOSTIC BIOMARKER FOR MALE FERTILITY." The Journal of Urology, 179(4S), p. 632 © 2008 by American Urological AssociationFiguresReferencesRelatedDetails Volume 179Issue 4SApril 2008Page: 632 Advertisement Copyright & Permissions© 2008 by American Urological AssociationMetricsAuthor Information Klaus Steger More articles by this author Jochen Wilhelm More articles by this author Lutz Konrad More articles by this author Thomas Stalf More articles by this author Robert Greb More articles by this author Thorsten Diemer More articles by this author Sabine Kliesch More articles by this author Martin Bergmann More articles by this author Wolfgang Weidner More articles by this author Expand All Advertisement PDF DownloadLoading ...

Smad6 and Smad7 are inhibitory Smad proteins, which are essential in the negative regulation of BMP and TGF-beta/activin-signalling. While Smad7 is ubiquitously expressed and inhibits BMP and TGF-beta signalling, Smad6 is expressed in a cell-specific fashion and preferentially inhibits BMP-signalling. Here, we identified in addition to the splice variant Smad6s, possessing a different N-terminus, a novel human Smad6 splice variant, termed Smad6B, in human prostatic and rodent testicular cell lines. Due to an alternative exon, the putative Smad6B protein exhibits a truncated C-terminus lacking the entire MH-2 domain and most parts of the linker region. While full-length Smad6 was expressed in almost all tissues and cell lines studied, expression of the splice variants Smad6s and Smad6B was more heterogenous. Species-specific sequence comparison revealed that Smad6 is found in most species, while Smad6s was found only in Homo sapiens and Pan troglodytes, and Smad6B was found only in Homo sapiens. In addition, Smad6 localisation was found in round spermatids of human testis. In conclusion, we propose that alternative splicing of Smad6s and Smad6B is restricted to higher mammals.

AIM:The multidrug resistance protein 1 (MDR1, P-gp, p-170) is a membrane glycoprotein that acts as an energy-dependent drug efflux pump. In various malignancies its expression is associated with resistance to diverse cytostatic drugs, and therefore predicts resistance to systemic treatment. The aim of this study was to investigate the prognostic value of MDR1 expression in primary tumor tissue to predict necrosis or viable cancer in residual tumor masses after systemic chemotherapy for advanced testicular germ cell cancer.MATERIALS AND METHODS:Out of 77 patients, histopathological characteristics of primary testicular cancer specimens and retroperitoneal lymph node dissection (RPLND) samples following chemotherapy were available from 72 and all 77 patients, respectively. Moreover, MDR1 expression was determined by immunohistochemistry in 47 primary tumors and corresponding 73 RPLND sections.RESULTS:After chemotherapy and subsequent RPLND, the examination of residual tumor masses revealed that mature teratoma and active viable tumor were predominantly found in patients with non-seminoma (NSGCT; p=0.048), especially in those with containing mature teratoma (p=0.001). Moreover, using univariate analysis the expression of MDR1 in the primary testicular tumor predicted viable tumor/teratoma residues in RPLND sections (p=0.003). However, in multivariate analysis including the tumors' histological subtype, MDR1 expression alone failed to reach statistical significance as an independent prognostic marker for residual vital tumor (p>or=0.16).CONCLUSIONS:With the limited number of patients given, the correlation between MDR1 expression in primary testis cancer and active residual retroperitoneal disease after chemotherapy failed to reach statistical significance as in independent marker. Therefore, up to now routine MDR1 staining of testicular germ cell cancer samples should not be performed in clinical practice. However, as there was a clear trend, a larger number of patients suffering from metastatic non-seminomas should be studied, as MDR1 expression might have significant prognostic value in this particular subgroup of patients.

PACE4, a member of the subtilisin-like proprotein convertase (SPC) family, is expressed at high levels in certain tumor cells and plays a role in metastatic progression through activation of matrix metalloproteinases. The mechanism leading to overexpression of PACE4 in tumor cells remains unclear. In this study, we show that the E2F1 transcription factor, which is implicated in carcinoma invasiveness, upregulates the expression of PACE4. HT1080 (highly tumorigenic and invasive) cells expressed much higher levels of PACE4 and E2F family (E2F1 and E2F2) transcripts than IMR90 (normal fibroblast) cells. Expression levels of other SPCs (furin and PC6) remained unchanged in these cells. Promoter analysis indicated that two E2F consensus binding sites (−117/−110 and −86/−79) in the 5′-flanking region of the human PACE4 gene function as positive regulatory elements. Mutation of these sites abolished PACE4 promoter response to E2F1 as well as binding of E2F1 in electrophoretic mobility-shift assays. Other E2F members, E2F2 and E2F3, also activated PACE4 expression, as in the case of E2F1. These results indicate a novel mechanism for E2F family-mediated promotion of carcinoma invasiveness through PACE4.

Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-βs) play an active role. Here we demonstrate that the three mammalian TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-β2 compared to TGF-β1 and TGF-β3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-β-induced apoptosis. The specificity of the TGF-β effects was proven by addition of recombinant latency-associated peptide against TGF-β1 (rLAP-TGF-β1) which completely abolished TGF-β1-induced and TGF-β3-induced germ cell apoptosis. Although TGF-β2-triggered germ cell death also was significantly reduced by rLAP-TGF-β1, inhibition was not maximal. Our results suggest that the three TGF-β isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty.