Effects of Pulsed-Wave Photobiomodulation Therapy on Human Spermatozoa.

BACKGROUND AND OBJECTIVES: Previous studies reported that photobiomodulation (PBM) positively affects the mitochondrial respiratory chain in sperm, resulting in improved motility and velocity. As laser settings are not yet fully established, the present study aimed at optimizing PBM on human sperm. In addition, possible side-effects of PBM on sperm DNA fragmentation level and acrosomal integrity have been analyzed. STUDY DESIGN/MATERIALS AND METHODS: A pulsed laser-probe (wavelength 655 nm, output power 25 mW/cm², impulse duration 200 nanoseconds) was used. Native fresh liquefied semen samples underwent radiation with energy doses of 0 (control), 4, 6, and 10 J/cm². Sperm parameters were assessed at 0, 30, 60, 90, and 120 minutes after radiation using a computer-assisted sperm analysis system. Motility and velocity of sperm from asthenozoospermic patients (n = 42) and normozoospermic controls (n = 22) were measured. The amount of DNA strand breaks was analyzed using ligation-mediated quantitative polymerase chain reaction in patients with asthenozoospermia (n = 18) and normozoospermia (n = 13). Post-irradiance acrosomal integrity was investigated using flow cytometry based on CD46 protein expression (n = 7). RESULTS: Exposure to laser energy-doses of 4 and 6 J/cm² improved sperm motility and velocity in asthenozoospermic patients. PBM exhibited no significant effect on DNA fragmentation level and expression of CD46 serving as a biomarker for acrosome integrity. CONCLUSION: PBM improves sperm motility parameters by maintaining DNA and acrosome integrity and, therefore, represents a promising new tool for assisted reproductive therapy. In particular, improving sperm motility in asthenozoospermic patients by PBM in future may contribute to increasing the chance for successful intrauterine insemination. The present trial has no clinical registration number, as only in vitro studies were performed. The study was approved by the local ethics committee and performed according to the Declaration of Helsinki. Lasers Surg. Med. © 2021 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.

Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2-DMR0 and its interaction with KLF4.

BACKGROUND: Human cancer cells often exhibit impaired IGF2 expression and the underlying mechanisms are multifaceted and complex. Besides the well-known imprinting control region IGF2/H19-ICR, the involvement of a differentially methylated region in the promoter P0 of IGF2 gene (IGF2-DMR0) has been suggested. Here, we evaluate several mechanisms potentially leading to up- and/or down-regulation of IGF2 expression in prostate cancer and present a novel role of Kruppel-like factor 4 (KLF4) as a transcriptional regulator of IGF2 binding in IGF2-DMR0. METHODS: Putative binding sites for transcription factors were identified in IGF2-DMR0 using JASPAR CORE database. Gene expressions were analyzed by RT-qPCR in prostate carcinoma and adjacent benign prostate hyperplasia samples obtained by radical prostatectomy (86 RP-PCa and 47 RP-BPH) and BPH obtained by transurethral prostate resection (13 TUR-BPH). Pyrosequencing and qMSP were used for DNA methylation studies in IGF2-DMR0, IGF2/H19-ICR and Glutathione-S-transferase-P1 (GSTP1) promoter. Loss of imprinting (LOI) was analyzed by RFLP. Copy number variation (CNV) test was performed using qBiomarker CNV PCR Assay. KLF4-binding and histone-modifications were analyzed by ChIP-qPCR in prostate cancer cell lines exhibiting differentially methylated IGF2-DMR0 (LNCaP hypomethylated and DU145 hypermethylated). KLF4 protein was analyzed by western blot. Statistical associations of gene expression to methylation, IGF2 LOI and CNV were calculated by Mann-Whitney-U-test. Correlations between gene expression and methylation levels were evaluated by Spearman's-Rank-Correlation-test. RESULTS: We found a significant reduction of IGF2 expression in the majority of RP-PCa and RP-BPH in comparison to TUR-BPH. Analyzing potential molecular reasons, we found in RP-PCa and RP-BPH in comparison to TUR-BPH a significant hypomethylation of IGF2-DMR0, which coincided with hypermethylation of GSTP1-promoter, a prominent marker of prostate tumors. In contrast, IGF2 LOI and CNV did not associate significantly with up- and/or down-regulation of IGF2 expression in prostate tumors. By analyzing IGF2-DMR0, we detected a consensus sequence for KLF4 with a z-score of 7.6. Interestingly, we found that KLF4 binds to hypomethylated (17%) IGF2-DMR0 enriched with H3K9me3 and H3K27me3 (LNCaP), but does not bind under hypermethylated (85%) and H3K4me3-enriched conditions (DU145). KLF4 expression was detected in TUR-BPH as well as in RP-BPH and RP-PCa and showed a highly significant correlation to IGF2 expression. CONCLUSIONS: Our study demonstrated that in human prostate cancer the impairment of IGF2 expression is accompanied by hypomethylation of IGF2-DMR0. We revealed that KLF4 is a putative transcriptional regulator of IGF2, which binds in IGF2-DMR0 in dependence of the prevailing epigenetic state in this region. Herewith we provide complementary new insights into IGF2 dysregulation mechanisms as a critical process in prostate tumorigenesis.

TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility.

STUDY QUESTION: Are ten-eleven-translocation (TET) 1-3 family enzymes involved in human spermatogenesis and do they impact male fertility? SUMMARY ANSWER: TET1, TET2 and TET3 are successively expressed at different stages of human spermatogenesis, and their expression levels associate with male fertility. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex cell differentiation process accompanied by a drastic epigenetic remodeling. TET1-3 dioxygenases are essential for active DNA demethylation in the paternal pronucleus and in embryonic stem cells. STUDY DESIGN, SIZE, DURATION: Expression of TET1-3 mRNAs and proteinss and 5-hydroxymethylcytosine (5-hmC) proteins were analyzed in human testis tissues from men with obstructive azoospermia and exhibiting histologically normal spermatogenesis. Ejaculated spermatozoa from normozoospermic healthy volunteers, the 'controls' (TET1: n = 58; TET2-3: n = 63), and subfertile men who participated with their female partners in an ICSI-program, the 'patients' (TET1: n = 66; TET2-3: n = 64), were analyzed concerning the stored TET1-3 mRNAs, and the values were correlated to semen parameters and ICSI-outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis sections were used for in situ hybridization (ISH) and immunohistochemical (IHC) studies to determine TET1-3 mRNA and protein expression, and for immunofluorescence (IF) detection of 5-hmC. Sperm samples from controls were analyzed by western blot, immunocytochemistry (ICC) and RT-PCR concerning the presence of non-degraded TET1-3 protein and mRNA. Sperm samples from controls and patients were used for quantitative TET1-3 mRNA analyses (reverse transcription-polymerase chain reaction) and for comparative statistical evaluations under consideration of semen parameters and ICSI-outcome (pregnancy). MAIN RESULTS AND THE ROLE OF CHANCE: During human spermatogenesis TET1-3 proteins are successively expressed: TET2 is expressed in the cytoplasm of late pachytene spermatocytes of Stage V, TET1 starts to be expressed in the nuclei of Step 1 round spermatids at Stage I, and TET3 starts to be expressed in the nuclei of Step 3 round spermatids at Stage III. Five-hmC appears only in Step 5 elongated spermatids. All three TETs are still detectable at the mRNA and protein level in sperm cells in considerable amounts. Control men generally exhibited higher levels of TET1-3 in sperm. TET1- and TET3-mRNA levels in sperm were significantly negatively correlated with age (P = 0.0025 and P = 0.0343) and positively correlated with progressive sperm motility (P = 0.0007 and P = 0.018). All TETs showed a significant association with sperm concentration (P < 0.03). Patients diagnosed with oligozoospermia and/or asthenozoospermia (TET1: n = 35; TET2-3: n = 32) showed significantly reduced TET1-3 in sperm in comparison to controls (P = 0.003, P = 0.041 and P = 0.028), but not compared with normozoospermic patients. Levels of TET3 in sperm was significantly associated with high-fertilization rates (P = 0.009). Concerning ICSI-outcome, the lowest levels of TET1-3 mRNAs in sperm were found in the non-pregnant group. Increased TET2 in sperm was significantly associated with pregnancy (P = 0.006). LIMITATIONS, REASONS FOR CAUTION: Our results concerning the association of the mRNA level of TETs in ejaculated sperm cells to different fertility parameters are descriptive. Further studies clarifying the reasons for decreased TET1-3 levels in subfertile men and their effect on their sperm methylome are essential. WIDER IMPLICATIONS OF THE FINDINGS: The study gives a substantial indication that in human spermiogenesis, an active DNA demethylation process occurs with an involvement of TET enzymes, and that the level of TET1-3 expression is pivotal for male fertility. STUDY FUNDING: Research grant from the German Research Foundation (DFG) to U.S. (SCHA1531/1-1 and SCHA1531/2-1). COMPETING INTERESTS: None.

PTPIP51­A New RelA-tionship with the NFκB Signaling Pathway.

The present study shows a new connection of protein tyrosine phosphatase interacting protein 51 (PTPIP51) to the nuclear factor κB (NFκB) signalling pathway. PTPIP51 mRNA and protein expression is regulated by RelA. If bound to the PTPIP51 promoter, RelA repress the mRNA and protein expression of PTPIP51. The parallel treatment with pyrrolidine dithiocarbamate (PDTC) reversed the suppression of PTPIP51 protein expression induced by TNFα. Using the intensity correlation analysis PTPIP51 verified a co-localization with RelA, which is also regulated by TNFα administration. Moreover, the direct interaction of PTPIP51 and RelA was established using the DuoLink proximity ligation assay. IκBα, the known inhibitor of RelA, also interacted with PTPIP51. This hints to the fact that in un-stimulated conditions PTPIP51 forms a complex with RelA and IκBα. The PTPIP51/RelA/IκBα complex is modulated by TNFα. Interestingly, the impact on the mitogen activated protein kinase pathway was negligible except in highest TNFα concentration. Here, PTPIP51 and Raf-1 interactions were slightly repressed. The newly established relationship of PTPIP51 and the NFκB signaling pathway provides the basis for a possible therapeutic impact.

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

Expression and role of leptin under hypoxic conditions in human testis: organotypic in vitro culture experiment and clinical study on patients with varicocele.

PURPOSE: We investigated the expression and the exact role of leptin under hypoxic conditions in the human testis. MATERIALS AND METHODS: Five testes from patients treated with orchiectomy for prostate cancer were used to construct an in vitro hypoxic culture system for human testicular tissue. Immunohistochemistry was performed to analyze leptin protein expression. Leptin, leptin receptor and HIF-1α mRNA were examined by quantitative reverse transcriptase-polymerase chain reaction. Serum and seminal plasma leptin, gonadal hormones and semen parameters were evaluated in 10 healthy donors and 42 infertile patients with varicocele before and after surgery. RESULTS: The viability of in vitro cultured testicular tissue was well maintained within 48 hours based on the results of morphological analysis, cell number and cell specific mRNAs. Immunohistochemistry demonstrated that leptin was mainly expressed in seminiferous tubules. Interestingly the optical density of leptin, leptin mRNA and HIF-1α mRNA was significantly increased under hypoxia. Leptin mRNA and HIF-1α mRNA correlated positively (Rs = 0.843, p <0.01). In the clinical study the concentration of seminal plasma leptin before varicocelectomy was markedly higher in patients (mean ± SD 3.01 ± 1.23 ng/l, p <0.01). It was highest in the grade 3 group (mean 3.95 ± 1.37 ng/l, p <0.01) and significantly decreased in the 6-month postoperative group (2.35 ± 0.78, p <0.05). Furthermore, negative correlations were observed between seminal plasma leptin and the sperm concentration (Rs = -0.187, p <0.05), and progressive motility (Rs = -0.234, p <0.05). CONCLUSIONS: Leptin expression was induced under hypoxia in the human testis, probably via the HIF-1α related response pathway. Seminal plasma leptin closely correlated with varicocele related spermatogenesis dysfunction. It might effectively reflect the testicular hypoxic environment.

Toxoplasma gondii decreases the reproductive fitness in mice.

Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for Toxoplasma gondii, which forms specialized vacuoles containing reproductive cysts in the formers' tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how Toxoplasma gondii can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of Toxoplasma gondii infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in Toxoplasma gondii positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in Toxoplasma infected mice, and differences in the DNA methylation of selected genes were detected between the Toxoplasma positive and control group. These findings demonstrate a direct relation between Toxoplasma gondii infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of idiopathic infertility in humans.

Sperm protamine mRNA ratio and DNA fragmentation index represent reliable clinical biomarkers for men with varicocele after microsurgical varicocele ligation.

PURPOSE: We investigated whether the sperm protamine-1/2 mRNA ratio and DNA fragmentation index are reliable biomarkers in patients with clinical varicocele. MATERIALS AND METHODS: We performed a prospective study in 42 subfertile patients with left clinical varicocele and 10 normozoospermic healthy donors with proven fertility. All patients and female partners were seen 3 and 6 months after varicocelectomy for fertility examination. Real-time quantitative reverse transcriptase-polymerase chain reaction and SCSA® were performed to analyze the sperm protamine-1/2 mRNA ratio and DNA fragmentation index. RESULTS: The protamine-1/2 mRNA ratio and DNA fragmentation index were significantly higher in the preoperative group than in the control group (p <0.01). After varicocelectomy in the pregnant group the protamine-1/2 mRNA ratio recovered to the normal value and the DNA fragmentation index was significantly lower than in the preoperative group (p <0.01). In the nonpregnant group the protamine-1/2 mRNA ratio and DNA fragmentation index did not differ (p >0.05). However, significant differences were present preoperatively according to varicocele severity in the protamine-1/2 mRNA ratio and DNA fragmentation index (p <0.05 and <0.01, respectively) with the highest values in the grade 3 group. Nevertheless, postoperatively no difference in varicocele severity was noted (p >0.05). The protamine-1/2 mRNA ratio was strongly related to preoperative and postoperative sperm concentration (Rs -0.238, p <0.01), progressive motility (Rs -0.327, p <0.01), total motility (Rs -0.206, p <0.05) and DNA fragmentation index (Rs 0.293, p <0.01) except for normal morphology (Rs -0.064, p >0.05). CONCLUSIONS: The sperm protamine-1/2 mRNA ratio and DNA fragmentation index can effectively be used to evaluate male fertility. Male infertility due to varicocele may be associated with protamine deficiency and sperm DNA damage. The post-varicocelectomy protamine-1/2 mRNA ratio and DNA fragmentation index are associated with the post-varicocelectomy pregnancy rate.

Combined trifocal and microsurgical testicular sperm extraction is the best technique for testicular sperm retrieval in "low-chance" nonobstructive azoospermia.

BACKGROUND: There is no consensus for the best testicular sperm extraction (TESE) technique in patients with "low-chance" nonobstructive azoospermia (NOA). OBJECTIVE: To determine sperm retrieval rates in an intraindividual comparison using three locations of the testicle with and without the assistance of a microscope (microsurgical TESE [M-TESE]). DESIGN, SETTING, AND PARTICIPANTS: A series of 65 patients with low-chance NOA presenting with low testicular volume (<8 ml) and high serum follicle-stimulating hormone (FSH) (>12.4 IU/l) underwent trifocal-TESE plus M-TESE bilaterally (four biopsies per testis). INTERVENTION: Sperm retrieval was performed as trifocal-TESE (upper, middle, and lower testicular pole) with and without the assistance of a microscope in the middle incision. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The number of evaluated tubules, the mean spermatogenetic scores, and the sperm retrieval rates were evaluated to determine retrieval locations and the use of the microscope. The Friedman and Cochrane Q tests were applied to determine statistical differences. Receiver operating characteristic curves were used for the analysis of serum FSH and testicular volume as preoperative prognostic factors. RESULTS AND LIMITATIONS: The sperm retrieval success of 66.2% using the combined technique, meaning the percentage of patients with at least one tubule containing elongated spermatids, was the highest in the combination of trifocal- and M-TESE (p<0.01), indicating this technique as optimal for patients with low-chance NOA. M-TESE and trifocal-TESE alone were not significantly better. The mean spermatogenetic score giving the number of tubules with elongated spermatids in relation to all tubules was significantly higher in M-TESE versus conventional TESE (p<0.01), indicating the superior quality of the tissue harvested using the microscope. These results are limited by the definition of "success" using "one" spermatid/tubule. Preoperatively, high serum FSH and low testicular volumes did not exclude successful sperm retrieval. CONCLUSIONS: The combination of trifocal- and M-TESE is the best technique to reach high sperm retrieval rates in patients with low-chance NOA.

Epigenetic down regulation of RASSF10 and its possible clinical implication in prostate carcinoma.

BACKGROUND: Ras association domain family (RASSF) comprises several tumor suppressor genes, which are often epigenetically inactivated in human tumors. Here, we aim to analyze the relevance of the recently identified member RASSF10 in prostate carcinogenesis. METHODS: RASSF10 promoter methylation and mRNA expression were investigated by bisulfite-pyrosequencing and qRT-PCR, respectively, in prostate carcinoma (PCa) cell lines (LNCaP, 22Rv1, DU-145) and in 83 primary PCa and 53 primary benign prostatic hyperplasia (BPH) tissues obtained after radical prostatectomy. Histological localization of RASSF10 was done by in situ hybridization. To prove the epigenetic nature of RASSF10 down regulation, PCa cell lines were treated with 5-aza-2-deoxycytidine and trichostatin A. Potential function of RASSF10 was analyzed in LNCaP by colony formation and apoptosis assays. RESULTS: RASSF10 mRNA was localized to cells of the basal layer of the prostatic gland. Absence (LNCaP) and decrease (22Rv1, DU-145) of RASSF10 expression was associated with promoter methylation and could be restored by demethylating agents. A link between RASSF10 mRNA reduction and promoter methylation was also detected in primary prostate tissues (P = 0.006), where PCa showed more frequently reduced RASSF10 levels when compared with BPH (33.7% vs. 13.2%, P = 0.009). RASSF10 methylation could be further associated with advanced tumor stage and advanced age (P-values < 0.05). Our preliminary functional assays revealed the ability of RASSF10 to inhibit colony formation (P = 0.018) and to increase apoptosis (P = 0.035). CONCLUSIONS: This is the first study, which demonstrates the frequent epigenetic inactivation of RASSF10 in PCa and its implication in clinical symptoms of PCa.

PTPIP51 is phosphorylated by Lyn and c-Src kinases lacking dephosphorylation by PTP1B in acute myeloid leukemia.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is known to be expressed in blood cells with restriction to the myeloid lineage. All myeloid progenitor cells are PTPIP51 positive except for the myeloblasts. To define the expression of PTPIP51 in acute myeloid leukemia (AML), we performed immunohistochemical experiments with peptide specific antibodies (C-terminus, N-terminus and aas 114-129) to PTPIP51 with samples of AML bone marrow trephine biopsy specimens. AML blasts reacted positive for PTPIP51 protein encompassing the C-terminal sequence. Healthy bone marrow displayed an exclusive staining for the N-terminal containing form of PTPIP51. Moreover, PTPIP51 protein was highly phosphorylated at its tyrosine 176 residue. Acquired confocal images of AML cells displayed an absence of PTP1B and revealed a co-localization of PTPIP51 and Lyn. Duolink proximity ligation assays (DPLA) corroborated an interaction for PTPIP51 with Lyn and c-Src. In AML blasts rarely an interaction of PTPIP51 with PTP1B and Raf-1 was seen. Furthermore, DPLA signals were also obtained for PTPIP51 and c-Kit in AML cells. Therefore, PTPIP51 was identified as a new signal molecule of the c-Kit signaling pathway. By the phosphorylation done by Lyn, c-Src and c-Kit, PTPIP51 is prevented to influence mitogen activated protein kinase pathway on Raf-1 level contributing to increased proliferation of AML cells.

NANOG promoter methylation and expression correlation during normal and malignant human germ cell development.

Testicular germ cell tumors are the most frequent malignant tumors in young Caucasian males, with increasing incidence. The actual model of tumorigenesis is based on the theory that a block in maturation of fetal germ cells lead to formation of the intratubular germ cell neoplasia unclassified. Early fetal germ cells and undifferentiated germ cell tumors express pluripotency markers such as the transcription factor NANOG. It has been demonstrated, that epigenetic modifications such as promoter DNA-methylation is able to silence gene expression in normal and cancer cells. Here we show, that OCT3/4-SOX2 mediated expression of NANOG can be silenced by methylation of promoter CpG-sites. We found that global methylation of DNA decreased from fetal spermatogonia to mature sperm. In contrast, CpGs in the NANOG promoter were found hypomethylated in spermatogonia and hypermethylated in sperm. This selective repression might reflect the cells need to suppress pluripotency in order to prevent malignant transformation. Finally, methylation of CpGs in the NANOG promoter in germ cell tumors and derived cell lines correlated to differentiation state.

14-3-3 beta in the healthy and diseased male reproductive system.

BACKGROUND: Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been implicated in several diseases including tumourigenesis. Here we investigated the expression of the 14-3-3 beta isoform in healthy testicular tissues of humans, rats and mice as well as in tissues of Sertoli-cell-only (SCO) syndrome, intratubular germ cell neoplasia (IGCN) and classical seminoma. METHODS: Samples of healthy and diseased testes from humans, rats and mice were analysed by immunohistochemistry. For PCR, human testis cell lysates were used. Immunoblot analyses of rats and humans healthy testes were performed. Duolink proximity ligation assay (PLA) and co-immunoprecipitation (Co-IP) were carried out to investigate interactions between 14-3-3 beta and vimentin in human, rat and mouse testes. RESULTS: In healthy testes and SCO syndrome, strong 14-3-3 beta-positive cells could be identified as Sertoli cells. Furthermore, 14-3-3 beta proteins were detected in cells of the peritubular stroma. In samples of IGCN and classical seminoma, the malignant transformed cells stained positive for 14-3-3 beta antigen. Immunoblot analyses revealed the presence of 14-3-3 beta in healthy testicular tissues. 14-3-3 beta mRNA transcripts were detected in cell lysates of healthy human testes. Interaction of 14-3-3 beta with the intermediate filament vimentin was revealed by Duolink PLA and Co-IP. Co-IP experiments identified tubulin as another 14-3-3 beta binding partner. CONCLUSIONS: Our data suggest that 14-3-3 beta expression is essential for normal spermatogenesis by interacting with vimentin in Sertoli cells. Additionally, 14-3-3 beta expression in malignant transformed cells in IGCN and classical seminoma may lead to tumourigenesis and cell survival.

PTPIP51-a myeloid lineage specific protein interacts with PTP1B in neutrophil granulocytes.

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) was identified as an in vitro interacting partner of protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP). The full-length form of PTPIP51 encompasses 470aas and has a molecular weight of 52kDa. The physiological function is poorly understood but an involvement in differentiation processes and apoptosis has been suggested. Preliminary observations suggested differences in PTPIP51 expression in blood cells. To analyze a possible involvement of PTPIP51 in hematopoietic processes, we studied its expression in samples of peripheral venous blood (PVB), umbilical cord blood (UCB) and human bone marrow (HBM). In both, PVB and UCB PTPIP51 expression was restricted to neutrophil granulocytes. In HBM samples, besides in mature neutrophil ganulocytes PTPIP51 protein and mRNA was present in myeloid precursor cells of neutrophils. The expression of PTPIP51 in neutrophil granulocytes was corroborated by immunoblot analysis exhibiting different molecular weight forms of PTPIP51 protein. Anti-peptide antibodies, identifying specific regions of the PTPIP51 protein (C-terminus, N-terminus and aas114-129) revealed a distinct isoform expression pattern in neutrophil granulocytes of different sources. In PVB and UCB neutrophil granulocytes reacted positive for all three peptide antibodies. In contrast, neutrophils of HBM express solely an N-terminal variant of PTPIP51 protein, lacking the C-terminal and aas114-129 sequence. Immunocytochemical results displayed a strict co-localization of PTPIP51 and PTP1B in PVB and UCB. The interaction of both proteins was verified by a proximity ligation assay. Neither proliferating cells, as identified by PCNA immunostaining, nor apoptotic cells, labeled by TUNEL assay, displayed an immunoreactivity for PTPIP51 in HBM. In fact, PTPIP51 expression was restricted to myeloid precursor cells undergoing differentiation. In blood cells therefore, PTPIP51 expression is restricted to differentiating and mature neutrophil granulocytes.

PTPIP51 mRNA and protein expression in tissue microarrays and promoter methylation of benign prostate hyperplasia and prostate carcinoma.

BACKGROUND: Protein tyrosine phosphatase interacting protein 51 (PTPIP51) shows a tissue-specific expression pattern and is associated with cellular differentiation and apoptosis in several mammalian tissues. Overexpression of the full-length protein enhances apoptosis. It is also expressed in various carcinomas. In this study the expression of PTPIP51 and its in vitro interaction partners was investigated in human benign prostate hyperplasia (BPH) and in prostate carcinoma (PCa). METHODS: Tissue microarrays of human BPH and PCa were analyzed by immunohistochemistry. For polymerase chain reaction (PCR), cryo samples of BPH and PCa were used. Bisulfite DNA treatment, followed by sequencing of PCR products was performed in order to analyze CpGs methylation within the promoter region of the PTPIP51 gene. RESULTS: PTPIP51 mRNA and protein expression was detected in prostatic epithelia of BPH and in tumor cells of PCa, respectively, and within smooth muscle cells of the stromal compartment. A stronger expression was present in nerve fibers, particularly in PCa, in immune cells and in smooth muscle and endothelial cells of vessels of BPH and PCa. On mRNA levels, a slightly elevated expression of PTPIP51 was observed in the PCa group as tested by real-time quantitative PCR analyses. Methylation experiments revealed that at least 70% of methylated CpGs in the CpG island of the PTPIP51 gene promoter region were identified in BPH samples. In contrast, a loss of methylation has been found in the PCa group. CONCLUSION: The promoter methylation status of PTPIP51 seems to influence the expression of PTPIP51, which was seen as elevated in the PCa.

Aberrant epigenetic modifications in the CTCF binding domain of the IGF2/H19 gene in prostate cancer compared with benign prostate hyperplasia.

Expression of the imprinted genes insulin-like growth factor 2 (IGF2) and H19 depends on the methylation pattern of their common imprinting control region (ICR) located on chromosome 11p15. As the somatic imprinting pattern may be lost during tumorigenesis due to epigenetic alterations, in the present study, we analyzed the DNA methylation and histone modifications in the differentially methylated region (DMR) of IGF2/H19 in benign prostate hyperplasia (BPH) and prostate carcinoma (PCa). Sodium bisulfite sequencing was performed on frozen tissue collected after radical prostatectomy. Thirty tumors and 17 non-cancerous tissue samples were analyzed. Histological diagnosis was, in addition, confirmed by amplification of the epithelial tumor marker alpha-methylacyl coenzyme-A racemase. Chromatin immunoprecipitation assay (ChIP) was carried out on sonificated chromatin from fresh tissue samples from 10 PCa, 10 BPH using antibodies against trimethyl histone H3K9, dimethyl histone H3K9, trimethyl H3K27 and acetyl H3K9. The methylation pattern of 17 CpGs within 227 bp of the H19 fragment was characterized from each DNA sample. All (BPH) samples demonstrated >80% methylation of CpGs. In contrast, we found 41% of CpGs methylated in 9 out of 30 PCa specimens. We observed statistically significant differences in the methylation state between PCa and BPH groups, especially in the DMR of H19 (p<0.0001) and in the ICR (p=0.0034), which corresponds to CTCF binding domain. ChIP assay revealed that dimethyl H3K9 is associated with the ICR of IGF2/H19 in BPH, but not in PCa (p<0.0001). Our data demonstrate that DNA methylation and histone methylation analysis of the ICR within the DMR of IGF2/H19 provides important insights into early steps of carcinogenesis and, therefore, may contribute to improving diagnosis of PCa.

Expression of BLIMP1/PRMT5 and concurrent histone H2A/H4 arginine 3 dimethylation in fetal germ cells, CIS/IGCNU and germ cell tumors.

BACKGROUND: Most testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency. RESULTS: In the present study, we show that BLIMP1 and PRMT5 were expressed and arginine dimethylation of histones H2A and H4 was detected in human male gonocytes at weeks 12-19 of gestation, indicating a role of this mechanism in human fetal germ cell development as well. Moreover, BLIMP1/PRMT5 and histone H2A and H4 arginine 3 dimethylation was present in IGCNU and most seminomas, while downregulated in embryonal carcinoma (EC) and other nonseminomatous tumors. CONCLUSION: These data reveal similarities in marker expression and histone modification between murine and human PGCs. Moreover, we speculate that the histone H2A and H4 arginine 3 dimethylation might be the mechanism by which IGCNU and seminoma maintain the undifferentiated state while loss of these histone modifications leads to somatic differentiation observed in nonseminomatous tumors.

Expression of connexin 43 in normal canine testes and canine testicular tumors.

In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis.

Cancer stem cells as targets for cancer therapy: selected cancers as examples.

It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer-(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered.

Both protamine-1 to protamine-2 mRNA ratio and Bcl2 mRNA content in testicular spermatids and ejaculated spermatozoa discriminate between fertile and infertile men.

BACKGROUND: Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although aberrant protamine ratios have been observed in infertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the protamine ratio or Bcl2 content represent a reliable biomarker to discriminate fertile and infertile men. METHODS: Real-time quantitative RT-PCR was used for P1, P2 and the apoptotic marker Bcl2 in testicular biopsies (TB; 74 infertile men versus 17 controls) and ejaculates (E; 95 infertile men versus 10 controls). RESULTS: The P1-P2 mRNA ratio differed significantly between groups, namely 1:4 versus 1:3.2 in TB (P = 0.0038) and 1:1.7 versus 1:1 in E (P = 0.0002), for infertile men and controls, respectively. Bcl2 mRNA content was correlated with protamine mRNA ratio (P = 0.0250 for TB; P = 0.0003 for E). Infertile men exhibit a more than 10-fold (P = 0.0155 for TB; P = 7.0 x 10(-6) for E) higher Bcl2 mRNA content versus controls. No correlation was found between absolute sperm density and the protamine mRNA ratio or Bcl2 mRNA content. No significant correlation was demonstrated with fertilization rate after ICSI and either protamine ratio or Bcl2 content. CONCLUSIONS: We found significantly aberrant protamine ratios and a higher Bcl2 content in TB and E of infertile men compared to controls, suggesting that these molecules may be useful biomarkers for predicting male infertility.