Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer.

In mammals, male germ cell development starts during fetal life and is carried out in postnatal life with the formation of sperms. Spermatogenesis is the complex and highly orderly process during which a group of germ stem cells is set at birth, starts to differentiate at puberty. It proceeds through several stages: proliferation, differentiation, and morphogenesis and it is strictly regulated by a complex network of hormonal, autocrine and paracrine factors and it is associated with a unique epigenetic program. Altered epigenetic mechanisms or inability to respond to these factors can impair the correct process of germ development leading to reproductive disorders and/or testicular germ cell cancer. Among factors regulating spermatogenesis an emerging role is played by the endocannabinoid system (ECS). ECS is a complex system comprising endogenous cannabinoids (eCBs), their synthetic and degrading enzymes, and cannabinoid receptors. Mammalian male germ cells have a complete and active ECS which is modulated during spermatogenesis and that crucially regulates processes such as germ cell differentiation and sperm functions. Recently, cannabinoid receptor signaling has been reported to induce epigenetic modifications such as DNA methylation, histone modifications and miRNA expression. Epigenetic modifications may also affect the expression and function of ECS elements, highlighting the establishment of a complex mutual interaction. Here, we describe the developmental origin and differentiation of male germ cells and testicular germ cell tumors (TGCTs) focusing on the interplay between ECS and epigenetic mechanisms involved in these processes.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Non-Coding RNAs and Splicing Activity in Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are the most common tumors in adolescent and young men. Recently, genome-wide studies have made it possible to progress in understanding the molecular mechanisms underlying the development of tumors. It is becoming increasingly clear that aberrant regulation of RNA metabolism can drive tumorigenesis and influence chemotherapeutic response. Notably, the expression of non-coding RNAs as well as specific splice variants is deeply deregulated in human cancers. Since these cancer-related RNA species are considered promising diagnostic, prognostic and therapeutic targets, understanding their function in cancer development is becoming a major challenge. Here, we summarize how the different expression of RNA species repertoire, including non-coding RNAs and protein-coding splicing variants, impacts on TGCTs' onset and progression and sustains therapeutic resistance. Finally, the role of transcription-associated R-loop misregulation in the maintenance of genomic stability in TGCTs is also discussed.

To Be or Not to Be a Germ Cell: The Extragonadal Germ Cell Tumor Paradigm.

In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features (embryonal carcinomas) or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types.

Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways.

Despite germ cell tumors (GCTs) responding to cisplatin-based chemotherapy at a high rate, a subset of patients does not respond to treatment and have significantly worse prognosis. The biological mechanisms underlying the resistance remain unknown. In this study, by using two TGCT cell lines that have acquired cisplatin resistance after chronic exposure to the drug, we identified some key proteins and mechanisms of acquired resistance. We show that cisplatin-resistant cell lines had a non-homologous end-joining (NHEJ)-less phenotype. This correlated with a reduced basal expression of TP53-binding protein 1 (53BP1) and DNA-dependent protein kinase (DNA-PKcs) proteins and reduced formation of 53BP1 foci after cisplatin treatment. Consistent with these observations, modulation of 53BP1 protein expression altered the cell line's resistance to cisplatin, and inhibition of DNA-PKcs activity antagonized cisplatin cytotoxicity. Dampening of NHEJ was accompanied by a functional increase in the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. As a result, cisplatin-resistant cells were more resistant to PARP inhibitor (PARPi) monotherapy. Moreover, when PARPi was given in combination with cisplatin, it exerted an additive/synergistic effect, and reduced the cisplatin dose for cytotoxicity. These results suggest that treatment of cisplatin-refractory patients may benefit from low-dose cisplatin therapy combined with PARPi.

Assessing Homologous Recombination and Interstrand Cross-Link Repair in Embryonal Carcinoma Testicular Germ Cell Tumor Cell Lines.

Testicular germ cell tumors (TGCTs) are typically exquisitely sensitive to DNA interstrand cross-link (ICLs) agents. ICLs covalently link both strands of the DNA duplex, impeding fundamental cellular processes like DNA replication to cause cell death. A leading drug used for the treatment of TGCTs is cisplatin, which introduces ICLs and leads to formation of double strand breaks (DSBs), a DNA lesion that can be repaired in the S/G2 phases of the cell cycle by homologous recombination (HR, also termed homology-direct repair). Although most TGCTs respond to cisplatin-induced ICLs, a fraction is resistant to treatment. One proposed mechanism of TGCT resistance to cisplatin is an enhanced ability to repair DSBs by HR. Other than HR, repair of the ICL-lesions requires additional DNA repair mechanisms, whose action might also be implemented in therapy-resistant cells. This chapter describes GFP assays to measure (a) HR proficiency following formation of a DSB by the endonuclease I-SceI, and (b) HR repair induced by site-specific ICL formation involving psoralen. These experimental approaches can be used to determine the proficiency of TGCT cell lines in DSB repair by HR in comparison to HR repair of ICLs, providing tools to better characterize their recombination profile. Protocols of these assays have been adapted for use in Embryonal Carcinoma (EC) TGCT cell lines. Assays only require transient introduction of plasmids within cells, affording the advantage of testing multiple cell lines in a relatively short time.

Sempervirine inhibits RNA polymerase I transcription independently from p53 in tumor cells.

In the search of small molecules that can target MDM2/p53 pathway in testicular germ cell tumors (TGCTs), we identified sempervirine (2,3,4,13-tetrahydro-1H-benz[g]indolo[2,3-a]quinolizin-6-ium), an alkaloid of Gelsemium sempervirens, that has been previously proposed as an inhibitor of MDM2 that targets p53-wildtype (wt) tumor cells. We found that sempervirine not only affects cell growth of p53-wt cancer cells, but it is also active in p53-mutated and p53-null cells by triggering p53-dependent and independent pathways without affecting non-transformed cells. To understand which mechanism/s could be activated both in p53-wt and -null cells, we found that sempervirine induced nucleolar remodeling and nucleolar stress by reducing protein stability of RPA194, the catalytic subunit of RNA polymerase I, that led to rRNA synthesis inhibition and to MDM2 block. As shown for other cancer cell models, MDM2 inhibition by nucleolar stress downregulated E2F1 protein levels both in p53-wt and p53-null TGCT cells with the concomitant upregulation of unphosphorylated pRb. Finally, we show that sempervirine is able to enter the nucleus and accumulates within the nucleolus where it binds rRNA without causing DNA damage. Our results identify semperivirine as a novel rRNA synthesis inhibitor and indicate this drug as a non-genotoxic anticancer small molecule.

Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells.

Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the "endocannabinoid system" (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate "eCBs tone", associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.

Revisiting DNA damage repair, p53-mediated apoptosis and cisplatin sensitivity in germ cell tumors.

Testicular germ cell tumors (TGCTs), ie, seminomas and nonseminomas, account for 1% to 3% of all neoplasms in men. They are the most common cancer in young white males and are unique in their responsiveness to cisplatin-based chemotherapy. For this reason, TGCTs are considered a model for curative disease. However, up to now, the molecular mechanisms behind this exceptional responsiveness to DNA-damaging agents have remained unclear. A hypersensitive apoptotic response, as well as a reduction in the proficiency to repair cisplatin-induced DNA damage might account for this behavior. In this review, building on recent findings of p53-induced apoptosis and DNA-repair mechanisms in TGCTs, we will discuss the molecular bases that drive tumor sensitivity to cisplatin, emphasizing the new therapeutic approaches proposed to eventually constrain tumor recurrence, and target TGCTs which are unresponsive to standard therapies.

Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to Cisplatin and poly (adp-ribose) polymerase inhibition.

Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.

Expression of a truncated form of KIT tyrosine kinase in human spermatozoa correlates with sperm DNA integrity.

BACKGROUND: TR-KIT, a truncated form of KIT (the KITL receptor), corresponding to the c-terminal half of the intracellular split tyrosine kinase domain, is expressed during the haploid stages of mouse spermatogenesis, and is one of the candidate sperm factors possibly involved in egg activation at fertilization. METHODS: Immunocytochemistry of adult human testis, and studies of human semen samples from volunteer donors through immunofluorescence, confocal microscopy, flow cytometry, western blot and RT-PCR analyses were performed. RESULTS: We show that the TR-KIT is expressed during spermiogenesis in the human testis, and that it is maintained in human ejaculated spermatozoa. TR-KIT is localized both in the equatorial segment and in the sub-acrosomal region of the human sperm head. The equatorial localization of the TR-KIT persists after the spontaneous acrosome reaction. Cytometric analysis of several sperm samples from volunteer donors, showed variable degrees of the TR-KIT-specific immunolabeling, and a significant inverse correlation (Pearson's coefficient, r = -0.76, P < 0.0001, n = 23) of the TR-KIT positivity with markers of sperm damage, i.e. DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) analysis and the intense clusterin positivity. We also found less significant inverse correlation with altered head morphology (r = -0.47, P < 0.05, n = 23) and direct correlation with sperm forward motility parameters (r = 0.59, P < 0.01, n = 23). CONCLUSIONS: The TR-KIT is present in the equatorial region of human spermatozoa, which is the first sperm component entering into the oocyte cytoplasm after fusion with the egg. This localization is consistent with the function previously proposed for this protein in mice. In addition, the TR-KIT represents a potential predictive parameter of human sperm quality.

Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis.

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68(-/-) male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68(-/-) mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

ATM promotes the obligate XY crossover and both crossover control and chromosome axis integrity on autosomes.

During meiosis in most sexually reproducing organisms, recombination forms crossovers between homologous maternal and paternal chromosomes and thereby promotes proper chromosome segregation at the first meiotic division. The number and distribution of crossovers are tightly controlled, but the factors that contribute to this control are poorly understood in most organisms, including mammals. Here we provide evidence that the ATM kinase or protein is essential for proper crossover formation in mouse spermatocytes. ATM deficiency causes multiple phenotypes in humans and mice, including gonadal atrophy. Mouse Atm-/- spermatocytes undergo apoptosis at mid-prophase of meiosis I, but Atm(-/-) meiotic phenotypes are partially rescued by Spo11 heterozygosity, such that ATM-deficient spermatocytes progress to meiotic metaphase I. Strikingly, Spo11+/-Atm-/- spermatocytes are defective in forming the obligate crossover on the sex chromosomes, even though the XY pair is usually incorporated in a sex body and is transcriptionally inactivated as in normal spermatocytes. The XY crossover defect correlates with the appearance of lagging chromosomes at metaphase I, which may trigger the extensive metaphase apoptosis that is observed in these cells. In addition, control of the number and distribution of crossovers on autosomes appears to be defective in the absence of ATM because there is an increase in the total number of MLH1 foci, which mark the sites of eventual crossover formation, and because interference between MLH1 foci is perturbed. The axes of autosomes exhibit structural defects that correlate with the positions of ongoing recombination. Together, these findings indicate that ATM plays a role in both crossover control and chromosome axis integrity and further suggests that ATM is important for coordinating these features of meiotic chromosome dynamics.

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells.

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Male infertility due to germ cell apoptosis in mice lacking the thiamin carrier, Tht1. A new insight into the critical role of thiamin in spermatogenesis.

A mouse model of thiamin-responsive megaloblastic anemia (diabetes mellitus, deafness, megaloblastic anemia) lacking functional Slc19a2 has been generated and unexpectedly found to have a male-specific sterility phenotype. We describe here the characterization of the testis-specific effects of absence of the high-affinity thiamin transporter, Tht1. Null males were found to have hypoplastic testes secondary to germ cell depletion. Morphologic and expression analysis revealed that under conditions of standard thiamin intake, tissues affected in the syndrome (pancreatic beta-cell, hematopoietic cells, auditory nerve) maintained normal function but pachytene stage spermatocytes underwent apoptosis. Under conditions of thiamin challenge, the apoptotic cell loss extended to earlier stages of germ cells but spared Sertoli cells and Leydig cells. Injection of high-dose thiamin was effective in reversing the spermatogenic failure, suggesting that the absence of the thiamin carrier could be overcome by diffusion-mediated transport at supranormal thiamin concentrations. These observations demonstrated that male germ cells, particularly those with high thiamin transporter expression beyond the blood-testis barrier, were more susceptible to apoptosis triggered by intracellular thiamin deficiency than any other tissue type. The findings described here highlight an unexpected and critical role for thiamin transport and metabolism in spermatogenesis.

Prolin-rich tyrosine kinase 2 (PYK2) expression and localization in mouse testis.

Prolin-rich kinase 2 (PYK2) is a nonreceptor tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). PYK2 is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha (TNF-alpha), changes in osmolarity, elevation in intracellular calcium concentration, angiotensin, and UV irradiation. PYK2 has ligand sequences for Src homology 2 and 3 (SH-2 and SH-3), and has binding sites for paxillin and p130(cas). Activation of PYK2 leads to modulation of ion channel function, phosphorylation of tyrosine residues, and activation of the MAP kinase signaling pathways. Immunocytochemistry shows that PYK2 is present in mouse germinal and Sertoli cells (ser). Northern blot and immunoprecipitation analysis demonstrate that, among germinal cells, PYK2 is more abundant in spermatocytes (spc) and spermatids (spt); in addition, immunofluorescence analysis clearly shows that the diffuse cytoplasmic localization of PYK2 changes in a specific cellular compartment in spt and spermatozoa.

Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo.

SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events.

Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase.

Microinjection in mouse eggs of tr-kit, a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cgamma1 (PLCgamma1) and Ca(2+) mobilization from intracellular stores. We show that the Src-like kinase Fyn phosphorylates Tyr161 in tr-kit and that this residue is essential for tr-kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr-kit and that the interaction requires Tyr161. The interaction between tr-kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCgamma1. Co-injection of tr-kit with the SH2 domain of Fyn, or pre-treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr-kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr-kit but still requires PLC activity. We suggest that the mechanism by which tr-kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCgamma1.

HMGA1 and HMGA2 protein expression in mouse spermatogenesis.

The high-mobility group A (HMGA) nonhistone chromosomal proteins HMGA1 and HMGA2 play a role in determining chromatin structure and in regulating the transcription of several genes. High levels of these proteins are characteristic of rapidly dividing cells in embryonic tissue and in tumors. The aim of this study was to determine the role of HMGA1 and HMGA2 throughout mouse spermatogenesis. Northern blot analysis and immunocytochemistry showed HMGA1 and HMGA2 expression during the progression from spermatocyte to spermatid. Interestingly, Western blot analysis with antibodies against the HMGA1 gene product revealed only the HMG1c isoform (27 kDa) in the testis; HMGA1a and HMGA1b were undetectable. These three isoforms are encoded by the HMGA1 gene through alternative splicing. Finally, few spermatids and complete absence of spermatozoa were observed in the testes of HMGA2-null mice, which suggests that the HMGA2 gene plays a critical role in male fertility.