Marco Barchi

Surveillance of Different Recombination Defects in Mouse Spermatocytes Yields Distinct Responses despite Elimination at an Identical Developmental Stage

ABSTRACT Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11 − / − mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1 − / − spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm − / − spermatocytes show similarities to Dmc1 − / − spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5 − / − mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.

Distinct properties of the XY pseudoautosomal region crucial for male meiosis.

Recombination between the sex chromosomes during sperm formation is controlled by a splicing isoform of the SPO11 protein. Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform–specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.

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 Cγ1 (PLCγ1) and Ca2+ 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 PLCγ1. 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 PLCγ1.

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.

Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication

Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand cross-links (ICLs) in mammalian cells. Cells from individuals with Fanconi anemia (FA) are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate.

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 (Pearsons 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.

Isolation and Analyses of Enriched Populations of Male Mouse Germ Cells by Sedimentation Velocity: The Centrifugal Elutriation

The studies of molecular events that occur in single cell types within a tissue often require the disaggregation of the tissue into a single cell suspension, followed by isolation of distinct cell populations. The germinal epithelium of mammals is composed of several cell types, which divide mitotically, before entering meiosis. In this chapter, we describe the isolation of five mouse germ-cell fractions by centrifugal elutriation, and characterize them by their DNA content (flow cytometry), cell morphology (DAPI staining of nuclei, Giemsa staining of squashed cells) and deposition of stage-specific meiotic markers (SYCP3, H1t, SAM68) on chromosome spreads and whole cells. Within 2 h it is possible to obtain enriched populations of elongated spermatids (up to approximately 50% of the fraction), round spermatids (up to approximately 80%), primary spermatocytes (up to approximately 89%), and secondary spermatocytes (up to approximately 17%). Furthermore, most of the collected spermatocytes of the primary spermatocyte fraction are in early-mid pachytene stage as judged by chromosome spreads, enriched up to approximately 89%. Elutriation and techniques used for characterization of germ cell fractions are described.