Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

The impact of percutaneous epididymal sperm aspiration on sperm quality in mice.

Abstract: In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits. Lay summary: In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes.

Localized fluxes, production, and/or degradation coupled to limited diffusion are well known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, together with localized membrane rupture, suffice for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that, in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion.

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice.

Background: The protein proAKAP4 is crucial for sperm motility and has been suggested as an indicator of male fertility. We determined the relationship between proAKAP4 concentration and sperm motility parameters in mice, and investigated the effects of cryopreservation on these variables. Methods: Computer-assisted sperm analysis and ELISA were applied to determine sperm motility and proAKAP4 concentration in fresh and frozen-thawed epididymal sperm of SWISS, B6D2F1, C57BL/6N, and BALB/c mice. Results: ProAKAP4 levels ranged between 12 and 89 ng/ml and did not differ between fresh and frozen-thawed samples, or between strains. We found a negative relationship between proAKAP4 levels and some sperm motility parameters. Sperm traits differed between strains, and cryopreservation negatively affected sperm velocity but not sperm direction parameters. Conclusion: ProAKAP4 levels in epididymal mouse spermatozoa were unaffected by cryopreservation, highlighting the robustness of this parameter as a potentially time-independent marker for sperm motility and fertility. The high individual variation in proAKAP4 levels supports the potential role of proAKAP4 as a marker for sperm quality, though we found no positive, and even negative relationships between proAKAP4 levels and some sperm motility parameters. Future studies have to investigate the significance of proAKAP4 as an indicator for fertility in mice.

Structural and functional consequences of the STAT5BN642H driver mutation.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

STAT5BN642H is a driver mutation for T cell neoplasia.

STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.

Microsurgical and Percutaneous Epididymal Sperm Aspiration for Sperm Collection from Live Mice.

Spermatozoa for in vitro fertilization of mouse oocytes and other methods of assisted reproduction typically are collected from the cauda epididymis of euthanized male mice. As an alternative to this terminal protocol, we developed and examined 2 methods for collecting sperm from anesthetized male mice without decreasing subsequent fertility: microsurgical epididymal sperm aspiration and, as a refinement, percutaneous epididymal sperm aspiration. Collected sperm was evaluated in terms of motility, concentration and in vitro fertilization ability. After recovery, both treated and untreated control male mice underwent in vivo fertility testing and subsequent histologic analysis of the treated male reproductive tract (epididymis and testis). In vitro fertilization using sperm recovered by the 2 collection methods was successfully achieved in all cases. The in vivo fertility test and the histologic analysis revealed no impairment of fertility and no permanent histologic alteration in the treated mice. Therefore, we recommend both techniques as simple and effective methods for recovering high-quality epididymal mouse sperm without having to euthanize fertile male mice.

In vivo functional requirement of the mouse Ifitm1 gene for germ cell development, interferon mediated immune response and somitogenesis.

The mammalian Interferon induced transmembrane protein 1 (Ifitm1) gene was originally identified as a member of a gene family highly inducible by type I and type II interferons. Based on expression analyses, it was suggested to be required for normal primordial germ cell migration. The knockdown of Ifitm1 in mouse embryos provided evidence for a role in somitogenesis. We generated the first targeted knockin allele of the Ifitm1 gene to systematically reassess all inferred functions. Sperm motility and the fertility of male and female mutant mice are as in wild type littermates. Embryonic somites and the adult vertebral column appear normal in homozygous Ifitm1 knockout mice, demonstrating that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. Proportions of leucocyte subsets, including granulocytes, monocytes, B-cells, T-cells, NK-cells, and NKT-cells, are unchanged in mutant mice. Based on a normal immune response to Listeria monocytogenes infection, there is no evidence for a dysfunction in downstream IFNγ signaling in Ifitm1 mutant mice. Expression from the Ifitm1 locus from E8.5 to E14.5 is highly dynamic. In contrast, in adult mice, Ifitm1 expression is highly restricted and strong in the bronchial epithelium. Intriguingly, IFITM1 is highly overexpressed in tumor epithelia cells of human squamous cell carcinomas and in adenocarcinomas of NSCLC patients. These analyses underline the general importance of targeted in vivo studies for the functional annotation of the mammalian genome. The first comprehensive description of the Ifitm1 expression pattern provides a rational basis for the further examination of Ifitm1 gene functions. Based on our data, the fact that IFITM1 can function as a negative regulator of cell proliferation, and because the gene maps to chromosome band 11p15.5, previously associated with NSCLC, it is likely that IFITM1 in man has a key role in tumor formation.

A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

EGFR ligands exert diverging effects on male reproductive organs.

While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Interestingly, male transgenic mice overexpressing EGF are sterile, and transgenic mice overexpressing TGFA, another EGFR ligand, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia. We studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression. Despite strong overexpression of BTC in different parts of the male reproductive tract, the gross appearance and histology of the reproductive organs of BTC-tg males were normal and the same were true for sperm parameters and the in vitro fertilization rate. Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction.

Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes.

In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.

Mitochondrial glutathione peroxidase 4 disruption causes male infertility.

Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.

Sperm cryopreservation and in vitro fertilization.

Since the mouse has become the most profound model system to investigate the genetics and pathogenetics of human diseases, a huge number of new mutant mouse strains has been generated and still a lot effort is being done to increase the number of suitable mouse models. In nearly all animal facilities the maintenance of breeding colonies is limited and the mouse strains have to be archived in a reliable way. Mouse sperm cryopreservation provides an efficient management of these genetic resources by reducing maintenance space and cost and by safeguarding them against, for example, disease, breeding failure, and genetic drift.The sperm archiving method has been proven extensively in large-scale ENU mutagenesis programs and in mouse repository and resource centers worldwide (Federation of International Mouse Resources, http://www.fimre.org ). Nevertheless, it is crucial to select accurately the most suitable archiving procedure, or combination of different archiving procedures, for each individual mouse mutant strain.

Risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes.

The aim of this study was to estimate the risk of mouse hepatitis virus (MHV) transmission by the in vitro fertilization and embryo transfer (IVF-ET) procedure. In addition, resistance to infection of zona-intact and laser-microdissected oocytes was compared. For this purpose, infectious mouse hepatitis virus, a common viral pathogen in mouse facilities, was used. Oocytes having an intact or laser-microdissected zona pellucida were incubated for fertilization in media containing MHV-A59 and resulting embryos were transferred to the oviduct of specific pathogen-free (SPF) Swiss recipients. The oocytes were divided into three experimental groups: 1) zona-intact oocytes continuously exposed to MHV in fertilization (HTF), culture (KSOM), and embryo transfer (M2) media; 2) zona-intact oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2; and 3) laser-microdissected oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2. Respective serum samples of embryo recipients and their offspring were tested for MHV antibodies using ELISA. In experiment 1, 10 out of 14 embryo recipients seroconverted to MHV and only their offspring (8 of 19) received maternal antibodies. In experiments 2 and 3, MHV antibodies were detected neither in the recipients nor in the offspring. These results indicate, for the first time, that even if the zona pellucida is partially disrupted by laser microdissection, the transmission of MHV-A59 can be avoided by correctly performed washing steps in the IVF-ET procedure.