SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.This work was supported by Inserm (Institut National de la Sante et de la Recherche Medicale), the Agence Nationale de la Recherche program ANR-12–JSV2-0005–01 (to JC), Labex ‘Who am I?’(ANR-11- LABX-0071 under program ANR-11-IDEX-0005-01) and a Marie Curie fellowship FP7-PEOPLE-2010-IEF-273143 (to JC

Impact of prepubertal exposure to vincristine and cyclophosphamide on long-term fertility and fertility restoration by in vitro maturation of mouse testicular tissue

Les traitements du cancer tels que la chimiothérapie s’accompagnent souvent d’effets secondaires et il n’est pas rare que les patients guéris d’un cancer pédiatrique présentent une infertilité à l’âge adulte. Des procédures de préservation de la fertilité se mettent en place : une congélation du tissu testiculaire peut être envisagée chez le garçon prépubère ne produisant pas de spermatozoïdes. Cependant, la majorité des enfants atteints de leucémie aiguë lymphoblastique ont reçu des cures de chimiothérapie considérées comme « peu gonadotoxiques » avant la cryoconservation de leur tissu testiculaire. Très peu d’études ont évalué l’impact, à court et à long terme, de ces traitements sur le testicule prépubère. De plus, l’impact de ces molécules sur la possibilité de restaurer leur fertilité par maturation in vitro du tissu testiculaire en culture organotypique n’a jamais été évaluée.Nos modèles expérimentaux montrent que l’exposition prépubère au cyclophosphamide entraîne une augmentation du pourcentage de spermatozoïdes présentant des anomalies de condensation de la chromatine et une oxydation de l’ADN associée à une diminution de la fertilité à l’âge adulte. Le traitement par vincristine chez les souris prépubères altère le tissu testiculaire à court et long terme et perturbe la progression de la spermatogenèse. Le pourcentage de flagelles isolés et de spermatozoïdes possédant un ADN fragmenté est significativement augmenté chez les souris exposées à la vincristine, entraînant une réduction de la fertilité. Dans le contexte de la restauration de la fertilité à partir de tissus testiculaires préalablement exposés à la chimiothérapie, ce projet a également permis de montrer la possibilité d’obtenir des spermatozoïdes murins in vitro à partir de tissus testiculaires prépubères frais exposés au cyclophosphamide et/ou à la vincristine, malgré une altération du tissu testiculaire observée avant mise en culture.Avant de pouvoir envisager une application clinique, il est indispensable de poursuivre ces travaux par une évaluation de l’impact des chimiothérapies sur la possibilité de produire des gamètes in vitro à partir de tissus testiculaires murins congelés/décongelés afin de reproduire, au mieux, la procédure de préservation/restauration de la fertilité qui sera utilisée chez les patients prépubères. Par ailleurs, la qualité nucléaire des gamètes générés in vitro ainsi que leur capacité à féconder un ovocyte et assurer un développement embryonnaire normal devront être étudiées.Cancer treatments such as chemotherapy often have side effects and it is not uncommon for patients cured of pediatric cancer to develop infertility in adulthood. Fertility preservation procedures are put in place: testicular tissue freezing may be considered in prepubertal boys who do not produce sperm. However, the majority of children with acute lymphoblastic leukemia have received chemotherapy cures considered "low gonadotoxic" prior to cryopreservation of their testicular tissue. Very few studies have evaluated the short- and long-term impact of these treatments on the prepubertal testis. Moreover, the impact of these molecules on the possibility of restoring their fertility by in vitro maturation of testicular tissue in organotypic culture has never been evaluated.Our experimental models show that prepubertal exposure to cyclophosphamide leads to an increase in the percentage of spermatozoa with chromatin condensation abnormalities and DNA oxidation associated with decreased fertility in adulthood. Vincristine treatment in prepubertal mice alters testicular tissue integrity in the short and long term and disturbs the progression of spermatogenesis. The percentage of isolated flagella and spermatozoa with fragmented DNA is significantly increased in mice exposed to vincristine, resulting in reduced fertility. In the context of restoring fertility from testicular tissue previously exposed to chemotherapy, this project also demonstrated the possibility of obtaining murine spermatozoa in vitro from fresh prepubertal testicular tissue exposed to cyclophosphamide and/or vincristine, despite an alteration of testicular tissue observed before culture.Before clinical application can be considered, it is essential to continue this work by assessing the impact of chemotherapies on the possibility of producing gametes in vitro from frozen/thawed murine testicular tissue in order to best replicate the fertility preservation/restoration procedure to be used in prepubertal patients. In addition, the nuclear quality of the gametes generated in vitro as well as their capacity to fertilize an oocyte and ensure normal embryonic development will have to be studied

Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility

International audienceOver the last decade, the number of cancer survivors has increased thanks to progress in diagnosis and treatment. Cancer treatments are often accompanied by adverse side effects depending on the age of the patient, the type of cancer, the treatment regimen, and the doses. The testicular tissue is very sensitive to chemotherapy and radiotherapy. This review will summarize the epidemiological and experimental data concerning the consequences of exposure to chemotherapy during the prepubertal period or adulthood on spermatogenic progression, sperm production, sperm nuclear quality, and the health of the offspring. Studies concerning the gonadotoxicity of anticancer drugs in adult survivors of childhood cancer are still limited compared with those concerning the effects of chemotherapy exposure during adulthood. In humans, it is difficult to evaluate exactly the toxicity of chemotherapeutic agents because cancer treatments often combine chemotherapy and radiotherapy. Thus, it is important to undertake experimental studies in animal models in order to define the mechanism involved in the drug gonadotoxicity and to assess the effects of their administration alone or in combination on immature and mature testis. These data will help to better inform cancer patients after recovery about the risks of chemotherapy for their future fertility and to propose fertility preservation options

Paradoxical risk of reduced fertility after exposure of prepubertal mice to vincristine or cyclophosphamide at low gonadotoxic doses in humans

Abstract Cancer treatment can have long-term side effects in cured patients and infertility is one of them. Given the urgency of diagnosis in children with cancer, the toxicity of treatments on the gonad was overshadowed for a long time. In the present study, prepubertal mice were treated by vincristine or cyclophosphamide commonly used in acute leukaemia treatment. The prepubertal exposure to cyclophosphamide, at a low gonadotoxic dose in humans (< 3.5 g/m 2 ), led to morphological alterations of prepubertal testicular tissue. An increased proportion of spermatozoa with hypocondensed chromatin and oxidized DNA associated with decreased fertility were uncovered at adulthood. Short- and long-term morphological alterations of the testicular tissue, disturbed progression of spermatogenesis along with increased proportions of isolated flagella and spermatozoa with fragmented DNA were evidenced in vincristine-treated mice. Moreover, the fertility of mice exposed to vincristine was severely affected despite being considered low-risk for fertility in humans. Paternal exposure to vincristine or cyclophosphamide before puberty had no impact on offspring development. Contrary to the current gonadotoxic risk classification, our results using a mouse model show that vincristine and cyclophosphamide (< 3.5 g/m 2 ) present a high gonadotoxic risk when administered before the initiation of spermatogenesis

Dynamics of epigenetic modifications in ICSI embryos from in vitro‐produced spermatozoa

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Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue

International audienceFreezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation

Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy

International audienceThe assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry