Impact of Endocrine Disruptors upon Non-Genetic Inheritance.

Similar to environmental factors, EDCs (endocrine-disrupting chemicals) can influence gene expression without modifying the DNA sequence. It is commonly accepted that the transgenerational inheritance of parentally acquired traits is conveyed by epigenetic alterations also known as "epimutations". DNA methylation, acetylation, histone modification, RNA-mediated effects and extracellular vesicle effects are the mechanisms that have been described so far to be responsible for these epimutations. They may lead to the transgenerational inheritance of diverse phenotypes in the progeny when they occur in the germ cells of an affected individual. While EDC-induced health effects have dramatically increased over the past decade, limited effects on sperm epigenetics have been described. However, there has been a gain of interest in this issue in recent years. The gametes (sperm and oocyte) represent targets for EDCs and thus a route for environmentally induced changes over several generations. This review aims at providing an overview of the epigenetic mechanisms that might be implicated in this transgenerational inheritance.

Diversity of Extracellular Vesicles in Human Follicular Fluid: Morphological Analysis and Quantification.

The oocyte microenvironment constituted by the follicular fluid (FF) is a key for the optimal development of female gametes. Its composition reflects the physiological state of the ovarian follicle. The particularity of FF is to contain a huge diversity of extracellular vesicles specific to women, in the same way as seminal plasma in men. Here, we described and compared morphological aspects of broad subcategories of human FF-related Extracellular Vesicles (EVs). EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. EVs isolated from FF are involved in different biological functions related to follicular growth, oocyte maturation, and embryo development. However, knowledge on the morphology of FF-derived EVs is limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. The aim of this study was to provide a comprehensive morphological description of EVs from FF of healthy subjects and quantification. EVs separation was realized by centrifugation, with comparison of the EV yield obtained from differential centrifugation and one-step ultracentrifugation. Cryo-Transmission Electron Microscopy was used to reveal the morphology, size, and phenotype of EVs. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) were used to quantify and analyze the size distribution for each centrifugation step. We performed a comprehensive inventory of human follicular fluid EVs. We show that human FF contains a huge diversity of EVs. This study brings novel insights on EVs from normal FF and provides a reference for further studies of EVs in ovarian diseases.

STAG3 homozygous missense variant causes primary ovarian insufficiency and male non-obstructive azoospermia.

Infertility, a global problem affecting up to 15% of couples, can have varied causes ranging from natural ageing to the pathological development or function of the reproductive organs. One form of female infertility is premature ovarian insufficiency (POI), affecting up to 1 in 100 women and characterised by amenorrhoea and elevated FSH before the age of 40. POI can have a genetic basis, with over 50 causative genes identified. Non-obstructive azoospermia (NOA), a form of male infertility characterised by the absence of sperm in semen, has an incidence of 1% and is similarly heterogeneous. The genetic basis of male and female infertility is poorly understood with the majority of cases having no known cause. Here, we study a case of familial infertility including a proband with POI and her brother with NOA. We performed whole-exome sequencing (WES) and identified a homozygous STAG3 missense variant that segregated with infertility. STAG3 encodes a component of the meiosis cohesin complex required for sister chromatid separation. We report the first pathogenic homozygous missense variant in STAG3 and the first STAG3 variant associated with both male and female infertility. We also demonstrate limitations of WES for the analysis of homologous DNA sequences, with this variant being ambiguous or missed by independent WES protocols and its homozygosity only being established via long-range nested PCR.

Pseudodicentric Chromosome Originating from Autosomes 9 and 21 in a Male Patient with Oligozoospermia.

Genetic factors are responsible for 15% of male infertility conditions. Numerical and structural chromosomal anomalies (related to the Y chromosome or to the autosomes) are validated genetic factors leading to spermatogenic quantitative defects with a frequency depending on the severity of the phenotype. The most frequent structural chromosomal rearrangements of autosomes are translocations and inversions, whereas dicentric chromosomes involving autosomes are rare. We report a man bearing a pseudodicentric chromosome (9;21) and presenting with oligozoospermia. Extensive cytogenetic analyses were necessary to determine the precise nature of the derivative chromosome and to discount the presence of interstitial telomeric sequences. Defects in spermatogenesis and abnormal segregation at meiosis for existing spermatozoa are proposed and are the likely cause of the reproductive phenotype of the patient.

Sperm DNA methylation dynamics after chemotherapy: a longitudinal study of a patient with testicular germ cell tumor treatment.

BACKGROUND: An important issue for young men affected by testicular germ cell tumor (TGCT) is how TGCT and its treatment will affect, transiently or permanently, their future reproductive health. Previous studies have reported that xenobiotics can induce changes on human sperm epigenome and have the potential to promote epigenetic alterations in the offspring. OBJECTIVES: Here, we report the first longitudinal DNA methylation profiling of frozen sperm from a TGCT patient before and up to 2 years after a bleomycin, etoposide, and cisplatin (BEP) chemotherapy. MATERIALS AND METHODS: A TGCT was diagnosed in a 30-year-old patient. A cryopreservation of spermatozoa was proposed before adjuvant BEP treatment. Semen samples were collected before and after chemotherapy at 6, 9, 12, and 24 months. The DNA methylation status was determined by RRBS to detect DNA differentially methylated regions (DMRs). RESULTS: The analysis revealed that among the 74 DMRs showing modified methylation status 6 months after therapy, 17 remained altered 24 months after treatment. We next associated DMRs with differentially methylated genes (DMGs), which were subsequently intersected with loci known to be important or expressed during early development. DISCUSSION AND CONCLUSION: The consequences of the cancer treatment on the sperm epigenome during the recovery periods are topical issues of increasing significance as epigenetic modifications to the paternal genome may have deleterious effects on the offspring. The altered methylated status of these DMGs important for early development might modify their expression pattern and thus affect their function during key stages of embryogenesis, potentially leading to developmental disorders or miscarriages.

LARS2 variants can present as premature ovarian insufficiency in the absence of overt hearing loss.

Premature ovarian insufficiency (POI) affects 1 in 100 women and is a leading cause of female infertility. There are over 80 genes in which variants can cause POI, with these explaining only a minority of cases. Whole exome sequencing (WES) can be a useful tool for POI patient management, allowing clinical care to be personalized to underlying cause. We performed WES to investigate two French sisters, whose only clinical complaint was POI. Surprisingly, they shared one known and one novel likely pathogenic variant in the Perrault syndrome gene, LARS2. Using amino-acylation studies, we established that the novel missense variant significantly impairs LARS2 function. Perrault syndrome is characterized by sensorineural hearing loss in addition to POI. This molecular diagnosis alerted the sisters to the significance of their difficulty in following conversation. Subsequent audiology assessment revealed a mild bilateral hearing loss. We describe the first cases presenting with perceived isolated POI and causative variants in a Perrault syndrome gene. Our study expands the phenotypic spectrum associated with LARS2 variants and highlights the clinical benefit of having a genetic diagnosis, with prediction of potential co-morbidity and prompt and appropriate medical care, in this case by an audiologist for early detection of hearing loss.

Extra-cellular vesicles of the male genital tract: new actors in male fertility?

Extracellular Vesicles (EVs) are membrane-limited particles containing proteins, lipids, metabolites and nucleic acids that are secreted by healthy and cancerous cells. These vesicles are very heterogeneous in size and content and mediate a variety of biological functions. Three subtypes of EV have been described in the male genital tract: microvesicles, myelinosomes and exosomes. Each type of EVs depends on the location of secretion such as the testis, prostate or epididymis. It has been shown that EVs can fuse together and deliver information to recipient cells, for example spermatozoa in the male genital tract. Cryo-electron microscopy remains the reference technique for determining EV morphology, but quantifying the absolute concentration of these EVs in biological fluids remains a challenge from a clinical point of view. The field of bio detection has considerably increased with the introduction of nanomaterials in biosensors and will provide a better understanding of the impact of these EVs. However, functional modifications of male gametes result from interactions with the components of the intraluminal fluid all along the genital tract and depend on the secretion and absorption of proteins and lipids from the local microenvironment. We cannot therefore exclude the possibility of epigenetic modulation of the information that will be transmitted to the embryo and therefore to the next generation via EVs.

RéSUMé: Les Vésicules Extracellulaires (VE) sont des constituants d'origine membranaire contenant des protéines solubles ou membranaires, des lipides, des métabolites ou des acides nucléiques. Ces vésicules sont très hétérogènes en taille et en contenu. Trois catégories de VE ont été décrites dans le tractus génital mâle: les microvésicules, les myélinosomes et les exosomes. Les types de VE sont différents selon le lieu de production testiculaire, prostatique ou encore épididymaire. Il a été montré que les VE peuvent fusionner et délivrer une information à la cellule réceptrice, en l'occurrence le spermatozoïde dans le tractus génital mâle. La cryo-microscopie électronique reste la technique de référence pour déterminer la morphologie des VE mais la quantification de la concentration absolue de ces VE dans les liquides biologiques reste un challenge dans le cadre d'une approche clinique. Le domaine de la biodétection s'est. considérablement développé avec l'introduction des nanomatériaux dans les biocapteurs et va permettre de mieux comprendre l'impact de ces VE. Or les modifications fonctionnelles des gamètes mâles résultent d'interactions avec les composants du liquide intraluminal tout le long du tractus génital et dépendent de la sécrétion et de l'absorption de protéines et de lipides du microenvironnement local. On ne peut donc pas exclure la possibilité d'une modulation épigénétique des informations qui seront transmises à l'embryon donc à la génération suivante via les VE.

[Myelinosomes: A new pathway of protein quality control]. / Les myélinosomes : une nouvelle voie du contrôle de qualité des protéines.

Maintenance of cell proteostasis relies on two degradation pathways: proteasome and autophagy. Here we describe a new proteostasis pathway avoiding degradation of abnormal proteins yet carrying them outside the cell using nanovesicles called myelinosomes. These myelinosomes are produced in pathological or stress situations in relation with genetic or environmental factors. Myelinosome vesicles are nano-sized multi-stacked membrane structures, resembling myelin sheath. It has recently been shown in two models of genetic diseases (Huntington's disease and cystic fibrosis) that myelinosomes are important for eliminating mutant proteins in an unusual secretory process, thus preventing their accumulation and aggregation in cells.

Title: Les myélinosomes : une nouvelle voie du contrôle de qualité des protéines. Abstract: Deux voies de dégradation des protéines mal repliées sont classiquement décrites : la voie du protéasome et la voie de l'autophagie. Nous décrivons ici une nouvelle voie de protéostase cellulaire ne dégradant pas la protéine anormale mais l'expulsant hors de la cellule grâce à des nanovésicules appelées myélinosomes. Ces myélinosomes sont produits par la cellule dans des situations pathologiques ou de stress en lien avec des facteurs génétiques ou environnementaux. Sur le plan morphologique, les myélinosomes sont caractérisés par des membranes osmiophiles denses aux électrons dont l'arrangement empilé est semblable à celui de la myéline et présente jusqu'à 30 feuillets selon le type de cellule. Dans deux modèles, au moins, de maladies génétiques (la maladie de Huntington et la mucoviscidose), les myélinosomes sont importants pour éliminer les protéines mutées par un processus sécrétoire inhabituel, évitant ainsi leur agrégation dans les cellules.

[Autophagy and spermatozoa]. / Autophagie et spermatozoïde.

Spermiogenesis, the ultimate stage of spermatogenesis, is a process involving autophagy. At this stage, the acrosome is generated by vesicular fusion and most of the cytoplasm disappears. Autophagy, literally "eating oneself", allowing the elimination and replacement of proteins and nonfunctional organelles, ensures the recycling of cellular constituents and is a highly conserved cellular mechanism within eukaryotic cells. The machinery of autophagy is present in the spermatozoon, regulating the vitality and mobility of the cells. The environmental and behavioral impact on autophagy and the consequences on spermatogenesis are beginning to be studied. The purpose of this review is to synthesize current knowledge about autophagy in the mature male gamete.

TITLE: Autophagie et spermatozoïde. ABSTRACT: La spermiogenèse, étape ultime de la spermatogenèse, est un processus qui fait intervenir des acteurs qui participe à l'autophagie. C'est en effet lors de cette étape que se forme l'acrosome par fusion vésiculaire et que disparaît la majeure partie du cytoplasme du spermatozoïde. L'autophagie (littéralement « se manger soi-même ¼), en permettant l'élimination et le remplacement continuel des protéines et des organites non fonctionnels, assure le recyclage des constituants de la cellule. C'est un mécanisme cellulaire très conservé au sein des cellules eucaryotes. La machinerie de l'autophagie est également présente dans les spermatozoïdes. Elle régule la vitalité de ces cellules et leur mobilité. Les conséquences environnementales et comportementales sur l'autophagie et sur la spermatogenèse commencent à être étudiées. Le but de cette revue est de synthétiser les connaissances actuelles concernant les processus d'autophagie dans le gamète mâle mature.