Analysis of the intratesticular control of spermatogenesis by ex-vivo approaching.

Spermatogenesis involves the realization of a particular genetic program which requires a specific environment ("niche"). Multiplication, differentiation and apoptosis of male germ cells are finely regulated by pituitary hormones (mainly LH and FSH), and by a complex network of factors originating from both the somatic cells and the germ cells of the testis. It is becoming clear that hormones and intra-testicular regulatory factors can compensate, at least in part, for the absence of some hormones or factors including FSH and LH or androgen receptors. Since, most of the growth factors, cytokines and neurotrophins produced within the testis are widely expressed in the organism, the attempts to understand their role in spermatogenesis by "classical" knock-out strategies have been often disappointing. Therefore an important aspect of our previous work was to settle and characterize carefully two systems of cocultures of testicular germ cells with somatic cells in bicameral chambers. For instance, we showed for the first time that the whole meiotic step could be performed in vitro in a mammalian species (the rat). Moreover, all our data indicate that our co-culture systems enable to highlight mechanisms pertinent to the physiological processes. Sperm parameters have been deteriorated considerably during the past 4-5 decades. There is now evidence that chemical exposure is at least partly responsible for these testicular diseases. If a large number of environmental pollutants are able to affect male fertility and to exert carcinogenic effects, their cellular and molecular mechanisms are still unidentified. The cultures in bicameral chambers that we settled can be used to study the effects of a toxicant when added in the basal compartment of the culture chamber, which appears relevant to the in vivo situation. Taken together our results indicate that our in vitro culture systems, which allow screening for the effect of biological activity of different physiological factors, can be also helpful to study that of any chemicals on both survival and multiplication/differentiation of somatic and/or spermatogenic cells on a relatively long time period

Etude histologique et immunocytochimique de la méiose chez trois patients porteurs d'une microdélétion complète de la région AZFc

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

Etude de la méiose chez trois patients présentant une microdélétion complète de la région AZFc [Azoospermia Factor] du chromosome Y

BUT DE L ETUDE : Nous avons étudié la méiose de trois patients infertiles porteurs d une microdélétion complète de type Azfc afin de tenter d expliquer l échec de la spermatogenèse chez ces patients. METHODES : Les spermatocytes au stade pachytène ont été étudiés à l aide de la technique d immunocytochimie avec les anticorps anti-SCP3 et anti H2AX. Pour chaque patient, nous avons étudié trois types d anomalies : les asynapsis, la fragmentation des bivalents et les images en pointillé; ainsi que le marquage avec H2AX. RESULTATS : Nous avons analysés avec les 2 anticorps 200 noyaux pour chacun des patients et des contrôles. Les fréquences moyennes des asynapsis, bivalents fragmentés et pointillés était respectivement de 22.3%, 37.5% et 25.3% chez les patients tandis que ces chiffres étaient de 9%, 19.7% et 4.7% chez les contrôles. La différence entre les 2 groupes était significative. Chez tous les patients, les asynaspis étaient courts et réduits à quelques bivalents. Tous nos patients présentaient une prédominance des sous-stades pachytènes précoces (I et II). La protéine H2AX était normalement phosphorylée chez tous les patients. CONCLUSION : Le taux modéré d asynapsis limités chez les patients porteurs de la microdélétion AZFc suggère que cette région ne soit pas une région critique dans le processus méiotique et confirme qu elle doit réguler les premières phases de la spermatogenèse, avant la méiose. Le phénotype méiotique observé est probablement un effet secondaire d un défaut primaire affectant la spermatogenèse.BACKGROUND: Meiosis in three infertile patients presenting complete AZFc microdeletion was studied to determine the mechanisms of spermatogenic failure in this microdeletion. METHODS: Pachytene spermatocytes were immunolabeled with SCP3 and gH2AX. For each patient we studied three types of abnormalities: asynapsis, fragmented and dotted bivalents, and gH2AX labeling. RESULTS: 200 pachytene nuclei from each patient and control were analyzed with SCP3 and H2AX. The mean frequency of asynapsed nuclei, fragmented bivalents and dotted bivalents were 22.3%, 37.5%, and 25.3% in patients respectively and 9%, 19.7%, and 4.7% in controls respectively. The difference between the two groups was significant. In all patients, the asynapsis were short and reduced to a few bivalents. All showed a prevalence of early pachytene substages (I and II). H2AX was normally phosphorylated in all patients. CONCLUSION: The mild level of limited asynapsis in the patients suggests that the AZFc region is not critical for the meiotic process and confirms that this region might regulate the first phases of spermatogenesis, before meiosis. The meiotic phenotype is believed to develop as a secondary effect of a primary defect that influences spermatogenesis.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Etude structurale et fonctionnelle de la vésicule sexuelle au cours de la méiose masculine

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Effets des facteurs environnementaux sur la spermatogenèse (déclin des paramètres du sperme chez l'homme et impact des métaux lourds sur la spermatogenèse du rat ex-vivo)

Au cours des dernières décennies, un contexte alarmant de déclin des paramètres du sperme etd accroissement constant des pathologies génitales masculines a été décrit dans de nombreuxpays industrialisés.Notre travail évalue dans un premier temps l évolution des paramètres spermatiques chez11 330 hommes ayant consulté pour infertilité conjugale au Centre de ProcréationMédicalement Assistée du CHU de Marseille entre 1988 et 2007. Les données ont étérecueillies de manière rétrospective et analysées par régression linéaire multi-variée afin detenir compte de l effet lié à l âge. Nous montrons une diminution significative des principauxparamètres du sperme: concentration en spermatozoïdes (-1.4% par an), numération totale enspermatozoïdes (-1.5% par an), mobilité progressive rapide (-5.6% par an) et morphologienormale (-1.9% par an). Les possibles biais de selection ont été discutés. L effet del environnement sur notre fonction de reproduction est l une des hypothèses principalesévoquées pour expliquer ce phénomène.Nous montrons ensuite comment un modèle de culture de tubes séminifères de rat en chambrebicamérale, couplé à des techniques d étude de la méiose en immunocytochimie peut êtreutilisé comme nouvel outil de reprotoxicologie. Nous décrivons tout d abord avec l anticorpsanti-SCP3 la prophase de première division méiotique chez le rat in-vivo et nous validons lemodèle de culture par comparaison entre les résultats des cellules obtenues in vivo et ceux descellules obtenues ex-vivo. Nous montrons ensuite grâce à ce système l impact du ChromeHexavalent et du Cadmium sur la méiose du rat. Ces 2 métaux lourds, largement présent dansnotre environnement, peuvent être responsables d atteintes importantes des cellulesméiotiques, y compris à de faibles doses pouvant correspondre à celles retrouvées chez desindividus exposés dans leur vie quotidienne et/ou professionnelle.In recent decades, an alarming decline of sperm parameters and a constant increase in malegenital diseases has been described in many industrialized countries.In a first time, our study evaluates the evolution of sperm parameters in 11 330 menconsulting for infertility of their couple in the Reproduction Biology Laboratory of anUniversity Hospital in Marseilles between 1988 and 2007. Data were collected retrospectivelyand analyzed by multivariate linear regression to take into account the effect related to age.We show a significant decrease of the main sperm parameters: sperm concentration (-1.4%per year), total sperm count (-1.5% per year), rapid progressive motility (-5.6% per year) andnormal morphology (-1.9 % per year). Possible selection bias were discussed. The effect ofenvironment on our reproductive function is one of the main hypothesis to explain thisphenomenon.Secondly, we show how a rat seminiferous tubule culture in a bicameral chamber system,coupled with study of meiosis by immunocytochemistry can be used as a new tool ofreprotoxicology. We first describe with an anti-SCP3 antibody the first prophase of ratmeiosis and we validate the model of culture by comparing the results obtained on cellsobtained after in vivo spermatogenesis and those of cells obtained ex-vivo. Then, we showusing this system, the impact of hexavalent chromium and cadmium on rat meiosis. These twoheavy metals, widely present in our environment, may cause substantial impairment ofmeiotic cells, including low doses which can fit with those found in individuals exposed intheir personnal life and / or occupational training.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Validation of a rat seminiferous tubule culture model as a suitable system for studying toxicant impact on meiosis effect of hexavalent chromium

International audienceThere is evidence that exposure to environmental factors is at least partly responsible for changes in semen quality observed over the past decades. The detection of reproductive toxicants under Registration, Evaluation and Authorisation of Chemicals (REACH) will impact animal use for regulatory safety testing. We first validated a model of culture of rat seminiferous tubules for toxicological studies on spermatogenesis. Then, using this model of culture, we assessed the deleterious effects of 1, 10, and 100 μg/l hexavalent chromium [Cr(VI)] on meiotic cells. The prophase I of meiosis was studied in vivo and ex vivo. Bromo-2′-deoxyuridine (BrdU) was used to describe the kinetics of germ cell differentiation. SCP3 labeling allowed to establish the distribution of the stages of the meiotic prophase I and to perform a qualitative study of the pachytene stage in the absence or presence of Cr(VI). The development of the meiotic step of pubertal rats was similar in vivo and ex vivo. The number of total cells appeared not affected by the presence of Cr(VI) irrespective of its concentration. However, the numbers of late spermatocytes and of round spermatids were decreased by Cr(VI) even at the lower concentration. The percentage of synaptonemal complex abnormalities increased slightly with the time of culture and dramatically with Cr(VI) concentrations. This model of culture appears suitable for toxicological studies. This study shows that Cr(VI) is toxic for meiotic cells even at low concentrations, and its toxicity increases in a dose-dependent manner

Exposure to Low-Dose bisphenol a impairs meiosis in the rat seminiferous tubule culture model: a physiotoxicogenomic approach

International audience[u]Background[/u]: Bisphenol A (BPA) is one of the most widespread chemicals in the world and is suspected of being responsible for male reproductive impairments. Nevertheless, its molecular mode of action on spermatogenesis is unclear. This work combines physiology and toxicogenomics to identify mechanisms by which BPA affects the timing of meiosis and induces germ-cell abnormalities. [u]Methods[/u]: We used a rat seminiferous tubule culture model mimicking the in vivo adult rat situation. BPA (1 nM and 10 nM) was added to the culture medium. Transcriptomic and meiotic studies were performed on the same cultures at the same exposure times (days 8, 14, and 21). Transcriptomics was performed using pangenomic rat microarrays. Immunocytochemistry was conducted with an anti-SCP3 antibody. [u]Results[/u]: The gene expression analysis showed that the total number of differentially expressed transcripts was time but not dose dependent. We focused on 120 genes directly involved in the first meiotic prophase, sustaining immunocytochemistry. Sixty-two genes were directly involved in pairing and recombination, some of them with high fold changes. Immunocytochemistry indicated alteration of meiotic progression in the presence of BPA, with increased leptotene and decreased diplotene spermatocyte percentages and partial meiotic arrest at the pachytene checkpoint. Morphological abnormalities were observed at all stages of the meiotic prophase. The prevalent abnormalities were total asynapsis and apoptosis. Transcriptomic analysis sustained immunocytological observations. [u]Conclusion[/u]: We showed that low doses of BPA alter numerous genes expression, especially those involved in the reproductive system, and severely impair crucial events of the meiotic prophase leading to partial arrest of meiosis in rat seminiferous tubule cultures