Bernard Sele

Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility

The World Health Organization conservatively estimates that 80 million people suffer from infertility worldwide. Male factors are believed to be responsible for 20–50% of all infertility cases, but microdeletions of the Y chromosome are the only genetic defects altering human spermatogenesis that have been reported repeatedly. We focused our work on infertile men with a normal somatic karyotype but typical spermatozoa mainly characterized by large heads, a variable number of tails and an increased chromosomal content (OMIM 243060). We performed a genome-wide microsatellite scan on ten infertile men presenting this characteristic phenotype. In all of these men, we identified a common region of homozygosity harboring the aurora kinase C gene (AURKC) with a single nucleotide deletion in the AURKC coding sequence. In addition, we show that this founder mutation results in premature termination of translation, yielding a truncated protein that lacks the kinase domain. We conclude that the absence of AURKC causes male infertility owing to the production of large-headed multiflagellar polyploid spermatozoa.

The Aurora Kinase C c.144delC mutation causes meiosis I arrest in men and is frequent in the North African population

Infertility concerns a minimum of 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. In a previous study, we demonstrated that a homozygous mutation (c.144delC) in the Aurora Kinase C (AURKC) gene led to the production of large-headed polyploid multi-flagellar spermatozoa, a primary infertility phenotype mainly observed in North Africans. We now want to estimate the prevalence of the defect, to improve our understanding of AURKC physiopathology in spermatogenesis and assess its implication in oogenesis. A carrier frequency of 1/50 was established from individuals from the Maghrebian general population, comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. A total of 62 patients were genotyped, all who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n = 32), whereas no AURKC mutations were detected in the others. Two homozygous females were identified; both were fertile indicating that AURKC is not indispensible in oogenesis. Previous FISH results had showed a great chromosomal heterogeneity in these patients spermatozoa. We demonstrate here by flow cytometry that all spermatozoa have in fact a homogeneous 4C DNA content and are thus all blocked before the first meiotic division. Our data thus indicate that a functional AURKC protein is necessary for male meiotic cytokinesis while its absence does not impair oogenesis.

No Long-Term Increase in Sperm Aneuploidy Rates after Anticancer Therapy: Sperm Fluorescence In situ Hybridization Analysis in 26 Patients Treated for Testicular Cancer or Lymphoma

Purpose: Lymphomas and testicular cancers are the most frequent malignancies among young men. With recent improvement of survival rates, for many patients, the question is raised of the consequences of the anticancer treatments on their fertility and more specifically of a potential genetic risk for the offspring. This article presents the study of sperm aneuploidy rates in the largest population of cancer-treated patients studied thus far. Experimental Design: In the present study, 38 patients were initially included 7 months to 5 years after a cancer treatment by chemotherapy and/or radiotherapy for testicular cancer (n = 19) or lymphoma (n = 19). Twelve of them were azoospermic. Sperm aneuploidy rates of chromosomes X, Y, 13, 18, and 21 were analyzed by multicolor fluorescent in situ hybridization in the 26 other patients. Results: In most cases, the disomy/diploidy rates after cancer therapy did not significantly differ from those observed in the group of control healthy donors. Only five patients (one lymphoma and four testicular cancer) showed significant but still moderate increases in disomic and/or diploid sperm. For the lymphoma patient, the short posttherapeutic delay after the treatment could explain the elevated aneuploidy rates, whereas no risk factor in the clinical, biological, or therapeutic records could be identified in any of the four testicular cancer patients with elevated sperm aneuploidy rates. Conclusions: These data suggest an absence of long-term effect of anticancer therapy on sperm aneuploidy rates, and therefore, no long-term increased risk of aneuploidy for the offspring obtained either spontaneously or after assisted reproductive techniques.

Facteurs impliqués dans le remodelage de la chromatine au cours de la spermiogenèse

ResumeLes spermatides rondes sont les cellules issues de la méiose dont le noyau haploïde présente initialement une structure similaire à celle d’une cellule somatique. Au cours de la maturation post-méiotique de la spermatide, ou spermiogenèse, la chromatine subit un remodelage au cours duquel le noyau de la spermatide s’allonge puis se condense pour former le noyau spermatique, dont la chromatine présente une structure très spécifique et très compacte. Lors de ce remodelage, les histones sont remplacées par les protéines de transition puis par les protamines, protéines nucléaires spécifiques du spermatozoïde. Immédiatement avant leur remplacement, les protéines histones sont hyperacétylées. Nous avons entrepris de caractériser précisément l’acétylation des histones cœur du nucléosome au cours de la spermatogenèse murine. Nous avons ainsi montré que, chez la souris, les histones cœur H2A, H2B, H3 et H4 sont hyperacétylées dans les spermatides en cours d’élongation. Nous avons ensuite montré qu’une dégradation des histones déacétylases (HDACs) est responsable de cette augmentation brutale du niveau d’acétylation en début d’élongation. Enfin, chez l’homme, nos données suggèrent qu’un processus semblable est mis en jeu lors de la spermatogenèse normale. De plus, une forte déplétion en cellules germinales, telle qu’elle est observée dans les syndromes des cellules de Sertoli isolées (SCO), est associée a une augmentation très importante de l’acétylation du noyau des cellules de Sertoli. L’ensemble de ces données apporte les bases nécessaires à la compréhension des mécanismes et à l’identification de facteurs impliqués dans les différentes étapes du remodelage postméiotique de la chromatine.AbstractRound spermatids are post-meiotic cells with a haploid genome contained in a nucleus, with a structure initially similar to that of the somatic cell nucleus. During spermatogenesis, the spermatid nucleus undergoes drastic remodelling during which it first elongates and then condenses into the very specific and tightly packaged structure of the sperm nucleus. During this remodelling dthe histones are replaced by transition proteins, which, in turn, are replaced by protamines, the specific nuclear proteins of the spermatozoa. Immediately prior to their replacement, the histones are hyperacetylated. The first part of our work was to precisely characterise the changes in histone acetylation during murine spermatogenesis. We have shown that the core histones H2A, H2B, H3 and H4 are hyperacetylated in the elongating spermatids. We have also shown that these changes in acetylation are associated with degradation of the enzymes responsible for histone deacetylation, histone deacetylases or HDACs, while histone acetyl transferases are still present in these cells. The histone acetylation pattern was also investigated during human spermatogenesis, revealing that histone hyperacetylation in the nucleus of elongating spermatids, which appears to be conserved during the course of evolution, also occurs during human spermatogenesis. Moreover, our data obtained from the testes of men with severely altered spermatogenesis, including SCO syndromes (Sertoli Cells Only Syndromes), show that a global hyperacetylation of the Sertoli cell nuclei is associated with an absence of meiotic and post-meiotic cells. This suggests that the global histone acetylation variations observed during spermatogenesis are part of a signalling pathway involving germ cell — Sertoli cell communication. Altogether, these data provide a basis for a better understanding of the mechanisms and identification of the factors involved in post-meiotic remodelling of chromatin.

Fertilité et aneuploïdies spermatiques après traitement par radiothérapie et/ou chimiothérapie pour cancer du testicule ou lymphome

ResumeLa chimiothérapie et la radiothérapie anticancéreuses ont fait des progrès considérables ces dernières années permettant ainsi d’améliorer le pronostic de nombreuses pathologies malignes. Les effets secondaires de ces traitements sur la fonction de reproduction masculine sont importants à considérer pour tout sujet en âge de procréer.Tout d’abord, ils entraînent une inhibition de la spermatogenèse, transitoire dans la plupart des cas. Certains facteurs, comme la qualité des paramètres de sperme avant traitement ainsi que la nature, la dose totale et le rythme d’administration des thérapeutiques anti-cancéreuses influencent la reprise de la spermatogenèse. Cependant, il est impossible à ce jour de prédire les chances et le délai de récupération au plan individuel, et la cryoconservation de sperme reste la seule méthode éprouvée pour pallier aux effets stérilisants de ces traitements.Par ailleurs, la chimiothérapie et la radiothérapie sont susceptibles d’altérer le contenu génétique des cellules germinales. Parmi ces altérations génétiques, les anomalies chromosomiques pourraient, si elles persistent après reprise de la spermatogenèse, être responsables de fausses-couches spontanées ou de malformations congénitales dans la descendance. Les effets à court terme (moins de 3 mois) sur le contenu chromosomique des spermatozoïdes sont maintenant assez bien évalués dans la littérature: les cassures de l’ADN et les anomalies de ségrégation des chromosomes au cours de la méiose sont responsables d’anomalies structurales et numériques. En revanche, à plus long terme, les conséquences ne sont pour l’instant pas clairement établies et les résultats des études réalisées sur le sujet sont contradictoires.Dans le cadre d’une étude rétrospective incluant des sujets traités par radiothérapie et/ou chimiothérapie entre 1995 et 2000 pour cancer du testicule ou pour lymphome, nous avons comparé les taux d’aneuploïdies spermatiques post-thérapeutiques de ces patients avec ceux de sujets fertiles sains. L’analyse par hybridationin situ de sondes marquées en fluorescence (FISH) de la ségrégation des chromosomes X, Y, 18 chez 13 patients a montré pour 4 d’entre eux une augmentation significative des taux d’aneuploïdes (disomies XY et diploïdies essentiellement). Ces anomalies ne semblent pas être corrélées au délai post-thérapeutique, à la numération spermatique ou à la tératozoospermie.Ainsi, les données de la littérature et les résultats préliminaires de notre étude montrent que les augmentations des taux d’aneuploïdies spermatiques à distance des traitements anticancéreux sont peu fréquentes et que ces taux sont variables d’un patient à l’autre sans que l’on retrouve de corrélation avec des paramètres clinico-biologiques.AbstractImprovements in cancer therapy have considerably modified patient survival rates over recent years. However, the side effects of these treatments especially the effects on fertility, must be taken into account. Anticancer therapy can transiently inhibit spermatogenesis. Factors such as pretreatment semen parameters and the type of chemotherapy or radiotherapy may influence recovery of spermatogenesis, but it is still impossible to predict the probability of and time to recovery for each patient. Sperm banking remains the only way to prevent the effects of cancer treatment on male fertility.Another possible effect of chemotherapy or radiotherapy is genetic damage to germ cells. For instance, chromosomal abnormalities in viable sperm produced by these patients after recovery of spermatogenesis may result in fetal death or congenital abnormalities in their offspring. It has been fairly well documented that, during the first three months after treatment, DNA breaks and abnormal chromosomal segregation induced by chemotherapy/radiotherapy lead to structural and numerical chromosomal abnormalities in spermatozoa, respectively. However, the long-term effects on genetic sperm content have not been clearly established. The results of published studies are contradictory and are based on limited numbers of patients (maximum of 6).We present the preliminary results of a retrospective study concerning patients treated for testicular cancer or lymphoma between 1995 and 2000. Fluorescence in situ hybridization (FISH) analysis of chromosomes X, Y and 18 was performed on sperm collected one to five years after treatment and compared to the data obtained for non-affected fertile men. For four out of 13 patients, we found a significantly increased frequency of aneuploidy rates (mainly XY disomy and diploidy), and these results did not appear to be correlated with sperm count, sperm morphology or post-treatment duration.In conclusion, increased sperm aneuploidy rates appear to only concern a small number of patients, to varying degrees and without any predictive factors. According to published data and our preliminary results, we recommend waiting at least two years before starting ART (Assisted Reproduction Therapy) for patients treated for testicular cancer or lymphoma. Moreover, FISH analysis could be helpful to choose between ART with post-treatment sperm or cryopreserved sperm.