Antoine Kerjean

In vitro follicular growth affects oocyte imprinting establishment in mice

In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.

Loss of parental-specific methylation at the IGF2 locus in human hepatocellular carcinoma

Significant production of the growth factor IGF2 has been reported in human hepatocellular carcinomas (HCCs). Disturbances associated with changes in methylation at this locus or affecting the 11p15.5 imprinting domain as a whole can be postulated in HCCs. In the present study, the methylation status of differentially methylated regions of the imprinted genes TSSC5, LIT1, and IGF2, which span the 11p15 domain, was analysed in 71 liver tissues from virus‐associated and non‐virus‐associated HCCs compared with six normal liver tissues. Altered methylation of TSSC5 and LIT1 was observed in only 6% and 8% of HCCs, respectively, compared with 89% at the IGF2 locus, suggesting that these loci were not concomitantly dysregulated. These observations suggest that loss of parental‐specific methylation at the IGF2 locus may be specifically associated with HCC, whether virus‐associated or non‐virus‐associated, and arising in cirrhotic or non‐cirrhotic livers. Copyright

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR—target of the CTCF insulator—is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

La biopsie testiculaire dans l’azoospermie secretoire: Aspects histopathologiques

ResumeL’avènement de la biopsie testiculaire à visée thérapeutique dans le cadre d’aide médicale à la procréation implique un examen anatomopathologique systématique lors de tout prélévement chirurgical. En particulier, en cas d’azoospermie sécrétoire, l’information apportée par le pathologiste complète le bilan de l’infertilité.La qualité de la lecdture anatomopathologique dépend d’une bonne fixation tissulaire et nécessite que l’échantillon contienne au moins une cinquantaine de tubes séminiféres. Les critéres d’évaluation morphologiques sont quantitatifs mais surtout qualitatifs et décrivent l’aspect des tubes séminifères et du tissu interstitiel. En cas d’azoospermie sécrétoire, trois grands tableaux histopathologiques sont observés: le syndrome des “Cellules de Sertoli Seules”, le blocage de la spermatogénése et l’hypospermatogénèse avec mosaïcisme histologique. Ce résultat, confronté à celui obtenu par le biologiste de la reproduction qui recherche spécifiquement les spermatozoïdes testiculaires, constitue une aide à la prise en charge de l’homme infertile.AbstractTesticular biopsy can allow much more precise diagnosis and treatment of male infertility disorders. The major issue for the diagnostician concerns grading of the defect of spermatogenesis. This practical paper describes technical handling of the specimen, and a stepwise approach to histological diagnosis.