New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice.

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.

FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder.

FGFR3 and TP53 mutations are frequent in superficial papillary and invasive disease, respectively. We used denaturing high-performance liquid chromatography and sequencing to screen for FGFR3 and TP53 mutations in 81 newly diagnosed urothelial cell carcinomas. Tumors were classified as follows: 31 pTa, 1 carcinoma in situ, 30 pT1, and 19 pT2-T4. Tumor grades were as follows: 10 G1, 29 G2, and 42 G3. FGFR3 mutations were associated with low-stage (P < 0.0001), low-grade (P < 0.008) tumors, whereas TP53 mutations were associated with high-stage (P < 0.003), high-grade (P < 0.02) tumors. Mutations in these two genes were almost mutually exclusive. Our results suggest that FGFR3 and TP53 mutations define separate pathways at initial diagnosis of urothelial cell carcinoma.

Tetranucleotide microsatellite instability in surgical margins for prediction of local recurrence of head and neck squamous cell carcinoma.

PURPOSE: Postoperative radiotherapy is used to prevent local recurrence of head and neck squamous cell carcinoma in patients with positive surgical margins. We sought to determine whether tetranucleotide microsatellite instability could be detected in surgical margins and used to predict local recurrence. EXPERIMENTAL DESIGN: We prospectively collected tumor and surgical margin specimens from patients with head and neck squamous cell carcinoma who had undergone surgical resection at Institut Gustave-Roussy during a 1-year period. Margins were considered positive if extensive pathological examination revealed either carcinoma within 5 mm or dysplasia. We tested five tetranucleotide microsatellite markers (UT5085, L17686, D9S753, ACTBP2, and CSF1R) in the tumor specimens and paired surgical margins of the patients whose margins were negative on pathological examination. RESULTS: Pathological examination revealed that among the 76 patients, 22 had positive margins; therefore, these patients were excluded. Of the 54 remaining patients, 26 (48%) had tumors informative for markers UT5085, L17686, or both; the other 3 markers were not informative. Seven (27%) of the 26 informative tumors had the same instability pattern in the surgical margins. At a median follow-up of 26 months, 5 of the 7 local recurrences occurred in patients with molecularly positive surgical margins. A strong, independent association was found between positive surgical margins and local recurrence (P = 0.01; hazard ratio, 6.49). CONCLUSIONS: Tetranucleotide microsatellite instability in surgical margins may be a useful biomarker to predict local recurrence of head and neck squamous cell carcinoma in patients with apparently disease-free margins.

Whole-body or isolated ovary (60)Co irradiation: effects on in vivo and in vitro folliculogenesis and oocyte maturation.

For the first time, the effects of low doses of gamma-radiation were studied on folliculogenesis and on isolated oocytes. After irradiation of adult mice, even at the lowest dose, a drastic loss of primordial follicles was observed in serial sections of ovaries, with, in opposite, no effect on the other follicle stages. Moreover, oocytes freshly recovered from the largest antral follicles of irradiated adult ovaries exhibited significantly less regular Ca(2+) oscillations than controls. Finally, in vitro folliculogenesis demonstrated a smaller diameter of preantral follicles recovered from irradiated juvenile ovaries compared to control, and an increase in follicle atresia. Further on, PLC-beta1 localization was not affected in the enclosed oocytes whereas chromatin configuration revealed that a quarter of them had prematurely resumed meiosis or was fragmented. These results raise the question of the risk of genetic and teratogenic effects on women submitted to chronic exposure even of very low radiation.

Similarities and differences in the in vivo response of mouse neonatal gonocytes and spermatogonia to genotoxic stress.

Neonatal gonocytes are the precursors of both spermatogonial stem cells and spermatogonia; thus, any persistent DNA damage in these cells may lead to heritable mutations. We investigated the response of male mouse neonatal germ cells to ionizing radiation. Both gonocytes and spermatogonia died in large numbers by apoptosis. However, we found that the gonocytes were significantly more sensitive than spermatogonia and somatic cells to radiation-induced double-strand breaks (DSBs), as assayed by the number of gamma-H2AFX foci. In contrast, gonocytes irradiated in G2 phase seemed to repair DSBs faster than spermatogonia. Moreover, when irradiated in S phase, gonocytes arrested their cell cycle at the G1/S phase transition, whereas spermatogonia were mostly blocked in G2/M phase. Despite these differences, both cell types expressed high levels of proteins involved in DSB signaling and repair. Within the first hours after irradiation, the expression of Atr, Mre11a, H2afx, Xrcc6, and Xrcc4 was downregulated in neonatal spermatogonia, whereas, in gonocytes, most gene expression was unaffected. Together, these results suggest that the response of neonatal testis to genotoxic stress is regulated by different mechanisms according to the cell type and the differentiation status.

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes.

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63gamma expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.