In vitro cytotoxic effects of 5-Fluorouracil on isolated murine ovarian preantral follicles.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

PURPOSE: 5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired  ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. METHODS: Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, ß-catenin immunofluorescence was assessed in mouse ovaries. RESULTS: The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in ß-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. CONCLUSION: Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.