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

Abstract

5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who mayhave impaired ovarian follicle pool reserve with lasting effectsto reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450mg/kg i.p) or saline and sacrificed 3days 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, 92.2mM). 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, β-catenin immunofluorescence was assessed in mouse ovaries. 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 β-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.