Juliana Z Almeida1, Laritza F Lima2, Luís A Vieira2, Carolina Maside2, Anna C A Ferreira2, Valdevane R Araújo2, Ana B G Duarte3, Ramon S Raposo4, Sônia N Báo5, Cláudio C Campello2, Luiz F S Oliveira6, Thayse P da Costa6, José Garcia Abreu6, José R Figueiredo2, Reinaldo B Oriá7. 1. Department of Morphology, Institute of Biomedicine, Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition, School of Medicine, Federal University of Ceara, 1315 Rua Cel. Nunes de Melo, Fortaleza, CE, 60430-270, Brazil. 2. Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral Follicles (LAMOFOPA), State University of Ceara, Fortaleza, CE, Brazil. 3. Department of Morphology, Faculty of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil. 4. Experimental Biology Core, University of Fortaleza, Fortaleza, CE, Brazil. 5. Laboratory of Electron Microscopy, Department of Cell Biology, University of Brasilia, Brasília, DF, Brazil. 6. Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 7. Department of Morphology, Institute of Biomedicine, Laboratory of the Biology of Tissue Healing, Ontogeny and Nutrition, School of Medicine, Federal University of Ceara, 1315 Rua Cel. Nunes de Melo, Fortaleza, CE, 60430-270, Brazil. oria@ufc.br.
Abstract
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, β-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 β-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.
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, β-catenin immunofluorescence was assessed in mouseovaries. 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 β-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.
Authors: Orleâncio Gomes R Azevedo; Renato André C Oliveira; Bruna Castro Oliveira; Snjezana Zaja-Milatovic; Celina Viana Araújo; Deysi Viviana T Wong; Tiê Bezerra Costa; Herene Barros Miranda Lucena; Roberto César P Lima; Ronaldo A Ribeiro; Cirle A Warren; Aldo Ângelo M Lima; Michael P Vitek; Richard L Guerrant; Reinaldo B Oriá Journal: BMC Gastroenterol Date: 2012-07-13 Impact factor: 3.067