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.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

High insulin concentrations promote the in vitro growth and viability of canine preantral follicles.

To determine whether the effects of different concentrations of insulin on the development of canine preantral follicles in vitro were associated or not with FSH, secondary follicles were isolated and cultured. In Experiment 1, follicles were cultured in the following media: modified minimum essential medium (CtrlMEM) alone; CtrlMEM plus 5 ng mL⁻¹ insulin (Ins5ng); CtrlMEM plus 10 ng mL⁻¹ insulin (Ins10ng); and CtrlMEM plus 10 µg mL⁻¹ insulin. In Experiment 2, follicles were cultured in the same media but in the presence of sequential FSH (i.e. CtrlFSH, Ins5ngF, Ins10ngF and 10µgF, respectively). Increasing concentrations of FSH (100, 500 and 1000 ng mL⁻¹) were added sequentially to the culture medium on Days 0, 6 and 12 of culture. Viability were assessed at the end of culture and follicular diameter and the antrum formation rate at four time points (Days 0, 6, 12 and 18). In Experiment 1, the high insulin concentration significantly increased follicular viability (P<0.05). In contrast, in Experiment 2, viability was not affected by the inclusion of insulin. In addition, viability was significantly better in follicles cultured in CtrlFSH (P<0.05). The diameter of follicles in the high-insulin group in Experiment 1 and high-insulin plus FSH group in Experiment 2 was superior to other groups tested. In experiment 2, the Ins10µg and Ins10µgF groups exhibited significantly higher antrum formation rates than the other groups. In conclusion, in the absence of FSH, high concentrations of insulin have beneficial effects on follicular viability. However, to promote the growth of canine preantral follicles in vitro, it is recommended that a combination of insulin and FSH be added to the medium.