title: Effect of lanosterol on the in vitro maturation in semi-defined culture system of prepubertal ewe oocytes.
abstract: The choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 10(6) and 7.4 ± 0.2 × 10(6) arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 10(6) arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.
authors.affiliation: Laboratory of Biotechnology of Reproduction, Institute of Science and Animal Technology (ICTA) at the Polytechnic University of Valencia, C/Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Marco-Jiménez
authors.affiliation: Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, 46022, Spain.
authors.forename: J S
authors.initials: JS
authors.lastname: Vicente
authors.affiliation: Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias, Segorbe, 12400, Spain.
authors.forename: M P
authors.initials: MP
authors.lastname: Viudes-de-Castro
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007810:Lanosterol / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects* / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D011627:Puberty; D012756:Sheep
chemical_list: D007810:Lanosterol; D010100:Oxygen