title: Expression of lysyl oxidase and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulosa cells cultured in serum-free media.
abstract: Subfertility that will respond to appropriate copper supplementation is a widespread problem in the UK dairy herd and, although characterized by reduced or absent oestrus and reduced conception rates, the exact cause remains unknown. The aim of this study was to investigate the expression of mRNA for the copper-dependent enzyme, lysyl oxidase, and the effect of copper and/or copper chelating thiomolybdates on FSH-induced differentiation of bovine granulosa cells cultured in serum-free media. Expression of lysyl oxidase mRNA was investigated using bovine specific primers and RT-PCR on cell lysates obtained from bovine granulosa cells cultured under optimum conditions for 0, 16, 24, 48, 96, 144 and 192 h. The effect of thiomolybdates and copper were investigated by supplementing optimized granulosa cell culture media with ammonium tetrathiomolybdate at 0, 0.1, 1, 10, 100 and 1000 micro g ml(-1), copper chloride at equimolar concentrations (0, 0.0516, 0.516, 5.16, 51.6, 516 micro g ml(-1)) or equimolar combinations of both media. Lysyl oxidase mRNA was expressed by the granulosa cells throughout the 192 h of culture. Thiomolybdate depressed oestradiol production in a dose-dependent manner at doses > 1 micro g ml(-1) and prevented the characteristic clumped appearance of granulosa cells in this serum-free system. Although the supplementation of copper alone had no effect at physiological doses, the use of the equimolar copper and thiomolybdate media ameliorated the effect of tetrathiomolybdates on both oestradiol production and cellular morphology. In conclusion, the results of the present study indicate that lysyl oxidase is expressed by granulosa cells, that thiomolybdates can prevent FSH-induced differentiation of bovine granulosa cells in vitro and that these effects can be reversed by copper supplementation. Overall, these data support the hypothesis that copper-responsive subfertility results from perturbation of the normal pattern of ovulatory follicle growth and development, an effect that may be mediated, at least in part, via lysyl oxidase activity.
authors.affiliation: Division of Reproductive Medicine, School of Human Development, D Floor, East Block, Queen's Medical Centre, Nottingham NG7 2UH, UK. Nigel.Kendall@Nottingham.ac.uk
authors.forename: N R
authors.initials: NR
authors.lastname: Kendall
authors.forename: P
authors.initials: P
authors.lastname: Marsters
authors.forename: R J
authors.initials: RJ
authors.lastname: Scaramuzzi
authors.forename: B K
authors.initials: BK
authors.lastname: Campbell
mesh_terms: D000818:Animals; D002417:Cattle / Q000502:physiology*; D002418:Cattle Diseases / Q000188:drug therapy; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology*; D016895:Culture Media, Serum-Free; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects; D007247:Infertility, Female / Q000188:drug therapy / Q000662:veterinary; D008982:Molybdenum / Q000494:pharmacology*; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*
chemical_list: D002614:Chelating Agents; D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D003300:Copper; D008982:Molybdenum; D005640:Follicle Stimulating Hormone; C020809:tetrathiomolybdate; D008249:Protein-Lysine 6-Oxidase