Kendall N R, Marsters P, Scaramuzzi R J, Campbell B K
Division of Reproductive Medicine, School of Human Development, D Floor, East Block, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Reproduction. 2003 May;125(5):657-65.
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.
在英国奶牛群中，对适当补充铜有反应的亚生育能力是一个普遍存在的问题。尽管其特征是发情减少或消失以及受孕率降低，但其确切原因仍然未知。本研究的目的是调查铜依赖性酶赖氨酰氧化酶的mRNA表达，以及铜和/或铜螯合硫代钼酸盐对在无血清培养基中培养的牛颗粒细胞FSH诱导分化的影响。使用牛特异性引物和RT-PCR对在最佳条件下培养0、16、24、48、96、144和192小时的牛颗粒细胞获得的细胞裂解物进行赖氨酰氧化酶mRNA表达的研究。通过在优化的颗粒细胞培养基中添加0、0.1、1、10、100和1000μg ml(-1)的四硫代钼酸铵、等摩尔浓度（0、0.0516、0.516、5.16、51.6、516μg ml(-1)）的氯化铜或两种培养基的等摩尔组合来研究硫代钼酸盐和铜的作用。在整个192小时的培养过程中，颗粒细胞都表达赖氨酰氧化酶mRNA。硫代钼酸盐在剂量>1μg ml(-1)时以剂量依赖性方式抑制雌二醇的产生，并阻止了无血清系统中颗粒细胞特有的聚集外观。尽管单独补充铜在生理剂量下没有效果，但使用铜和硫代钼酸盐等摩尔培养基可改善四硫代钼酸盐对雌二醇产生和细胞形态的影响。总之，本研究结果表明颗粒细胞表达赖氨酰氧化酶，硫代钼酸盐可在体外阻止FSH诱导的牛颗粒细胞分化，并且这些作用可通过补充铜来逆转。总体而言，这些数据支持以下假设：铜反应性亚生育能力是由排卵卵泡生长和发育的正常模式受到干扰引起的，这种影响可能至少部分通过赖氨酰氧化酶活性介导。
