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.

Effects of parenteral supplementation of Cu in female cattle with different levels of cupremia

“…During hypocuprosis, the activity of the enzyme lysyl oxidase (LOX), which is Cu dependent, suffers a considerable reduction, thereby causing a severe perturbation in the growth rate of pre-ovarian follicles, leading to reduction in the secretion of ovarian estradiol, a lower peak of estradiol in the pre-ovarian follicle, which is an endocrine sign that responsible for estrus induction, which in turn leads to perturbations of estrus and ovulation respectively (Kendall et al 2003) During Cu deficiency, the activity of the enzyme 15'15' dioxygenase is reduced; it is Cu dependent and responsible for the rupture of the central double bond of the carbon 15 of the carotene molecule, an indispensable factor in the formation of vitamin A (Izaguirre & Shimada 2001). Sharma et al (2003) have reported a correlation of vitamin A with the Cu serum status of the animals with a marked significant deficiency of vitamin A in the Cudeficient animals.…”

“…There are various clinical symptoms associated with hypocupremia, even though it has been suggested that reproductive problems are the principal manifestation of hypocuprosis in adult animals (Kendall et al 2003, Ahola et al 2005. Reports have shown a negative correlation between cupremia in the range of 9.8±1.0 to 14.0±0.9 µmo/L and reproductive indicators, as much in animals with spontaneous cupremia as with the induced form.…”

The effect of molybdenum on thein vitrodevelopment of mouse preimplantation embryos

“…Similar phenomena were observed in other types of cells cultured in vitro. For example, ammonium tetrathiomolybdate at 10 µg/ml resulted in a marked reduction in cellular clumping, and at 100 µg/ml and higher in absence of clumping [Kendall et al 2003]. This showed that ammonium tetrathiomolybdate can prevent induced differentiation of bovine granulosa cells cultured in vitro.…”

“…Zygotes were flushed from oviducts 18 h post-hCG injection, removed their cumulus cells by a brief exposure to 0.01% hyaluronidase, and then cultured in 50 µl droplets of potassium simplex optimized medium (KSOM) [Lawits and Biggers 1993] with 0,5,10,20,40,80,120, and 160 µg/ml of molybdenum (sodium molybdate dihydrate) covered under mineral oil at 377°C and 5% CO 2 in air for 5 d. The selection of those doses were based on previous reports [Kendall et al 2003;Kendall et al 2006;Kolesarova et al 2009] and the concentration that humans may be exposed [Huang et al 2011].…”

Ovarian Biomarkers in Infertility

“…TGF-1 and GDF9 increase lox mRNA expression in rat granulosa cells (Kendall et al, 2003). The GDF9-induced preantral follicular growth in vitro involves increased mural granulosa cell lox mRNA expression (Assou et al, 2010), and similarly the investigations on rat granulosa cells showed that lox transcripts were significantly suppressed 48h after eCG injection compared with untreated controls (Jiang et al, 2010).…”