Effect of copper and thiomolybdates on bovine theca cell differentiation in vitro

Abstract

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17 -hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions.The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0-100 µg/ml, copper (chloride) at equimolar concentrations (0-51·6 µg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17 -hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 µg/ml dose of thiomolybdate and equimolar copper thiomolybdate.Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 µg/ml at 96 h (P<0·05); doses above 20 µg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0·001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 µg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17 -hydroxylase expression was significantly increased (P<0·05) by tetrathiomolybdate, possibly due to a local regulatory system.In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitro and that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.

The Anti-Epileptic Drug Valproic Acid (VPA) Inhibits Steroidogenesis in Bovine Theca and Granulosa Cells In Vitro

“…Using well defined in vitro model systems based on primary cultures of bovine TC [26], [27], [28], [29] and GC [30], [31], [32], [33] the present study shows that VPA has a direct, dose-dependent inhibitory action on ovarian steroidogenesis, suppressing both basal and LH-induced androgen production by TC as well as FSH- and IGF-induced estrogen production by GC. With regard to TC, our findings concur with those of a recent study [22] documenting a VPA-induced inhibition of basal and hCG-induced androgen secretion by rat theca-interstitial cells.…”

The effect of molybdenum on thein vitrodevelopment of mouse preimplantation embryos

“…This showed that ammonium tetrathiomolybdate can prevent induced differentiation of bovine granulosa cells cultured in vitro. A follow-up study showed that 5-100 µg/ml of ammonium tetrathiomolybdate can prevent induced differentiation of bovine theca cells in culture, but apparently not affect cellular morphology and cell number [Kendall et al 2006]. In addition, ammonium molybdate at 330 µg/ ml can inhibit cell division and differentiation in vitro [Kolesarova et al 2009].…”

“…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].…”

<b>Efeitos da administração de um suplemento de microminerais injetável contendo cobre, manganês, selênio e zinco sobre a saúde uterina de vacas leiteiras</b>

“…O cobre, magnésio e zinco regulam enzimas que participam da produção de progesterona (GOTTSCH; VAN KIRK;MURDOCH, 2000;KENDALL et al, 2006). A concentração de progesterona tem sido associada com a perda precoce embrionária (MANN;LAMMING, 2001;FORDE et al, 2011).…”

“…A concentração de progesterona tem sido associada com a perda precoce embrionária (MANN;LAMMING, 2001;FORDE et al, 2011). É necessário para formação de enzimas esteroidogênicas, tais como citocromo P450, 17 alfa-hidroxilase, citocromo P450 de clivagem de cadeia lateral e lisil oxidase, a presença do cobre (KENDALL et al, 2006). O zinco é um mineral que age nas enzimas endopeptidases e está envolvido na reorganização dos folículos ovarianos em um corpo lúteo eficiente, que são é responsável pela produção de progesterona, já o manganês atua como um co-fator para a síntese de colesterol, precursor da síntese de progesterona (OLSON, 1965).…”