Effects of the mycotoxin, deoxynivalenol, and its major metabolite, de-epoxy deoxynivalenol, on bovine reproduction
Deoxynivalenol (DON) is a major mycotoxin found in animal feed and is known to reduce fertility in pigs by inhibiting progesterone secretion from granulosa cells. In cattle, it is metabolized to de-epoxy DON (DOM-1) in the rumen, and DOM-1 can reach high concentrations in blood and follicular fluid. One of the major pathways activated by DON is the ribotoxic stress response (RSR), which involves autophosphorylation of protein kinase R (PKR) and downstream activation of MAP kinases including MAPK3/1. It is not known if these mycotoxins affect bovine reproduction. The objectives of present thesis were (1) to determine how and at what doses DON affects ovarian granulosa cell function and to elucidate its mechanism of action; and (2) to determine how and at what doses major mycotoxin DON and its metabolite DOM-1 affect theca cell function in cattle. The results are separated into three articles. In the first article the effects of DON on granulosa cells were explored; treatment with DON resulted in a significant inhibition of estradiol and progesterone (P4) secretion, and an increase in the proportion of apoptotic cells after 4 days of treatment. Western blot demonstrated significant upregulation of ERK1/2 and MAPK14 phosphorylation within 15-30 minutes of adding DON. We then determined the effect of DON on ERK1/2 target genes; EGR1 and FOS mRNA levels were transiently stimulated with maximum levels at 1 h of adding DON, whereas COX2 and GADD45B mRNA levels were upregulated but not until 24 h after DON treatment. In the second article, the effects of DON and DOM-1 on theca cells were assessed. Treatment with DOM-1 resulted in a dose-dependent inhibition of P4 and testosterone secretion, and an increase in the proportion of apoptotic cells, while DON inhibited P4 but did not alter testosterone secretion or the percentage of dead cells. Both ii DON and its metabolite were maximally effective at concentrations of 1 ng/ml (in contrast, the effects of DON on occur at 100ng/ml). Western blot demonstrated rapid phosphorylation of MAPK3/1, PKR and of JUN kinase after addition of DOM-1 or DON. Interestingly, phosphorylation of MAPK14 was significantly increased by DOM-1 but decreased by DON. The addition of a PKR inhibitor abrogated the ability of DON and DOM-1 to increase phosphorylation of MAPK3/1, and partly abrogated the inhibitory effect of DON on MAPK14 phosphorylation, however, the PKR inhibitor further increased the phosphorylation of MAPK14 caused by DOM-1. Together, these results suggest that DON activates the RSR in bovine granulosa and theca cells, and that theca cells are more sensitive than granulosa cells to the effects of DON. The data also demonstrate for the first time in any cell type the ability of DOM-1 to affect cell function and health.
Deoxynivalenol Ribotoxic stress response Mycotoxin De-epoxy deoxynivalenol Granulosa cells