IN VITRO EFFECTS OF FUMONISIN B1 ALONE AND COMBINED WITH DEOXYNIVALENOL OR ZEARALENONE ON PORCINE GRANULOSA CELL PROLIFERATION AND STEROIDOGENESIS

Fumonisins, zearalenone and trichothecenes, such as deoxynivalenol (DON) and T-2 toxin, are the major Fusarium mycotoxins occurring throughout the world in cereal grains and animal feeds. Direct ovarian effects of fumonisin B1 (FB1) and its interaction with DON or α-zearalenol (α-ZOL), zearalenone major active metabolite, have so far not been investigated. Thus, the goal of this thesis was to determine if FB1, alone or combined with DON or α-ZOL, can impair swine reproductive activity via affecting granulosa cell function. To this aim, two different studies were designed. In the first study the effects of FB1 alone and combined with DON or α-ZOL on granulosa cell proliferation were evaluated. Porcine granulosa cells from small ovarian follicles (1-5 mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing FB1 at various doses (0, 0.01, 0.4, 10 and 14 μM) and combinations. At the end of the experiments, numbers of granulosa cells were determined using a Coulter counter. The results revealed that FB1 had inhibitory effects on granulosa cell proliferation at doses ≥ 10 μM. DON (3.4 μM) strongly inhibited (by 80%; P < 0.0001) granulosa cell proliferation and no significant difference was detected in combination with FB1 (10 μM). α-ZOL (9.4 μM) showed a stimulatory effect (P < 0.01) on granulosa cell numbers, even when treated in combination with FB1 (10 μM). In the second study the effects of FB1 alone and combined with DON or α-ZOL on granulosa cell steroid production and gene expression were investigated. Porcine granulosa cells from small follicles (1-5 mm) were cultured as described above. At the end of the experiments, concentrations of progesterone and estradiol in culture medium were determined by radioimmunoassays. Real-time RT-PCR was used to elucidate the effects of FB1 on gene expression of P450scc (CYP11A1) and aromatase (CYP19A1). All doses of FB1 (i.e., 0.01, 0.4, 10 and 14 µM) had no significant effect on estradiol production, whereas FB1 showed a stimulatory effect on progesterone production induced by FSH plus insulin-like growth factor-I (IGF-I) at 10 and 14 µM. α-ZOL (9.4 µM) increased (P < 0.0001) FSH plus IGF-I-induced progesterone production by 51%. Combination of FB1 with α-ZOL resulted in an increase of progesterone production (91%; P < 0.0001) that was significantly higher than that induced by either Fusarium mycotoxin alone. DON drastically inhibited (by 74%; P < 0.0001) progesterone production and FB1 had little effect on this response. α-ZOL had no effect on FSH plus IGF-I-induced estradiol production, whereas decreased (P < 0.05) estradiol production when co-treated with FB1. DON (3.4 µM) strongly inhibited (by 67%; P < 0.0001) estradiol production and no difference was detected in combination with FB1 (10 µM). FB1 (10 µM) had no effect on granulosa cell CYP19A1 mRNA abundance, whereas decreased (by 23%; P < 0.0001) granulosa cell CYP11A1 mRNA abundance induced by FSH plus IGF-I. In conclusion, the present thesis indicates that FB1 has direct effects on porcine granulosa cell proliferation, steroid production and gene expression and provides information on the interactions between FB1 and DON or α-ZOL in granulosa cells. These interactions and direct ovarian effects should be considered in swine reproductive failures.