Effects of the mycotoxin metabolite de-epoxy-deoxynivalenol (DOM-1) on embryo development and sperm motility in cattle.

Contamination of animal feed with Fusarium spp results in accumulation of mycotoxins including deoxynivalenol. In animals, deoxynivalenol is metabolized to de-epoxy deoxynivalenol (DOM-1), which is generally considered to be a non-toxic metabolite; however, recent studies demonstrated that DOM-1 can reduce steroid production and induce apoptosis in the bovine ovary. The objectives of this study were to assess the effects of DOM-1 on applied aspects of reproductive function in cattle, specifically sperm function and embryo development in vitro and follicle growth and superovulatory responses in vivo. The effect of naturally contaminated feed on superovulatory responses was assessed; a dose of 6 ppm deoxynivalenol increased blood DOM-1 concentrations to 20 ng/ml, but this did not alter the number of viable embryos recovered on day 7. However, intrafollicular injection of DOM-1 (100 ng/ml) directly into the growing dominant follicle resulted in cessation of follicular growth over the subsequent 3 days. Treatment with DOM-1 reduced motility of bull spermatozoa over a 10-h period in vitro. Addition of DOM-1 to oocytes in vitro during IVM did not alter rates of cumulus expansion and nuclear maturation, but treatment during IVF reduced the rate of blastocyst formation. These data illustrate that DOM-1 is more biologically active than previously thought and negatively impacted reproductive outcomes in cattle.

The mycotoxin metabolite deepoxy- deoxynivalenol increases apoptosis and decreases steroidogenesis in bovine ovarian theca cells.

The mycotoxin deoxynivalenol (DON) has been shown to inhibit ovarian granulosa cell function in cattle in vitro, but it is not known whether DON or its metabolite deepoxy-DON (DOM-1) affects theca cell function. The objectives of this study were to determine the effects of DON and of DOM-1 on theca cell steroidogenesis and apoptosis, and to determine the main pathways through which they act. Bovine theca cells were cultured in a nonluteinizing serum-free culture system, and challenged with DON or DOM-1 for 4 days to measure steroidogenesis and apoptosis, for 1-8 h to measure immediate-early genes, and for 5-60 min to measure phosphorylation of intracellular signaling proteins. Addition of DON decreased progesterone secretion at doses as low as 0.5 ng/ml but had no effect on testosterone secretion. Addition of DOM-1 inhibited progesterone and testosterone secretion at 0.5 ng/ml. Treatment of cells with 1 ng/ml DOM-1 increased the proportion of apoptotic cells, whereas DON had no effect. Addition of DON or DOM-1 stimulated phosphorylation of EIF2AK2, MAPK3/1, and AKT. However, DON inhibited and DOM-1 stimulated MAPK14 phosphorylation. DON increased the levels of mRNA encoding early-immediate genes EGR1, EGR3, and FOS, whereas DOM-1 was without effect. DOM-1 but not DON increased abundance of mRNA of the endoplasmic reticulum (ER) stress-related proteins, PRKRA and ATF4. We conclude that DOM-1 has a major impact on theca function in cattle, and possibly induces theca cell apoptosis through ER stress.

The role of fibroblast growth factor-18 in follicular atresia in cattle.

Although the various members of the fibroblast growth factor (FGF) family are generally mitotic, one member, FGF18, has been shown to increase the rate of apoptosis of ovarian granulosa cells. In the present study, we first determined whether granulosa cells express FGF18 and we then explored the mechanism through which FGF18 increases apoptosis in vitro. Under culture conditions that favored estradiol secretion and CYP19A1 expression, granulosa FGF18 mRNA levels were barely detectable; however, withdrawing gonadotropic support (follicle-stimulating hormone or insulin-like growth factor 1) reduced levels of CYP19A1 mRNA and increased abundance of mRNA encoding the death ligand FASLG and FGF18. Addition of FGF18, but not FGF2, FGF10, or EGF, increased the proportion of apoptotic cells and frequency of caspase 3 activation, and these effects were abrogated by coculture with estradiol. Addition of FGF18 decreased abundance of mRNA encoding the antiapoptotic proteins GADD45B and MDM2, and increased that encoding the proapoptotic protein BBC3; these effects were reversed by coculture with estradiol. The physiological relevance of FGF18 was determined using an in vivo model: injection of FGF18 directly into growing bovine dominant follicles caused cessation of follicle growth by 24 h after injection. Collectively, these data demonstrate that FGF18 is proapoptotic in vivo and may act through a mechanism involving the BBC3-MDM2 pathway.

Effects of the mycotoxin deoxynivalenol on steroidogenesis and apoptosis in granulosa cells.

Mycotoxins can reduce fertility and development in livestock, notably in pigs and poultry, although the effect of most mycotoxins on reproductive function in cattle has not been established. One major mycotoxin, deoxynivalenol (DON), not only targets immune cells and activates the ribotoxic stress response (RSR) involving MAPK activation, but also inhibits oocyte maturation in pigs. In this study, we determined the effect of DON on bovine granulosa cell function using a serum-free culture system. Addition of DON inhibited estradiol and progesterone secretion, and reduced levels of mRNA encoding estrogenic (CYP19A1) but not progestogenic (CYP11A1 and STAR) proteins. Cell apoptosis was increased by DON, which also increased FASLG mRNA levels. The mechanism of action of DON was assessed by western blotting and PCR experiments. Addition of DON rapidly and transiently increased phosphorylation of MAPK3/1, and resulted in a more prolonged phosphorylation of MAPK14 (p38) and MAPK8 (JNK). Activation of these pathways by DON resulted in time- and dose-dependent increases in abundance of mRNA encoding the transcription factors FOS, FOSL1, EGR1, and EGR3. We conclude that DON is deleterious to granulosa cell function and acts through a RSR pathway.

Real-life exposure to Fusarium toxins deoxynivalenol and zearalenone triggers apoptosis and activates NLRP3 inflammasome in bovine primary theca cells.

Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 µM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1ß, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary.

The Mycotoxin De-Epoxy-Deoxynivalenol (DOM-1) Increases Endoplasmic Reticulum Stress in Ovarian Theca Cells.

Deoxynivalenol (DON) is a major mycotoxin present in animal feed and negatively affects growth and reproduction in farm species, including pigs and cattle. The mechanism of DON action involves the ribotoxic stress response (RSR), and it acts directly on ovarian granulosa cells to increase cell death. In ruminants, DON is metabolized to de-epoxy-DON (DOM-1), which cannot activate the RSR but has been shown to increase cell death in ovarian theca cells. In the present study, we determined if DOM-1 acts on bovine theca cells through endoplasmic stress using an established serum-free cell culture model and to assess whether also DON activates endoplasmic stress in granulosa cells. The results show that DOM-1 increased the cleavage of ATF6 protein, increased the phosphorylation of EIF2AK3, and increased the abundance of cleaved XBP1 mRNA. Activation of these pathways led to an increased abundance of mRNA of the ER stress target genes GRP78, GRP94, and CHOP. Although CHOP is widely associated with autophagy, inhibition of autophagy did not alter the response of theca cells to DOM-1. The addition of DON to granulosa cells partially increased ER stress pathways but failed to increase the abundance of mRNA of ER stress target genes. We conclude that the mechanism of action of DOM-1, at least in bovine theca cells, is through the activation of ER stress.

Divergence of intracellular signaling pathways and early response genes of two closely related fibroblast growth factors, FGF8 and FGF18, in bovine ovarian granulosa cells.

Fibroblast growth factors (FGFs) modulate ovarian function, including FGF8 and FGF18. These FGFs activate the same receptors, although FGF18 is unusual in that it increases apoptosis in ovarian granulosa cells whereas the 'typical' response to FGF is increased proliferation. The objective of the present study was to determine which early response genes and pathways are activated by FGF8 and FGF18 in bovine granulosa cells. FGF8 increased abundance of mRNA encoding the FGF-responsive genes SPRY1, SPRY2, SPRY4, NR4A1 and NR4A3 whereas FGF18 did not. FGF8 increased but FGF18 decreased levels of mRNA encoding the growth arrest associated protein, GADD45B. FGF8 increased ERK1/2 phosphorylation but FGF18 did not. Microarray analysis identified EGR1, FOS, FOSL1, BAMBI, XIRP1 and PLK2 as other FGF8 immediate-early response genes, and FGF18 stimulated EGR1, FOSL1, BAMBI and PLK2, but not FOS or XIRP1. This study demonstrates that FGF8 and FGF18 signal through divergent pathways in ovarian granulosa cells, despite reportedly similar receptor activation patterns.