Abstract
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.
Introduction
Fungal contamination of animal feed is a significant problem in many parts of the world (Marin et al. 2013). Contamination with Fusarium spp is common and results in significant accumulation of the mycotoxins zearalenone (ZEN) and deoxynivalenol (DON) among others (Rodrigues & Naehrer 2012). The actions of ZEN are well known; it is estrogenic and affects the female reproductive system, particularly in pigs where symptoms include nymphomania, pseudopregnancy, and ovarian atrophy (reviewed in Cortinovis et al. (2013)). In cattle, ZEN intoxication is reported to result in reduced conception rates, possibly owing to toxic effects on the oocyte (Minervini et al. 2001).
Less is known about the effects of DON, a non-estrogenic compound, on the female reproductive system. In pigs, DON inhibited cumulus expansion and oocyte maturation in vitro (Alm et al. 2002, Malekinejad et al. 2007, Schoevers et al. 2010). The potential effects of DON on granulosa cells are unclear; DON has been reported to either increase or decrease progesterone secretion and to have a biphasic effect on estradiol (E2) secretion from porcine granulosa cells in vitro (Ranzenigo et al. 2008, Medvedova et al. 2011). In cattle, there are preliminary data to suggest that DON increased levels of mRNA coding for the rate-limiting progestagenic enzyme cytochrome P450 cholesterol side-chain cleavage (CYP11A1), but had no effect on abundance of mRNA encoding the main estrogenic enzyme cytochrome P450 aromatase (CYP19A1) in cultured granulosa cells (Pizzo et al. 2014); any effect of DON on abundance of mRNA encoding StAR protein (STAR), the protein involved in the transport of cholesterol across the mitochondrial membrane, was not reported. To our knowledge, no other information is available on the effects of DON on ovarian function in cattle.
The generally accepted mechanism of action of DON is through binding to ribosomes and initiation of the ribotoxic stress response (RSR). This involves activation of the p38 (MAPK14), ERK1/2 (MAPK3/1), and c-Jun N-terminal kinase (MAPK8) members of the MAP kinase (MAPK) family (Pestka 2008). As all these pathways are active in bovine granulosa cells (Evans & Martin 2000, Uzbekova et al. 2009, Abedini et al. 2015), we hypothesize that DON may activate one or more of these pathways to alter granulosa cell function. The objectives of this study were to determine the effects of DON on granulosa cell steroidogenesis and apoptosis in a non-luteinizing serum-free culture system, and to determine whether DON acts through typical RSR intracellular signaling pathways, including early response genes.
Materials and methods
Cell culture
All materials were obtained from Life Technologies, Inc. Bovine granulosa cells were cultured in serum-free conditions that maintain E2 and progesterone secretion and responsiveness to follicle-stimulating hormone (FSH; Gutiérrez et al. 1997, Silva & Price 2000, Sahmi et al. 2004). Bovine ovaries were obtained from adult cows, independently of the stage of the estrous cycle, at the slaughterhouse and transported to the laboratory at 30 °C in PBS containing penicillin (100 IU) and streptomycin (100 μg/ml). Granulosa cells were harvested from follicles 2–5 mm in diameter, and the cell suspension was filtered through a 150 mesh steel sieve (Sigma–Aldrich Canada). Cell viability was assessed by Trypan blue dye exclusion.
Cells were seeded into 24-well tissue plates (Sarstedt, Inc., Newton, NC, USA) at a density of 500 000 viable cells in 500 μl DMEM/F12 containing sodium bicarbonate (10 mM), 25 mM HEPES, sodium selenite (4 ng/ml), BSA (0.1%; Sigma–Aldrich), penicillin (100 U/ml), androstenedione (10−6 M), and bovine FSH (10 ng/ml starting on day 2, AFP5346D; National Hormone and Peptide Program, Torrance, CA, USA). Cultures were maintained at 37 °C in an atmosphere of 5% CO2 and 95% air for up to 6 days.
Experimental treatments
To determine the effects of DON on granulosa cell steroidogenesis, cells were treated from day 2 with 0, 1, 10, or 100 ng/ml DON (in methanol) or vehicle (methanol alone), and cells and media were recovered on day 6; these doses were based on concentrations of 2–14 ng/ml DON reported in serum of cattle fed a contaminated concentrate (Keese et al. 2008). Apoptosis was measured using an Annexin V–FITC apoptosis detection kit (Sigma–Aldrich) after treating cells with an effective dose of DON for 4 days. To assess the effect of DON on intracellular pathway activation, cells were treated on day 5 of culture with an effective dose of DON for 0, 5, 15, 30, and 60 min, and cells were recovered in RIPA buffer to measure the phosphorylation status of key protein kinases. The dose- and time-dependent effects of DON on abundance of mRNA of early response genes were determined by treating cells on day 5 of culture with an effective dose of DON for 0, 1, 2, 4, 8, and 24 h, and by treating cells for 1 h with 0, 1, 10, or 100 ng/ml DON. Cells were recovered for RNA extraction. All experiments were carried out with three different pools of cells collected on different occasions.
Steroid assay
E2 concentrations in a conditioned medium were measured in duplicate as described (Jiang & Price 2012) using an antibody raised in rams (Sanford 1987). Intra- and inter-assay coefficient of variation (CV) values were 6 and 9% respectively. Progesterone concentration was measured in a conditioned medium in duplicate as described (Bélanger et al. 1990, Price et al. 1995) with mean intra- and interassay CV values of 7.2 and 18% respectively. Steroid concentrations in the culture medium were corrected for cell number by expressing the data per unit mass of total cell protein. The sensitivities of these assays were 10 and 4 pg/tube for E2 and progesterone, equivalent to 0.3 and 20 ng/μg protein respectively.
Total RNA extraction and real-time PCR
After treatments, the medium was removed and total RNA was extracted using TRIzol according to the manufacturer's instructions. Total RNA (0.5 μg) was quantified by absorbance at 260 nm and treated with 1 U DNase (Invitrogen). RNA was reverse transcribed in the presence of 1 mmol/l oligo (dT) primer and 4 U Omniscript RTase (Qiagen), and 0.25 mmol/l ddNTP mix and 19.33 U RNase inhibitor (GE Healthcare Canada, Baie D'Urfé, QC, Canada) in a volume of 20 μl at 37 °C for 1 h. The reaction was terminated by incubation at 93 °C for 5 min.
Real-time PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems) with Power SYBR Green PCR Master Mix. The bovine-specific primers have been published previously (Jiang et al. 2013). Common thermal cycling parameters (3 min at 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 59 °C, and 30 s at 72 °C) were used to amplify each transcript. Melting curve analyses were performed to verify product identity. Samples were run in duplicate and were expressed relative to histone H2AFZ as a housekeeping gene. Data were normalized to a calibrator sample using the ΔΔCt method with correction for amplification efficiency (Pfaffl 2001).
Western blot
After challenge with DON, cells were washed with cold PBS and lysed in 100 μl/well cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and protease inhibitor cocktail). The homogenate was centrifuged at 6000 g for 5 min at 4 °C. The resulting supernatant was retained and stored at −20 °C. Protein concentrations were determined by BCA Protein Assay (Pierce, Rockford, IL, USA).
Samples were resolved on 12% SDS–polyacrylamide gels (15 μg total protein/lane) and electrophoretically transferred onto nitrocellulose membrane in a Bio-Rad wet Blot Transfer Cell apparatus (transfer buffer: 39 mM glycine, 48 mM Tris-base, 1% SDS, 20% methanol, and pH 8.3). After transfer, the membranes were blocked in TTBS (10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20, and pH 7.5) for 1 h. Membranes were incubated overnight with the primary antibody (rabbit anti-rat MAPK3/1, #9102, 1:2000; rabbit anti-human phospho-MAPK3/1, #9101, 1:1000; rabbit anti-human MAPK14 #9211, 1:1000; rabbit anti-human phospho-MAPK14 #9215, 1:1000; rabbit anti-human MAPK8 #9251, 1:1000; and rabbit anti-human MAPK8 #9252, 1:1000; Cell Signaling Technology, Danvers, MA, USA) diluted in 5% BSA (total MAPK8) or TTBS (all other antibodies) at 4 °C. The loading control was COX4I1 (#69359, 1:1000; Santa Cruz Biotechnology). After washing three times with TTBS, membranes were incubated for 2 h at room temperature with 1:10 000 anti-rabbit HRP-conjugated IgG (GE Healthcare Canada) diluted in TTBS. After five washes in TTBS, protein bands were revealed by ECL (Millipore, Billerica, MA, USA) using a gel imaging system (ChemiDoc XRS system, Bio-Rad). Semiquantitative analysis was performed using the Bio-Rad ChemiDoc XRS Software.
Statistical analysis
All statistical analyses were performed using the JMP Software (SAS Institute, Cary, NC, USA). Data were transformed to logarithms if they were not normally distributed (Shapiro–Wilk test). At instances where main effects were significant, the effect of time or treatment was tested using the Tukey–Kramer honest significant difference (HSD) test. The data are expressed as least square means±s.e.m.
Results
DON suppressed steroid secretion and steroidogenic enzyme gene expression
We first assessed the effect of DON on steroidogenesis. Cultured bovine granulosa cells were challenged with 1, 10, and 100 ng/ml DON for 4 days. At the dose of 100 ng, DON significantly inhibited E2 and progesterone secretion (Fig. 1), and potently suppressed CYP19A1 mRNA levels but did not alter CYP11A1 and STAR mRNA levels (Fig. 2).
Effect of DON on estradiol and progesterone secretion from bovine granulosa cells in a serum-free medium. Cells were cultured for 4 days with the doses of DON shown, and steroid in the medium measured by RIA. Concentrations were corrected for cell number (total cell protein) and represent the amount secreted during the last 2 days of culture. Data are expressed as means (±s.e.m.) of four independent cultures, and bars without common letters are significantly different (P<0.05, Tukey–Kramer HSD). Vehicle, methanol.
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
Effect of DON on abundance of mRNA encoding steroidogenic proteins in bovine granulosa cells. Cells were cultured for 4 days with 100 ng/ml DON or methanol (vehicle), and RNA collected for real-time PCR. Data are expressed relative to a calibrator sample using the ΔΔCt method with correction for amplification efficiency and are presented as means (±s.e.m.) of three independent cultures. Asterisk denotes treatment significantly different from control (P<0.05, Tukey–Kramer HSD).
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
DON increases granulosa cell apoptosis
The ability of DON to inhibit E2 secretion prompted us to determine the effect of DON on granulosa cell health. Addition of an effective dose of DON (100 ng/ml) for 4 days increased the proportion of apoptotic cells by 15% (Fig. 3). In a subsequent experiment, DON was added for 24 h and increased levels of mRNA encoding the apoptosis-related genes FASLG and GADD45B (Fig. 3).
Addition of DON increased the proportion of dead cells and abundance of FASLG and GADD45B mRNA in bovine granulosa cells. Cells were cultured for 4 days with 100 ng/ml DON and recovered for either the measurement of apoptosis by flow cytometry (Annexin-V apoptosis kit) or for RNA measurement by real-time PCR. Data are expressed as means (±s.e.m.) of three independent cultures. Asterisk denotes treatment significantly different from control (P<0.05, Student's t-test).
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
DON activates pathways related to RSR in granulosa cells
The main mechanism of action of DON is through RSR pathways; therefore, we assessed the activation of these pathways in granulosa cells. The addition of DON caused a rapid and transient increase in MAPK3/1 phosphorylation within 15 min and an increase in MAPK14 phosphorylation that was significant at 30 and 60 min (Fig. 4A and B). Furthermore, DON increased both MAPK8 (Fig. 4C) and phospho-MAPK8 levels (Fig. 4D), such that the ratio of phosphorylated to total MAPK8 did not change (not shown).
Intracellular pathways activated by DON in granulosa cells. Bovine granulosa cells were cultured in a serum-free medium and on day 5 were challenged with DON (100 ng/ml) for the times shown. Total cell protein was recovered for western blotting with antibodies against total and phosphorylated forms of (A) MAPK3/1, (B) MAPK14, and (C and D) MAPK8. Representative blots from one replicate are shown above the graphs, and samples were loaded in the same order as in the graphs. Data are represented as the ratio of phosphorylated:total protein for MAPK3/1 and MAPK14, and of each form of MAPK8:COX4I1 (housekeeping protein), and are means (±s.e.m.) of three independent cultures; bars without common letters are significantly different (P<0.05, Tukey–Kramer HSD).
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
Our next step was to determine whether the activation of these pathways by DON affected expression of specific target genes. Addition of DON increased EGR1, EGR3, FOS, and FOSL1 mRNA levels within 1–2 h, and levels declined to control values by 8 h for all genes. DON also increased PTGS2 mRNA levels, and this was not significant until 24 h of treatment (Fig. 5). We confirmed the effect on the early response genes with a dose–response study at 1 h of treatment, and mRNA levels of all four target genes were increased at the dose of DON that inhibited E2 secretion (Fig. 6).
Acute effect of DON on early response genes in bovine granulosa cells. Cells were cultured in a serum-free medium and on day 5 were challenged with DON (100 ng/ml) for the times shown. Cells were recovered for RNA measurement by real-time PCR. Data are expressed relative to a calibrator sample using the ΔΔCt method with correction for amplification efficiency, and are presented as means (±s.e.m.) of three independent cultures. Bars without common letters are significantly different (P<0.05, Tukey–Kramer HSD).
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
Effect of DON on early response genes is dose dependent. Bovine granulosa cells were cultured in a serum-free medium and on day 5 were challenged with DON for 1 h at the doses shown. Cells were recovered for RNA measurement by real-time PCR. Data are expressed relative to a calibrator sample using the ΔΔCt method with correction for amplification efficiency and are presented as means (±s.e.m.) of three independent cultures. Bars without common letters are significantly different (P<0.05, Tukey–Kramer HSD). Vehicle, methanol.
Citation: REPRODUCTION 149, 6; 10.1530/REP-15-0018
Discussion
The results of this study clearly demonstrate that DON can have a significant negative impact on granulosa cell health and function, notably on E2 secretion and CYP19A1 mRNA levels, and that DON acts through typical RSR pathways involving activation of MAPK3/1, MAPK8, and MAPK14 kinases.
Reports on the effects of DON on steroidogenesis are contradictory. In pigs, DON increased E2 secretion and CYP19A1 mRNA levels at a concentration of 10 ng/ml and inhibited both at a concentration of 100 ng/ml (Ranzenigo et al. 2008), whereas, in bovine granulosa cells, DON (1000 ng/ml) increased CYP19A1 mRNA levels (Pizzo et al. 2014). In this study, no stimulatory effect of DON on E2 secretion or CYP19A1 mRNA levels was observed, although the decrease in E2 secretion and CYP19A1 mRNA levels with 100 ng/ml DON was consistent with the study by Ranzenigo et al. (2008). Similarly, for progesterone, 100 ng/ml DON increased progesterone secretion in one study carried out on pigs (Medvedova et al. 2011), but inhibited secretion from granulosa cells in another study (Ranzenigo et al. 2008) and in this study with bovine cells. One difference between the previous and present studies is the use of serum in the culture medium in all previous studies; serum is known to alter granulosa cell steroidogenesis in vitro (Gutiérrez et al. 1997).
In pig granulosa cell cultures, DON at the doses used increased cell numbers and abundance of the proliferation marker PCNA without increasing apoptosis (Ranzenigo et al. 2008, Medvedova et al. 2011), whereas, in this study, DON increased the rate of apoptosis. This difference is again likely to be owing to the absence of serum in the current culture system, as this reduces the rate of proliferation of granulosa cells (Gutiérrez et al. 1997). As an increase in the incidence of apoptotic granulosa cells (Irving-Rodgers et al. 2001) and a decrease in E2 secretion (McNatty et al. 1984) are characteristics of atretic follicles in vivo, the ability of DON to increase apoptosis and decrease E2 secretion suggests that it may be able to cause or promote follicle atresia.
The addition of DON increased the abundance of FASLG and GADD45B mRNAs, both of which have been linked to apoptosis. While FASLG is well known to induce apoptosis in a variety of cell types including granulosa cells (Porter et al. 2000), the role of GADD45B is much less clear. Granulosa cells of atretic bovine follicles contain less GADD45B mRNA than do those of healthy follicles (Mihm et al. 2008), and pro-apoptotic factors such as fibroblast growth factor 18 (FGF18) decrease GADD45B mRNA levels in granulosa cells in vitro (Portela et al. 2010), whereas mitogenic factors such as FGF2 increase GADD45B mRNA levels (Jiang et al. 2011). Thus, the increase in GADD45B mRNA levels with increased apoptosis is not consistent with the previous data, and supports the suggestion that the regulation of GADD45B mRNA abundance is context (ligand?) specific (Salvador et al. 2013). It has been suggested that GADD45B may enhance or mitigate FAS-mediated apoptosis, depending on the cell type (Zazzeroni et al. 2003, Cho et al. 2010), thus the increase in GADD45B mRNA levels occurring with increased FASLG mRNA abundance may be part of either the apoptotic mechanism or a DNA repair mechanism.
The intracellular pathways activated by DON in a variety of non-reproductive cell types include MAPK3/1, MAPK8, and MAPK14. In this study, we demonstrate for the first time that DON activates these MAPKs in granulosa cells. The time-course of DON-induced phosphorylation observed herein is similar to that observed in a number of cell types, including murine macrophages (Moon & Pestka 2002, Pan et al. 2013), human intestine epithelial cells (Moon et al. 2007), and mouse skin (Mishra et al. 2014) among others. The activity of these pathways was demonstrated by the time- and dose-dependent increase in levels of mRNA encoding the transcription factors EGR1 and FOS; DON has previously been shown to increase Egr1 and Fos mRNAs in various cell lines (Moon et al. 2007, Nielsen et al. 2009) and mouse spleen (Kinser et al. 2004). In this study, we also identified FOSL1 and EGR3 as targets of DON activity, which are novel findings. EGR3 is a zinc finger-containing transcription factor that has been reported in breast cancer cells and in the mouse oocyte (Inoue et al. 2004, Shin et al. 2014). Although it is well known that EGR1 mRNA abundance in granulosa cells is increased by ligands including gonadotropins and growth factors (Espey et al. 2000, Russell et al. 2003, Sayasith et al. 2006, Jiang et al. 2013), we are unaware of any reports demonstrating the regulation of EGR3 mRNA abundance in granulosa cells. Interestingly, EGR3 but not EGR1 was shown to increase FASLG expression in T cells and fibroblasts (Mittelstadt & Ashwell 1998, Yoo & Lee 2004); therefore, the effect of DON on FASLG expression may be mediated in part through EGR3.
E2 is a major determinant of follicle development and decreases granulosa cell apoptosis in rodents in vivo (Billig et al. 1993). Studies with bovine granulosa cells have demonstrated that E2 can overcome the apoptotic effect of ligands such as FASLG and FGF18 (Quirk et al. 2006, Portela et al. 2015). Of the main RSR pathways, MAPK3/1 is known to alter CYP19A1 mRNA levels; inhibition of MAPK3/1 phosphorylation increased CYP19A1 mRNA levels in bovine and rodent granulosa cells (Moore et al. 2001, Silva et al. 2006) and reduced the ability of tumor necrosis factor alpha to increase apoptosis (Morales et al. 2006). Therefore, one mechanism for the action of DON might be via MAPK3/1 inhibition of CYP19A1 expression and E2 secretion, which then predisposed cells to apoptosis.
In conclusion, this study demonstrates that, in vitro, the mycotoxin DON has a negative impact on granulosa cell steroidogenesis and survival, and that the mechanism of action probably involves activation of the RSR. The potential impact of natural intoxication with DON on fertility in cattle warrants investigation.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Medi-Vet, Inc.
Acknowledgements
The authors thank Dr Raynald Dupras for criticism of the experimental design.
References
Abedini A, Zamberlam G, Boerboom D & Price CA 2015 Non-canonical WNT5A is a potential regulator of granulosa cell function in cattle. Molecular and Cellular Endocrinology 403 39–45. (doi:10.1016/j.mce.2015.01.017)
Alm H, Greising T, Brussow KP, Torner H & Tiemann U 2002 The influence of the mycotoxins deoxynivalenol and zearalenol on in vitro maturation of pig oocytes and in vitro culture of pig zygotes. Toxicology In Vitro 16 643–648. (doi:10.1016/S0887-2333(02)00059-0)
Bélanger A, Couture J, Caron S & Roy R 1990 Determination of nonconjugated and conjugated steroid levels in plasma and prostate after separation on C-18 columns. Annals of the New York Academy of Sciences 595 251–259. (doi:10.1111/j.1749-6632.1990.tb34299.x)
Billig H, Furuta I & Hsueh AJ 1993 Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133 2204–2212. (doi:10.1210/endo.133.5.8404672)
Cho HJ, Park S-M, Hwang EM, Baek KE, Kim I-K, Nam I-K, Im M-J, Park S-H, Bae S & Park J-Y et al. 2010 Gadd45b mediates Fas-induced apoptosis by enhancing the interaction between p38 and retinoblastoma tumor suppressor. Journal of Biological Chemistry 285 25500–25505. (doi:10.1074/jbc.M109.091413)
Cortinovis C, Pizzo F, Spicer LJ & Caloni F 2013 Fusarium mycotoxins: effects on reproductive function in domestic animals – a review. Theriogenology 80 557–564. (doi:10.1016/j.theriogenology.2013.06.018)
Espey LL, Ujioka T, Russell DL, Skelsey M, Vladu B, Robker RL, Okamura H & Richards JS 2000 Induction of early growth response protein-1 gene expression in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin. Endocrinology 141 2385–2391. (doi:10.1210/en.141.7.2385)
Evans AC & Martin F 2000 Kinase pathways in dominant and subordinate ovarian follicles during the first wave of follicular development in sheep. Animal Reproduction Science 64 221–231. (doi:10.1016/S0378-4320(00)00210-4)
Gutiérrez CG, Campbell BK & Webb R 1997 Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics. Biology of Reproduction 56 608–616. (doi:10.1095/biolreprod56.3.608)
Inoue A, Omoto Y, Yamaguchi Y, Kiyama R & Hayashi S 2004 Transcription factor EGR3 is involved in the estrogen-signaling pathway in breast cancer cells. Journal of Molecular Endocrinology 32 649–661. (doi:10.1677/jme.0.0320649)
Irving-Rodgers HF, van Wezel IL, Mussard ML, Kinder JE & Rodgers RJ 2001 Atresia revisited: two basic patterns of atresia of bovine antral follicles. Reproduction 122 761–775. (doi:10.1530/rep.0.1220761)
Jiang Z & Price C 2012 Differential actions of fibroblast growth factors on intracellular pathways and target gene expression in bovine ovarian granulosa cells. Reproduction 144 625–632. (doi:10.1530/REP-12-0199)
Jiang ZL, Ripamonte P, Buratini J, Portela VM & Price CA 2011 Fibroblast growth factor-2 regulation of Sprouty and NR4A genes in bovine ovarian granulosa cells. Journal of Cellular Physiology 226 1820–1827. (doi:10.1002/jcp.22509)
Jiang Z, Guerrero-Netro HM, Juengel JL & Price CA 2013 Divergence of intracellular signaling pathways and early response genes of two closely related fibroblast growth factors, FGF8 and FGF18, in bovine ovarian granulosa cells. Molecular and Cellular Endocrinology 375 97–105. (doi:10.1016/j.mce.2013.05.017)
Keese C, Meyer U, Valenta H, Schollenberger M, Starke A, Weber I-A, Rehage J, Breves G & Dänicke S 2008 No carry over of unmetabolised deoxynivalenol in milk of dairy cows fed high concentrate proportions. Molecular Nutrition & Food Research 52 1514–1529. (doi:10.1002/mnfr.200800077)
Kinser S, Jia Q, Li M, Laughter A, Cornwell PD, Corton JC & Pestka JJ 2004 Gene expression profiling in spleens of deoxynivalenol-exposed mice: immediate early genes as primary targets. Journal of Toxicology and Environmental Health. Part A 67 1423–1441. (doi:10.1080/15287390490483827)
Malekinejad H, Schoevers EJ, Daemen IJ, Zijlstra C, Colenbrander B, Fink-Gremmels J & Roelen BA 2007 Exposure of oocytes to the Fusarium toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal embryo development in pigs. Biology of Reproduction 77 840–847. (doi:10.1095/biolreprod.107.062711)
Marin S, Ramos AJ, Cano-Sancho G & Sanchis V 2013 Mycotoxins: occurrence, toxicology, and exposure assessment. Food and Chemical Toxicology 60 218–237. (doi:10.1016/j.fct.2013.07.047)
McNatty KP, Heath DA, Henderson KM, Lun S, Hurst PR, Ellis LM, Montgomery GW, Morrison L & Thurley DC 1984 Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary. Journal of Reproduction and Fertility 72 39–53. (doi:10.1530/jrf.0.0720039)
Medvedova M, Kolesarova A, Capcarova M, Labuda R, Sirotkin AV, Kovacik J & Bulla J 2011 The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro. Journal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes 46 213–219. (doi:10.1080/03601234.2011.540205)
Mihm M, Baker PJ, Fleming LM, Monteiro AM & O'Shaughnessy PJ 2008 Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes. Reproduction 135 253–265. (doi:10.1530/REP-06-0193)
Minervini F, Dell'Aquila ME, Maritato F, Minoia P & Visconti A 2001 Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17β-estradiol levels in mural granulosa cell cultures. Toxicology In Vitro 15 489–495. (doi:10.1016/S0887-2333(01)00068-6)
Mishra S, Tripathi A, Chaudhari BP, Dwivedi PD, Pandey HP & Das M 2014 Deoxynivalenol induced mouse skin cell proliferation and inflammation via MAPK pathway. Toxicology and Applied Pharmacology 279 186–197. (doi:10.1016/j.taap.2014.06.003)
Mittelstadt PR & Ashwell JD 1998 Cyclosporin A-sensitive transcription factor Egr-3 regulates Fas ligand expression. Molecular and Cellular Biology 18 3744–3751.
Moon Y & Pestka JJ 2002 Vomitoxin-induced cyclooxygenase-2 gene expression in macrophages mediated by activation of ERK and p38 but not JNK mitogen-activated protein kinases. Toxicological Sciences 69 373–382. (doi:10.1093/toxsci/69.2.373)
Moon Y, Yang H & Lee SH 2007 Modulation of early growth response gene 1 and interleukin-8 expression by ribotoxin deoxynivalenol (vomitoxin) via ERK1/2 in human epithelial intestine 407 cells. Biochemical and Biophysical Research Communications 362 256–262. (doi:10.1016/j.bbrc.2007.07.168)
Moore RK, Otsuka F & Shimasaki S 2001 Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells. Biochemical and Biophysical Research Communications 289 796–800. (doi:10.1006/bbrc.2001.6052)
Morales V, Gonzalez-Robayna I, Santana MP, Hernandez I & Fanjul LF 2006 Tumor necrosis factor-α activates transcription of inducible repressor form of 3′,5′-cyclic adenosine 5′-monophosphate-responsive element binding modulator and represses P450 aromatase and inhibin α-subunit expression in rat ovarian granulosa cells by a p44/42 mitogen-activated protein kinase-dependent mechanism. Endocrinology 147 5932–5939. (doi:10.1210/en.2006-0635)
Nielsen C, Lippke H, Didier A, Dietrich R & Märtlbauer E 2009 Potential of deoxynivalenol to induce transcription factors in human hepatoma cells. Molecular Nutrition & Food Research 53 479–491. (doi:10.1002/mnfr.200800475)
Pan X, Whitten DA, Wu M, Chan C, Wilkerson CG & Pestka JJ 2013 Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage. Toxicology and Applied Pharmacology 268 201–211. (doi:10.1016/j.taap.2013.01.007)
Pestka JJ 2008 Mechanisms of deoxynivalenol-induced gene expression and apoptosis. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment 25 1128–1140. (doi:10.1080/02652030802056626)
Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29 e45. (doi:10.1093/nar/29.9.e45)
Pizzo F, Caloni F, Schreiber N, Cortinovis C, Totty M & Spicer LJ 2014 The in vitro effects of Fusarium mycotoxins on bovine granulosa cell CYP11A1 and CYP19A1 mRNA. Toxicology Letters 229 (Supplement) S57. (doi:10.1016/j.toxlet.2014.06.232)
Portela VM, Machado M, Buratini J Jr, Zamberlam G, Amorim RL, Goncalves P & Price CA 2010 Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle. Biology of Reproduction 83 339–346. (doi:10.1095/biolreprod.110.084277)
Portela VM, Dirandeh E, Guerrero-Netro HM, Zamberlam G, Barreta MH, Goetten AF & Price CA 2015 The role of fibroblast growth factor-18 in follicular atresia in cattle. Biology of Reproduction 92 14 1–8 doi:10.1095/biolreprod.114.121376)
Porter DA, Vickers SL, Cowan RG, Huber SC & Quirk SM 2000 Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia. Biology of Reproduction 62 62–66. (doi:10.1095/biolreprod62.1.62)
Price CA, Carrière PD, Bhatia B & Groome NP 1995 Comparison of hormonal and histological changes during follicular growth, as measured by ultrasonography, in cattle. Journal of Reproduction and Fertility 103 63–68. (doi:10.1530/jrf.0.1030063)
Quirk SM, Cowan RG & Harman RM 2006 The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle. Journal of Endocrinology 189 441–453. (doi:10.1677/joe.1.06549)
Ranzenigo G, Caloni F, Cremonesi F, Aad PY & Spicer LJ 2008 Effects of Fusarium mycotoxins on steroid production by porcine granulosa cells. Animal Reproduction Science 107 115–130. (doi:10.1016/j.anireprosci.2007.06.023)
Rodrigues I & Naehrer K 2012 A three-year survey on the worldwide occurrence of mycotoxins in feedstuffs and feed. Toxins 4 663–675. (doi:10.3390/toxins4090663)
Russell DL, Doyle KM, Gonzales-Robayna I, Pipaon C & Richards JS 2003 Egr-1 induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone: combinatorial regulation by transcription factors cyclic adenosine 3′,5′-monophosphate regulatory element binding protein, serum response factor, SP1, and early growth response factor-1. Molecular Endocrinology 17 520–533. (doi:10.1210/me.2002-0066)
Sahmi M, Nicola ES, Silva JM & Price CA 2004 Expression of 17β- and 3β-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro. Molecular and Cellular Endocrinology 223 43–54. (doi:10.1016/j.mce.2004.05.010)
Salvador JM, Brown-Clay JD & Fornace AJ Jr 2013 Gadd45 in stress signaling, cell cycle control, and apoptosis. Advances in Experimental Medicine and Biology 793 1–19. (doi:10.1007/978-1-4614-8289-5_1)
Sanford LM 1987 Luteinizing hormone release in intact and castrate rams is altered with immunoneutralization of endogenous estradiol. Canadian Journal of Physiology and Pharmacology 65 1442–1447. (doi:10.1139/y87-226)
Sayasith K, Brown KA, Lussier JG, Doré M & Sirois J 2006 Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation. Journal of Molecular Endocrinology 37 239–250. (doi:10.1677/jme.1.02078)
Schoevers EJ, Fink-Gremmels J, Colenbrander B & Roelen BA 2010 Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle. Theriogenology 74 968–978. (doi:10.1016/j.theriogenology.2010.04.026)
Shin H, Kwon S, Song H & Lim HJ 2014 The transcription factor Egr3 is a putative component of the microtubule organizing center in mouse oocytes. PLoS ONE 9 e94708. (doi:10.1371/journal.pone.0094708)
Silva JM & Price CA 2000 Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro. Biology of Reproduction 62 186–191. (doi:10.1095/biolreprod62.1.186)
Silva JM, Hamel M, Sahmi M & Price CA 2006 Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. Reproduction 132 909–917. (doi:10.1530/REP-06-0058)
Uzbekova S, Salhab M, Perreau C, Mermillod P & Dupont J 2009 Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation. Reproduction 138 235–246. (doi:10.1530/REP-09-0136)
Yoo Y-G & Lee M-O 2004 Hepatitis B virus X protein induces expression of Fas ligand gene through enhancing transcriptional activity of early growth response factor. Journal of Biological Chemistry 279 36242–36249. (doi:10.1074/jbc.M401290200)
Zazzeroni F, Papa S, Algeciras-Schimnich A, Alvarez K, Melis T, Bubici C, Majewski N, Hay N, De Smaele E & Peter ME et al. 2003 Gadd45β mediates the protective effects of CD40 costimulation against Fas-induced apoptosis. Blood 102 3270–3279. (doi:10.1182/blood-2003-03-0689)
