The mycotoxins alternariol and alternariol methyl ether negatively affect progesterone synthesis in porcine granulosa cells in vitro.

Mycotoxins as contaminants of animal food can impair fertility in farm animals. In the regulation of female fertility the ovarian steroid hormone progesterone (P(4)) plays an important role. In the present study we have investigated the influence of the mycotoxins alternariol (AOH), alternariol mono-methyl ether (AME), and tenuazonic acid (TeA) on cell viability, P(4) synthesis, abundance of the key enzymes of P(4) synthesis, P450 cholesterol side-chain cleavage enzyme (P450SCC) and 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD), and of the corresponding Cyp11a1 and Hsd3b transcripts in cultured pig granulosa cells. Already 0.8 microM, AOH and AME inhibited P(4) secretion and 1.6 microM also significantly reduced cell viability. The abundance of P450scc protein but not of Cyp11a1 or Hsd3b transcripts was already significantly reduced by 0.8 microM AOH and AME. 1.6 microM AOH but not AME significantly reduced the abundance of alpha-tubulin and also clearly affected actin protein concentrations. TeA neither impaired viability nor P(4) secretion. Also mycotoxin extracts isolated from naturally occurring Alternaria strains by HPLC purification inhibited cell viability and P(4) synthesis, however at higher concentrations compared to AOH and AME. In conclusion, AOH and AME, but not TeA specifically inhibited P(4) secretion in cultured porcine granulosa cells. Alternaria toxin contaminated food may therefore affect reproductive performance in pig and other mammalian species.

Influence of alternariol (AOH) on regulator proteins of cap-dependent translation in porcine endometrial cells.

The molecular mechanisms that control the mycotoxin-mediated effects in porcine endometrial cells are far from being completely understood. Recent results show that they could inhibit cell proliferation. The present study investigated the effects of the mycotoxin alternariol (AOH) on the viability of porcine endometrial cells. In this context, the abundance and phosphorylation state (activity) of the eukaryotic initiation factor 4E (eIF4E) and its repressor protein 4E-BP1 (4E binding protein) were investigated. The results show that AOH has an influence on the viability of porcine endometrial cells. A significant reduction of cells in the S phase together with the arrest of the cells in the G(0)/G(1) phase after treatment with 12.5 microM AOH could be indicated. The cell number was also decreased by culturing the cells with 12.5 microM AOH. However, the metabolic activity of endometrial cells was already influenced by AOH concentrations of 3.12 microM. These data are in agreement with the detected dephosphorylation of 4E-BP1 and eIF4E and let assume that AOH effects gene expression on translational level. Furthermore, a binding of AOH to estrogen receptor (ER) or an influence on the abundance of the ER alpha could not be detected.

In vivo and in vitro effects of the organochlorine pesticides DDT, TCPM, methoxychlor, and lindane on the female reproductive tract of mammals: a review.

The present paper summarizes the toxicological data on the effects of the organochlorine pesticides DDT, its metabolites, TCPM, methoxychlor and lindane on folliculogenesis, ovulation, fertilization, and implantation of female reproductive organs in human, laboratory, and farm animals. These compounds possess the ability to disrupt endogenous hormone synthesis, storage or metabolism. Cells of the ovary, oviduct, and uterus are susceptible to the disruptive effects of organochlorine compounds (OCCs). This review discusses that the exposure to OCC causes an impairment of (1) female fertility by altering ovarian development and function and (2) implantation by altering endometrial function through their estrogenic activity. The main focus of this review is to provide an overview on data which support that assumption that OCC can substitute for estradiol in regulating the microanatomy of the female reproductive tract. The data indicate the potential of these compounds act as endocrine disrupting agents, but in a different extent.

Changes in the spleen and liver of pregnant sows and full-term piglets after feeding diets naturally contaminated with deoxynivalenol and zearalenone.

Wheat contaminated naturally with the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) was fed to pregnant Landrace sows for 35days. On day 110, caesarean section was carried out, the offspring were killed immediately after birth, and their livers and spleens examined. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of tissues from sows of the treated group revealed changes in liver and spleen tissues, whereas no significant changes were observed in these tissues in their piglets. Liver damage, as measured by prominent elevated transaminase activities, was not detected in the serum of the sows. In pregnant sows there were individual variations in sensitivity to the Fusarium toxins. In conclusion, it can be assumed that there are no adverse effects on the liver and spleen of full-term piglets when their mothers consumed diets containing up to 9570 and 358mug DON/ZON per kg diet.

In vitro exposure of porcine granulosa cells to the phytoestrogens genistein and daidzein: effects on the biosynthesis of reproductive steroid hormones.

Since a discrepancy concerning the effects of phytoestrogens on steroidogenesis exists in the literature we investigated the effects of genistein and daidzein on progesterone and estradiol synthesis in cultured primary granulosa cells derived from follicles of porcine ovaries. In this context, the investigation was performed to test the hypothesis that isoflavones can reduce hydroxysteroid dehydrogenase/isomerase (3beta-HSD) activity by down-regulation of its transcription. We found that daidzein did not impair the viability of cultured granulosa cells in the concentration range from 0.1 to 100 microM, but genistein inhibited the cell viability at 50 microM compared to the unexposed controls. Forskolin (10 microM) and pregnenolone (2.5 microM) enhanced the basal progesterone secretion in the absence of both phytoestrogens. Daidzein or genistein at non-toxic concentrations alone or combined with forskolin or pregnenolone significantly reduced progesterone synthesis. This reduction was not due to changes of the abundance of P450scc protein, but the gene hydroxysteroid dehydrogenase/isomerase (3beta-HSD) was significantly decreased at a non-toxic concentration of daidzein (50 microM) in non-stimulated and pregnenolone-stimulated cells. Moreover, genistein (1, 10 microM) significantly inhibited the 3beta-HSD-mRNA only in pregnenolone-stimulated granulosa cells. It can be suggested that the effect of genistein on steroidogenesis only partly results from the impairment of 3beta-HSD gene expression. In non-toxic concentrations daidzein and genistein did not change the androstenedione- or testosterone-stimulated estradiol-17beta synthesis. In summary, genistein and daidzein have direct effects on porcine granulosa cell progesterone synthesis which involve the inhibition of 3beta-HSD enzyme activity across the post-cyclic AMP pathway.

On the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from sows to their fetuses during days 35-70 of gestation.

Eleven pregnant sows with a body weight between 153 and 197 kg were fed a control diet (CON, 0.15 mg DON and 0.0035 mg ZON/kg diet) or a diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON/kg diet) in the period of day 35 and 70 of gestation. The indirect effect of feed intake was separated from the direct effects of the Fusarium toxins by the restricted feeding regimen where all sows were fed the same amount of feed (2000 g/d) over the whole study. At the end of experiment, fetuses were delivered by Caesarian section and samples of serum, bile, urine, liver, kidney and spleen of euthanatized sows and fetuses were taken to analyze the concentrations of DON, ZON and their metabolites. Feeding the Fusarium toxin contaminated diet to pregnant sows caused neither adverse effects on performance, organ weights and maintenance of pregnancy of sows nor on fetus weight and length. Furthermore, no teratogenic or embryolethal effects could be observed in the MYCO group. Hematological and clinical-chemical parameters of sows and fetuses were not affected by feeding, with the exception of significantly lower GLDH (glutamate dehydrogenase) serum activities in MYCO sows. The carry over of DON and ZON from the diet to the sow or fetus tissues was calculated by the diet ratio (sum of concentrations of all metabolites in the physiological specimen divided by the dietary toxin concentration), while the fetus ratio was evaluated by the sum of concentrations of all metabolites in the physiological specimen of the fetus divided by that of the sows. DON and deepoxy-DON were found in urine, bile, serum, liver, kidney and spleen of sows of the MYCO group, but not in the bile of fetuses (spleen not analyzed). ZON and its metabolite alpha-zearalenol (alpha-ZOL) were detected in urine and bile of sows, while all specimens of fetuses as well as serum and liver of sows were negative for ZON metabolites. The maximum diet ratios for urine and bile in sows of the MYCO group were 0.84 and 0.05 for DON metabolites and 1.2 and 3.8 for ZON metabolites, underscoring the differences in metabolism and excretion of both toxins. The maximum diet ratio of DON and deepoxy-DON into liver, kidney and spleen of MYCO sows were 0.003, 0.007 and 0.003, respectively. The maximum fetus ratio of DON and deepoxy-DON into urine, bile, serum, liver and kidney of fetuses were 0.006, 0, 0.5, 0.88, and 0.33, while the maximum placental ratio (sum of toxin concentrations in the physiological specimen of the fetus divided by the toxin serum concentration of the sow) were 0.64, 0, 0.50, 0.70 and 0.52, respectively. Therefore, it can be concluded that the developing fetus is exposed to DON between the gestation days 35 and 70 when the sows are fed a Fusarium toxin contaminated diet. ZON concentration in the MYCO diet was too low to get reliable results for fetus or placental ratios.

The embryonic pregnancy signal oestradiol influences gene expression at the level of translational initiation in porcine endometrial cells.

In the pig, conceptus-derived oestrogens (days 11 and 12 of pregnancy) seem to be a critical component of the signalling mechanism for maternal recognition of pregnancy. Embryonic oestrogens can mediate effects on endometrial function by interactions with epithelial and stromal oestrogen receptors (ER). Recent data demonstrate that cell membrane ER interacts with the phosphatidylinositol 3-kinase/Akt pathway in several types of cells. The protein kinase Akt is involved in the control of cell growth, survival and proliferation. One distinct function of the Akt signalling cascade is its ability to phosphorylate the eukaryotic initiation factor-4E (eIF-4E)-binding protein 1 (4E-BP1). This phosphorylation suppresses the inhibitory effect of 4E-BP1 on the translation initiation factor eIF4E and in such a way potentially stimulates gene expression at the level of translational initiation. The aim of the present study was to examine if embryonic oestradiol (E(2)) transmits its effect by such a mechanism. Endometrial cells of cyclic gilts (day 13 of the oestrous cycle, n = 4) were cultured and supplemented with vehicle (control), E(2) (50 and 100 pm/l) or with the selective ER modulator raloxifen (10 and 1000 nm/l), and incubated for 24 h. The cell viability was detected by MTT assay, the abundance and phosphorylation of Akt, 4E-BP1 and ERalpha was analysed by Western blotting. Incubation with E(2) or raloxifen did not alter endometrial cell viability. The phosphorylation of Akt at Ser(473) seems to be increased by E(2) (p < 0.05) and decreased by raloxifen (p > 0.05). Raloxifen (1000 nm/l) induced a band shift in 4E-BP1 to the highest electrophoretic mobility which reflects a decrease in phosphorylation (p < 0.05), whereas an influence of E(2) on 4E-BP1 phosphorylation could not be detected. The decrease (p < 0.05) of the abundance of the 80 kDa ERalpha form both by E(2) and raloxifen indicates that the E(2)-stimulated Akt phosphorylation and the inhibition of 4E-BP1 phosphorylation by raloxifen is an E(2) ER-transmitted process. Therefore, embryonic oestrogens can potentially transmit their effect by influencing signalling cascades which modulate gene expression at the level of translational initiation.

Influence of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected enzymatic and histological parameters of liver in gilts.

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of enzymatic and histopathological effects of Fusarium toxins on porcine liver cells. A total of 36 prepuberal gilts were divided into four groups and fed diets with increasing proportions of Fusarium toxin-contaminated wheat at a total wheat proportion of 40% over a period of 35 days. The concentrations of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON) which were analyzed by HPLC methods were 210/4, 3070/88, 6100/235, and 9570/358 microg/kg in the diets fed to groups I-IV, respectively. The feeding of mycotoxin-contaminated diets did not cause gross pathological findings in the livers of the animals. Liver tissues were subjected to enzymatic, histological, and ultrastructural examinations. The percentages of the stained areas in periodic acid-Schiff (PAS), Berlin-Blue, and Masson Goldner's trichrome stainings were calculated using the AnalySIS 3.4-system. Significant histopathological findings of alterations with varying degrees in glycogen reduction and increase of hemosiderin particles were found in the liver cells of groups II, III and IV. The thickness of interlobular connective tissue septum in liver cells was significantly increased in groups III and IV. Qualitative ultrastructural alterations were observed in hepatocytes of gilts in groups III and IV. Dependent upon the mycotoxin concentration in the diet, the hepatocytes developed a dose-dependent, extensive, smooth endoplasmic reticulum, exhibited loss of ribosomes, and acquired an increased number of fatty and autophagic vacuoles. However, liver damage as measured by prominent elevated transaminase activities in serum was not detected. Together, the histopathological results provide evidence of liver dysfunction in the absence of clinical signs, especially in pigs fed higher concentrations of Fusarium toxin-contaminated wheat.

In vitro and in vivo effects of deoxynivalenol (DNV) on regulators of cap dependent translation control in porcine endometrium.

Deoxynivalenol (DNV) is the most frequently encountered trichothecene in grain-based foods, and is able to produce toxic effects resulting in various diseases in farm and laboratory animals. The molecular mechanisms that control this mycotoxin mediated effects in porcine endometrial cells are far from being completely understood. Recent results show that DNV inhibits protein synthesis in actively proliferating tissues. Therefore, the present study investigated the effects of this mycotoxin on a cellular level in an in vivo and in vitro system. The abundance and phosphorylation state (activity) of the cell cycle dependent kinases MAPk and Akt (PKB) and their potential targets eIF-4E (eukaryotic initiation factor 4E) and 4E-BP1 (4E binding protein, eIF4E repressor protein) were examined. In previous investigations it was found that these factors are involved in initiation of mRNA translation. The results show that DNV in vitro strongly reduce the abundance of p38 MAPk, protein kinase Akt and the alpha- and beta-4E-BP1 bands. The phosphorylation state of these proteins was obviously not modulated. In contrast, the eIF4E phosphorylation was strongly reduced in DNV treated cells. In summary, our in vitro results let assume that DNV potentially influences gene expression, but this work does not present a direct proof that DNV alters processes, which are involved in the initiation of mRNA translation. Surprisingly in vivo, an influence of DNV feeding on the investigated molecular events could not be demonstrated.

Influence of ovarian steroid hormones or platelet-activating factor on mRNA of platelet-activating factor receptor in endometrial explant perfusion cultures from ovariectomized bovine.

Platelet-activating factor (PAF) and its receptors are involved in inflammatory-like processes of the uterus associated with increased vascular permeability. PAF is supposed to be influenced by ovarian steroid hormones. The present study was undertaken to examine whether progesterone (P(4)), estradiol (E(2)) or PAF influence the PAF receptor gene expression in perfused endometrial explants derived from ovariectomized bovine. Furthermore, we identified the cell types in which the PAF receptor gene and protein are expressed. In endometrial explants, applications of 10 nM P(4) or 10nM P(4) plus 10 nM E(2) for 24 h induced elevated transcript levels of PAF receptor in comparison to the controls or after treatment with 1 nM E(2). When explants were administered 10 nM E(2), a slight decrease in the transcript level was recorded. After treatment of explants with PAF, no significant changes in PAF receptor mRNA expression was observed compared to the control group. We demonstrate that PAF receptor immunoreactivity and mRNA are detected mainly in the luminal epithelium, epithelial cells of the superficial glands and to a lesser degree in stroma. Levels of PAF receptor mRNA in bovine endometrial explants were correlated with PAF receptor protein localization assessed by immunohistochemistry. The regulation of PAF receptor by progesterone in bovine endometrial explants suggests that PAF is involved in the physiological process of reproduction.

Influence of the mycotoxins alpha- and beta-zearalenol (ZOL) on regulators of cap-dependent translation control in pig endometrial cells.

The molecular mechanisms that control the mycotoxin-mediated effects in porcine endometrial cells are far from being completely understood. Recent results show that they could inhibit cell proliferation. Therefore, the present study investigated the effects of the mycotoxins alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) on a cellular level. Mainly, the abundance and phosphorylation state (activity) of the cell cycle-dependent kinases MAPK and Akt (PKB) and their potential targets eIF4E (eukaryotic initiation factor 4E) and 4E-BP1 (4E binding protein, eIF4E repressor protein) were investigated. The results show that alpha-ZOL has apparently only a slight influence on the phosphorylation state of MAP kinases, Akt and on eIF4E and 4E-BP1. In contrast, their phosphorylation was strongly reduced in beta-ZOL-treated cells in a concentration-dependent manner. Therefore, our results indicate that beta-ZOL potentially not only influences transcription but also effects gene expression on translational level. The effect of alpha- and beta-ZOL on endometrial cell proliferation and their toxicology are discussed.

Effects of the mycotoxins alpha- and beta-zearalenol on regulation of progesterone synthesis in cultured granulosa cells from porcine ovaries.

Mycotoxins as contaminants of animal food can impair fertility and can cause abnormal fetal development in farm animals. Therefore, the present study has investigated whether derivatives of the mycotoxin zearalenone, alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL), influence progesterone synthesis via cytochrome p450 side chain cleavage enzyme (p450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) in cultured porcine granulosa cells. Both enzymes are essential for the conversion of cholesterol to progesterone. No differences in basal progesterone levels and numbers of viable cell were observed between untreated granulosa cells and those treated with alpha- or beta-ZOL (15 and 30 microM). FSH (0.01 microg/ml) or forskolin (10 microM) enhanced the basal progesterone secretion in the absence of mycotoxins. The addition of alpha- or beta-ZOL (7.5, 15 and 30 microM) to cultures stimulated with FSH (0.01 microg) or forskolin (10 microM) reduced progesterone synthesis and the levels of p450scc and 3beta-HSD transcripts in a dose-dependent manner (P<0.05). The enzymatic activity of 3beta-HSD and the abundance of p450scc protein were also reduced by these mycotoxins. In conclusion, effects of mycotoxins on FSH receptor-dependent and receptor-independent pathways indicate that adenylate cyclase activity and/or regulatory pathways further downstream are targets of mycotoxin actions. The apparent dose-dependent reduction of p450scc and 3beta-HSD transcripts implies an effect of alpha- and beta-ZOL on transcriptional regulation of these enzymes.

Forskolin-induced cyclic AMP signaling in single adherent bovine oviductal cells: effect of dichlorodiphenyltrichloroethane (DDT) and tris(4-chlorophenyl)methanol (TCPM).

The influence of tris(4-chlorophenyl)methanol (TCPM) and dichlorodiphenyltrichloroethane (o,p'DDT) on forskolin induced cAMP signalling in single adherent bovine oviductal cells was investigated. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520/580 nm fluorescence emission ratio of the protein kinase A fluorosensor (FICRhR). FICRhR was microinjected into single cells, and the 520/580 nm fluorescence emission ratio was monitored by image cytometry with an image analysis system as a measure of intracellular cAMP concentration ([cAMP](i)). Applications of dibutyryl cAMP and forskolin caused time- and dose-dependent effects on [cAMP](i) in single oviductal cells. The addition of 16 or 32 microM TCPM or DDT for 1 h to the culture medium decreased the intracellular cAMP concentration significantly, whereas 8 microM was not able to influence the [cAMP](i). In the presence of both pesticides at 16 microM the forskolin (30 microM)-induced [cAMP](i) was significantly reduced after 1 h of incubation. It is suggested that TCPM can have the same influence compared with DDT on cells responsible for reproduction.

Influence of the mycotoxins alpha- and beta-zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells.

The present study investigated the effects of the mycotoxins alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) at concentrations of 7.5, 15, and 30 microM, and deoxynivalenol (DON) at concentrations of 0.94, 1.88, and 3.76 microM on cell cycle distribution (propidium iodide, PI staining) in combination with the proliferating cell nuclear antigen (PCNA) by flow cytometry. The viability of porcine uterine cells was not impaired at 30 microM alpha-ZOL, whereas beta-ZOL at this concentration and 3.76 microM DON significantly decreased cell number. Some cells showed ultrastructural features of cell death indicated by swollen mitochondria, disrupted cell membranes, and many vacuoles. After 24 and 48h of exposure to alpha-ZOL (7.5, 15, or 30 microM), the cell cycle distribution was still comparable to the control groups. An anti-proliferative effect of beta-ZOL and DON was detected by a significant reduction in the S-phase together with arrest of cells in the G(0)/G(1)-phase. The results show that beta-ZOL (7.5, 15, or 30 microM) and DON (0.94, 1.88, or 3.76 microM) control the progression of cells through the cycle by decreasing S-phase and arresting cells in the G(0)/G(1)-phase of the cell cycle. A significant decrease in the expression of the proliferation marker PCNA amounts indicates that beta-ZOL and DON disengaged cells from active cycling. We confirm that alpha-ZOL possesses a relative binding affinity to porcine uterine cytoplasmic estrogen receptor.

Regulation of the VEGF-system in the endometrium during steroid-replacement and early pregnancy of pigs.

Vascular endothelial growth factor (VEGF) and its specific receptors FLT-1 and FLK-1 represent an important ligand-receptor system involved in angiogenesis and permeability. These factors are supposed to be influenced by ovarian steroids involved in developmental changes in female reproductive tissue as oviduct and uterus. The aims of this study were to assess the expression of VEGF and its receptor mRNAs during the early implantation period in porcine endometrium using real-time RT-PCR. Furthermore, effects of estradiolbenzoate (EB) and progesterone (P) on endometrium of ovariectomized (ovx) pigs were examined by RT-PCR and immunohistochemistry. A complete VEGF system was found in endometrial tissue using RT-PCR detecting the main VEGF isoform 188 aa, FLT-1 and FLK-1. A significant upregulation of the mRNAs of VEGF and its receptors was observed in the endometrium during the peri-implantation when compared with the pre-implantation period. Regarding endometrium of non-pregnant ovx-pigs an application of P led to elevated transcript levels of VEGF whereas mRNA-expression was reduced after EB treatment compared to non-treated ovx-animals. When pigs were administrated EB and P simultanously, a decrease in VEGF mRNA concentration was recorded. For FLT-1, none of the steroids increased mRNA expression compared to the ovx-group. Analysis of FLK-1 receptor mRNA demonstrated that only after EB + P treatment mRNA-expression was stimulated but stayed unchanged after P and EB when compared with the ovx-group. Immunohistochemistry revealed FLK-1 and VEGF proteins in glandular and luminal epithelia of the endometrium with emphasized staining after P and P + EB treatment of ovx-pigs. Summarized, altered VEGF and FLK-1 expression during the implantation period as well as under steroid hormones suggest this growth factor as a potent regulator of hyperpermeability supporting the angiogenic process in porcine endometrium.

Effects of ovarian steroids and epidermal growth factor (EGF) on expression and bioactivation of specific regulators of transcription and translation in oviductal tissue in pigs.

The epidermal growth factor receptor (EGF receptor) system is involved in regulation of proliferation and differentiation in oviductal and endometrial tissues. In this study the influence of ovarian steroids and EGF on the expression and activity of specific markers of transcription (mitogen-activated protein kinase; MAP42k) and translation (a potential repressor of eukaryotic initiation factor 4E; 4E-BP1) in pig oviducts was investigated. Furthermore, determination of the distribution of translationally active (polysomal) and repressed (free) mRNA, and cell cycle analysis were performed. Oviductal tissue collected at two points of the oestrous cycle (days 12 and 20) from gilts and tissues from ovariectomized gilts with or without steroid replacement treatment were analysed. The influence of EGF was detected by culture of oviductal explants. MAP42k activity was stimulated by oestrogen treatment, whereas progesterone treatment appeared to decrease its activity. High oestrogen but not high progesterone concentrations resulted in reduced mobility of 4E-BP1 on polyacrylamide gels, indicating its inactivation. EGF and oestrogen treatment of oviductal explants further reduced the mobility of 4E-BP1 on polyacrylamide gels. High concentrations of oestrogen in the plasma promoted cell cycle activity. Progesterone treatment alone did not stimulate the rate of DNA synthesis. There were no significant differences in the distribution of free oviductal poly (A+) mRNA, but the amount of polysomal mRNA was downregulated by oestrogen and progesterone. Increased oestrogen concentrations are involved in the regulation of MAP42k and 4E-BP1 activation in the oviductal tissue of pigs. The effect of oestrogen and EGF in reducing the mobility of 4E-BP1 on gels in oviductal explants indicates that EGF may mediate the effect of oestradiol in the oviducts.

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system. A significant increase (P < 0.05) in the portion of PAF-R-positive oviductal epithelial and stromal cells was detected in both non-pregnant and pregnant cattle on days 2 to 4 in comparison to day 12 and 20. In endometrial stromal cells derived from day 20 pregnant bovine, a significant increase (P < 0.05) in PAF-R staining was observed in comparison to the day 20 non-pregnant and days 2 to 4 or 12 pregnant and non-pregnant animals. The PAF-R was detected in oviductal cells by using immunoblotting and immuno-gold postembedding method. Positive binding of the anti-PAF-R antibody was found on the cell membrane and in the cytoplasm. We concluded that the increased PAF-R concentration measured in cultured oviductal epithelial and stromal cells of cyclic and pregnant heifers on days 2 to 4 was hormonally regulated. The increased PAF-R in endometrial stromal cells on day 20 of pregnant heifers was a pregnancy-specific effect and may mediate a local increase in endometrial vascular permeability known to precede the implantation.

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy.

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.

Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells.

The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and gamma-hexachlorocyclohexane (gammaHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37 degrees C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 microM DDT, MXC, or gammaHCH compared with nonexposed controls. A significant inhibition began at 32 microM DDT, MXC, or gammaHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 microM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 microM DDT, 16 microM MXC, and 32 microM gammaHCH. The 128 microM concentration of the pesticides was toxic. At pesticide concentration of 64 microM, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 microM DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or gammaHCH was not associated with a detectable increase in [Ca2+]i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 microM DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

Influence of organochlorine pesticides on ATPase activities of microsomal fractions of bovine oviductal and endometrial cells.

In the present study the effects of organochlorine pesticides: o,p'-DDT, p,p''-DDT, methoxychlor, and lindane on ATPase activities of microsomal fractions of bovine oviductal and endometrial cells were investigated. We were not able to characterize a Ca2+ stimulated ATPase, whereas 57 and 15% of the total ATPase activity were sensitive to Mg2+ in the oviductal and endometrial fractions, respectively. After 10 min preincubation with the four organochlorines, a significant inhibition was found only with o,p''-DDT at 32 microM (27.9%) and 64 microM (35.6%) in the oviductal microsomal fraction and at 64 microM (32.2%) in that of the endometrium. Increasing the preincubation time to 30 min, the Mg2+ ATPase in the endometrial fraction was significantly inhibited by all four pesticides at 64 microM, but in the oviductal fraction only at 64 microM o,p''-DDT. It is suggested that organochlorine pesticides can have an influence on cells responsible for reproduction.