Incidence of apoptosis and transcript abundance in bovine follicular cells is associated with the quality of the enclosed oocyte.

The close contact and interaction between the oocyte and the follicular environment influence the establishment of oocyte developmental competence. Moreover, it is assumed that apoptosis in the follicular cells has a beneficial influence on the developmental competence of oocytes. The aim of this study was to investigate whether bovine oocytes with varied developmental competence show differences in the degree of apoptosis and gene expression pattern in their surrounding follicular cells (cumulus and granulosa cells). Oocytes and follicular cells from follicles of 3 to 5 mm in diameter were grouped as brilliant cresyl blue (BCB)+ and BCB- based on glucose-6-phosphate dehydrogenase (G6PDH) activity in the ooplasm by BCB staining. In the follicular cells initial, early and late apoptotic events were assessed by analyzing caspase-3 activity, annexin-V and TUNEL, respectively. Global gene expression was investigated in immature oocytes and corresponding follicular cells. BCB+ oocytes resulted in a higher blastocyst rate (19.3%) compared to the BCB- group (7.4%, P < 0.05). Moreover, the analysis of apoptosis showed a higher caspase-3 activity in the follicular cells and an increased degree of late apoptotic events in granulosa cells in the BCB+ compared with the BCB- group. Additionally, the global gene expression profile revealed a total of 34 and 37 differentially expressed genes between BCB+ and BCB- cumulus cells and granulosa cells, respectively, whereas 207 genes showed an altered transcript abundance between BCB+ and BCB- oocytes. Among these, EIF3F, RARRES2, RNF34, ACTA1, GSTA1, EIF3A, VIM and CS gene transcripts were most highly enriched in the BCB+ oocytes, whereas OLFM1, LINGO1, ALDH1A3, PTHLH, BTN3A3, MRPS2 and PPM1K were most significantly reduced in these cells. Therefore, the follicular cells enclosing developmentally competent oocytes show a higher level of apoptosis and a different pattern of gene expression compared to follicular cells enclosing non-competent bovine oocytes.

Truncation of the mRNA cap-binding protein eIF4E is specific for the non-invasive implantation in pigs.

The mechanism of implantation is species specific (pig: epitheliochorial, bovine: synepitheliochorial, mouse: hemochorial). Recently, we have shown that proteolytical cleavage of the prototypical 25 kDa mRNA cap-binding protein eIF4E (eukaryotic initiation factor 4E) produces a stable variant with a molecular mass of approximately 23 kDa in porcine endometrium at the time of implantation. Here, we investigate if an eIF4E truncation also takes place in the endometrium of species with other implantation forms. Thus, eIF4E and its repressor protein 4E-BP1 were investigated in porcine, murine and bovine endometrium during the time of implantation. Our results show that eIF4E truncation is specific for the porcine implantation. In bovine and mouse uterine tissue, no cleavage of eIF4E was observed. Whereas no difference of bovine 4E-BP1 was found, in murine samples, increased phosphorylation during implantation was observed. However, porcine samples exhibit an opposite behaviour, the abundance and mainly the phosphorylation of 4E-BP1 decrease. We propose that the translation initiation in the endometrium is differently regulated by the two eIF4E forms with regard to different 4E-BP1 abundance and phosphorylation as well as different eIF4E/4E-BP1 binding dynamic depending on the type of implantation.

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.

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer.

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.

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.

A milk diet partly containing soy protein does not change growth but regulates jejunal proteins in young goats.

Soy protein is known to alter intestinal function and structure. We determined in young goats whether a diet partly containing soy protein differently affects intestinal morphology and the jejunal and hepatic proteome as compared with a milk diet. Fourteen male 2-wk-old White German dairy goat kids were fed comparable diets based on whole cow's milk in which 35% of the crude protein was casein (milk protein group; MP) or soy protein supplemented by indispensable AA (SPAA) for 34 d (n = 7/group). Body weight gain and food efficiency were not different. Jejunal and hepatic tissue was collected to determine intestinal morphology by microscopy and protein repertoire by 2-dimensional gel electrophoresis and mass spectrometry. Jejunal crypt depth was reduced and villus height to crypt depth ratio was higher in SPAA than in milk protein. Out of 131 proteins identified, 32 proteins were found to be differently expressed in both groups. In SPAA, down-regulated jejunal proteins were involved in processes related to cytoskeleton generation, protein, lipid, and energy metabolism. Downregulated hepatic proteins were related to glycolysis and Krebs cycle. Thirteen proteins were upregulated in SPAA. Among these, 2 hepatic proteins were related to carbohydrate breakdown. The other 11 jejunal proteins were involved in cytoskeleton assembly, proteolysis, and carbohydrate breakdown. In addition, glutathione-S-transferase was found to be upregulated in the medial jejunum. In conclusion, a SPAA diet as compared with a milk diet was related to changes in jejunal morphology and jejunal proteins relevant for protein turnover, energy metabolism, and cytoskeleton assembly with no apparent impact on animal BW gain.

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.

Comparison of the molecular effects of the mycotoxins beta-zearalenol and deoxynivalenol in porcine endometrial cells--a review.

The mycotoxins beta-zearalenol (beta-ZOL) and deoxynivalenol (DON) produce toxic effects that result in diseases in humans and animals. The molecular mechanisms that control the mycotoxin-mediated effects are far from being completely understood. Various results show that these mycotoxins could inhibit cell proliferation. In the present short communication, the influence of beta-ZOL and DON on the abundance and phosphorylation state of kinases that are included in regulation of the initiation of mRNA translation (which is correlated with cell proliferation) was compared in porcine endometrial cells (PEC). Our results indicate that these mycotoxins modulate the expression and phosphorylation of these factors in a different manner. Whereas beta-ZOL mainly had an impact on the biological activity of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E (eIF4E) and its repressor 4E binding protein 1 (4E-BP1), DON reduced the abundance of p38 MAPk, Akt and specific 4E-BP1 bands. In summary, these results indicate that beta-ZOL influences molecular events that are included in the initiation of mRNA translation in the porcine endometrium but DON does not alter such processes clearly.

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer.

Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.

Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of gilt oocytes.

Mycotoxins are contaminants of animal feed that can impair fertility and cause abnormal fetal development in farm animals. The aim of the present study was to investigate the influence of Fusarium-toxin contaminated feed on cumulus morphology and maturation of pig oocytes. Naturally with the Fusarium-toxins deoxynivalenol (DON) and zearalenone (ZON) contaminated wheat was included in feed for gilts at increasing proportions which resulted in increasing dietary concentrations of both toxins (in mgtoxin/kg feed: Group 1 (control), 0.21 and 0.004; Group 2, 3.07 and 0.088; Group 3, 6.1 and 0.235; Group 4, 9.57 and 0.358, for DON and ZON, respectively). Oocytes were recovered from gilt ovaries by follicle aspiration after ovario-hysterectomy. Granulosa cells were analyzed for the expression of the P450(SCC) and 3beta-HSD mRNA by RT-PCR and additionally for P450(SCC) protein by Western blotting. Neither the expression of the P450(SCC) nor of the 3beta-HSD mRNA or the abundance of the P450(SCC) protein was significantly influenced by the mycotoxin application. The distribution of different cumulus cell morphologies was not influenced by group. At the time of recovery, oocytes with compact cumuli in Groups 3 and 4 showed a reduced proportion having immature chromatin in comparison to that for Groups 1 and 2. The proportion of oocytes having degenerated meiotic chromatin was significantly higher in Group 4 than in the other groups. The proportion of oocytes reaching metaphase II in culture was significantly lower in Groups 3 and 4 than in Group 1, and tended to be lower in Group 2 than in Group 1. We conclude that oocyte quality is significantly reduced by feeding of Fusarium-toxins to gilts.

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.

Comparative analysis of protein synthesis, transcription and cytoplasmic polyadenylation of mRNA during maturation of bovine oocytes in vitro.

In many species, large numbers of macromolecules are accumulated during oocyte growth. The messenger and ribosomal RNAs produced in these cells are far in excess of those necessary to support protein synthesis. Thus, the aim of this study was to elucidate the processes of translational regulation during meiotic maturation. The relationship between transcription, translation and polyadenylation of mRNA during in-vitro maturation (IVM) of bovine oocytes was investigated. The results presented here show that overall protein synthesis is stimulated during meiotic maturation (approximately three times) concomitantly with the onset of germinal vesicle breakdown after 6 to 10 h of IVM. However, in metaphase II, the incorporation of [35S]methionine into proteins showed only basal levels, as in the germinal vesicle (GV) stage. Furthermore, in the course of IVM, de-novo transcription strongly declines as determined by measuring the incorporation of [3H]uridine into RNA. In contrast to this finding, the incorporation of [3H]adenosine increased and showed a peak during the time interval from 6 to 10 h of IVM, parallel with the onset of germinal vesicle breakdown (GVBD) and translation. In the further course of maturation, only a moderate decrease of [3H]adenosine incorporation was observed. These results indicate that (i) translation increased at the time of GVBD; (ii) these processes were accompanied by polyadenylation of mRNA; and (iii) although transcription declines, polyadenylated mRNA is accumulated until metaphase II (as shown by poly(U)-hybridization), in which protein synthesis is low. The correlation of these processes is discussed here. A detailed knowledge of the biochemical and molecular processes which occur during oocyte maturation can be useful for the improvement of IVM conditions.

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.

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.

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.

Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size.

Oocyte developmental competence depends on the size of the original follicle and is affected by compounds like prolactin. We wished to investigate nuclear and cytoplasmic maturation of bovine oocytes correlated to their origin and response to prolactin treatment, by monitoring at frequent intervals meiotic configuration of chromosomes and activity of histone H1 and MAP-kinase. Bovine ovaries were obtained from a slaughterhouse and oocytes were recovered by follicle isolation. Oocytes (n = 1,397) with a compact cumulus were selected from small (2 to 3 mm) and large (4 to 5 mm in diameter) follicles and cultured up to 28 h in TCM 199+20% bull serum with or without 50 ng/mL bovine prolactin. Four groups of oocytes were formed: originating from small or large follicles, and treated or not treated with prolactin. At the scheduled time intervals for in vitro maturation, cumulus oocyte complexes from the 4 groups were randomly selected and the oocytes were analyzed for histone H1 and MAP-kinase, and for chromatin configuration. The first meiotic division took longer to complete in oocytes from large follicles (P < 0.01). Under the influence of prolactin the meiosis was prolonged in oocytes both from small and large follicles (P < 0.05). Histone H1 and MAP-kinases started to be activated at approximately the same time, around 6 h after beginning maturation. But after this time, significantly lower levels of both kinase activities were found in oocytes treated with prolactin, especially those treated during Meiosis I (P < 0.05). Our results indicate a correlation of chromatin configuration and histone H1/MAP-kinase activities.

Ultrastructure, protein phosphorylation and mRNA status of equine oocytes matured in vivo and in vitro.

Equine oocytes were collected by follicle aspiration in vivo or by dissection of material obtained from an abattoir, and the ultrastructure, protein phosphorylation and mRNA status of the oocytes were evaluated. Electron microscopy studies indicated that the nucleus had a smooth membrane in oocytes with a compact cumulus, whereas the nuclear membrane was undulated in all other groups. Oocytes with compact cumuli had only a few microvilli, whereas those with expanded cumuli had more microvilli. There were only small numbers of cortical granules close to the oolemma in oocytes with compact cumuli and clusters of mitochondria were in the peripheral ooplasm. The number of mitochondria and cortical granules increased in oocytes with expanded cumuli and the Golgi complexes were smaller than in other oocytes. Oocytes were observed at 10, 20 and 30 h of in vitro maturation. During maturation, the mitochondria migrated centrally and the number of cortical granules immediately below the oolemma increased progressively. Membrane-bound smooth endoplasmic reticulum became progressively less predominant. Phosphorylated proteins of molecular mass ranging from 20 to 150 kDa were found in oocytes and cumulus cells. The pattern of phosphorylated proteins was different in oocytes developed in vivo compared with oocytes cultured for 16 and 32 h in vitro. Cells of different cumulus types did not have distinct bands of phosphorylated proteins. Oocytes with compact cumuli had mainly repressed mRNAs, whereas the translationally active form was found in oocytes with expanded cumuli.

Glycosylation and deglycosylation of proteasomes (prosomes) from calf-liver cells: high abundance of neuraminic acid.

Proteasomes (prosomes) of calf-liver cells were probed with three different biotinylated lectins: Limulus polyphemus agglutinin (LPA), specific for neuraminic acid; Solanum tuberosum agglutinin (STA), specific for GlcNac; and concanavalin A (Con A), specific for Man/Glc. While only one proteasomal protein reacted with STA, most of the proteasomal proteins reacted with LPA and several with Con A. Deglycosylation with N-glycosidase F showed that the detected glycan residues were asparagine-linked. Finally we demonstrate an alternative method for the isolation of proteasomes based on the affinity of certain proteasomal proteins to Con A.

Large-scale purification of prosomes from calf's liver.

Prosomes, cytoplasmatic ribonucleoprotein complexes containing small ribonucleic acid (19S small cytoplasmic RNPs), are ubiquitous in eukaryotic organisms. A new method for the preparation of prosomes in large amounts, starting with ca. 2 kg of calf's liver, is described. A combination of centrifugation and low- and high-pressure chromatography was used to purify intact particles. An alternative purification of prosomes with Solanum tuberosum agglutinin bound to divinyl sulphone-activated agarose is discussed. Calf's liver prosomes have a similar protein composition and RNA content to prosomes isolated from other tissues.