Association between progesterone and estradiol-17beta treatment and protein expression of pgr and PGRMC1 in porcine luminal epithelial cells: a real-time cell proliferation approach.

The correct functionality (sensitivity and receptivity) of endometrial tissue is regulated by paracrine and endocrine pathways that activate several mediators or metabolic pathways and gene cascades. This study aimed to investigate the influence of E2 and P4 on progesterone receptor (PGR) and progesterone receptor membrane component 1 (PGRMC1) protein expression in porcine luminal epithelial cells and their influence on the proliferation of these cells in real-time. Surface uterine luminal epithelial cells were removed using sterile surgical blades from uterine horns of ten crossbred anestrus gilts. Following treatment with collagenase I, cells were separated and transferred into 48-well E-Plates for use in a realtime cell analyzer (RTCA). The luminal epithelial cells were cultured in vitro (IVC) in standard DMEM cell culture medium and incubated with E2 (10 pg/ml, 40 pg/ml, 500 pg/ml) and P4 (10 ng/ml, 40 ng/ ml, 500 ng/ml). The cell proliferation index was analyzed after 0-240 h, 0-120 h, 120-240 h. After using the RTCA analysis we found increased proliferation of luminal epithelial cells after treatment of low doses of P4 (10 and 40 ng/ml), (P < 0.001). Higher doses of P4 led to decrease of proliferation (P < 0.001). Conversely, higher doses of E2 (500 pg/ml) increased the proliferation index as compared to low doses (10 pg/ml) and control (P < 0.001). Confocal microscopic observations revealed that higher concentrations of E2 upregulate the expression of both PGR and PGRMC1. Additionally, P4 used in lower concentrations stimulated the expression of these receptors, too. Our study presents a new influence of E2 and P4 on the expression of PGR and PGRMC1 and on the real-time proliferation of porcine luminal epithelial cells. The relationship between PGR or PGRMC1 expression and the proliferation of luminal epithelial cells may be influenced (up- or down regulated) by E2 or P4 in a steroid type- and dose-dependent manner.

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.

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 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.

The B-chain of mistletoe lectin I efficiently stimulates calcium signaling in human Jurkat T-cells.

Mistletoe lectin I (ML I), a heterodimeric disulfide-linked type II ribosome inactivating protein, exhibits immunomodulatory potency in stimulating the cytokine release in vitro and in vivo. However, data concerning early activation events in T-cells induced by ML I and its A and B chain preceding cytokine secretion and the receptors involved are of limited availability. Here we show by flow cytometric measurements that human T-lymphoblastoid Jurkat cells express surface glycoprotein receptors for ML I. One of which is shown to be the CD2 antigen involved in a variety of T-cell signaling events. The lectin induces in Jurkat T-cells an increase of the cytosolic calcium concentration ([Ca(2+)](i)) consisting of both, the transient release of Ca(2+) from internal stores and a sustained influx of extracellular Ca(2+). Studies with isolated A- and B-chains provided evidence that the lectin-induced increase in [Ca(2+)](i) is mediated by ML IB. The ML I and ML IB stimulated cellular calcium responses are inhibited by saccharidic competitors. In transiently transfected E6.1 cells ML IB stimulated the expression of the luciferase reporter construct pNFAT-TA-Luc that is activated through the nuclear factor of activated T-cells (NFAT). The ML IB stimulated expression of the reporter luciferase (Luc) is completely inhibited by cyclosporin A (0.2 microM) and by FK 506 at 0.05 microM. Pretreatment of Jurkat E6.1 cells with 1-deoxymannojirimycin (dMJ), an inhibitor of cis-Golgi alpha-mannosidase I, strongly reduced cell binding of ML IB-FITC and the ML IB induced calcium response. Benzyl-alpha-GalNAc, an inhibitor of O-linked glycosylation, has slightly decreasing effects in ML IB-FITC binding and was without effects on the lectin stimulated increase in [Ca(2+)](i). Inhibition of the lectin induced calcium responses by cholera toxin and by inhibitors of protein kinases as well as the absence of calcium responses in CD3- and CD45- Jurkat T-cell clones suggest that ML IB has the potency to induce early T-cell activation events.

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Developmental and hormonal regulated gene expression of fibroblast growth factor 2 (FGF-2) and its receptors in porcine endometrium.

This study examined the mRNA levels of the fibroblast growth factor 2 (FGF-2) and two of its receptors, FGFR1IIIc and FGFR2IIIc, at days 12 and 20 of the ovarian cycle (DC 12 and DC 20), days 1 and 12 of pregnancy (DP 1 and DP 12) as well as the influence of progesterone (P) and estradiolbenzoate (EB) on their expression in the endometrium of ovariectomized (ovx) gilts by real-time PCR. Proteins of FGF-2 and FGFR1 were immunolocalized. FGF-2 and FGFR2IIIc mRNAs were always found with a 5- to 30-fold higher absolute concentration compared to FGFR1IIIc. The latter transcript significantly declined between DP 1 and DP 12, whereas FGF-2 and FGFR2IIIc showed no significant changes at that time. FGF-2 transcription was greater at DC 20 than at DC 12, but significantly most transcripts were found in ovx gilts. EB induced a significant suppression of FGF-2 mRNA, an effect which was antagonized by P and even prevented by P+EB. FGFR1IIIc mRNA was significantly increased at DC 20, that of FGFR2IIIc at DC 12 displaying a 10 times higher absolute mRNA amount. Suppression of FGFR1IIIc mRNA by P was abolished by EB while P+EB attenuated this effect. FGFR2IIIc transcripts were equally restrained by P or EB while a combination of both slightly reduced such declines. Localization of FGF-2 and FGFR1 proteins in stromal, glandular and vascular compartments was effected by sex steroids. Both proteins were strongly expressed at DP 12 but not at DP 1. Summarized, differential temporal and spatial localization of FGF-2 and FGFR1 after response to sex steroids support a complex regulation of this ligand receptor system important for proliferation and differentiation of uterine cells including angiogenic processes. While FGFR1IIIc is presumed to be promoted by estradiol FGFR2IIIc appears to be dominated by progesterone implicating different biological importance for a functional endometrium.

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.

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.

Identification of the EGF/EGF-R system in the oviduct and endometrium of pigs in early stages of pregnancy and early conceptus.

In the oviduct and endometrium, higher mRNA amounts encoding for EGF were found on day 6 and 12 in cyclic and on day 6 in pregnant pigs compared to signals on day 1. Reproductive state related changes could also be detected for TGFalpha mRNA in cyclic and pregnant pig oviduct but not in the endometrium. EGF-R protein concentration was higher (P < 0.05) in oviductal membranes on day 1 of the pregnancy (42.6 +/- 16.5 fmol mg(-1) protein) compared to day 6 and 12 (21.6 +/- 6.0 fmol mg(-1) protein and 17.1 +/- 3.8 fmol mg(-1) protein). In the endometrium EGF-R protein concentrations increased (P < 0.05) on day 1 (14.7 +/- 4.8 fmol mg(-1) protein) to day 6 (29.0 +/- 6.8 fmol mg(-1) protein) and day 12 (27.5 +/- 7.0 fmol mg(-1) protein) of pregnancy. The mature EGF-R protein (170 kDa) was verified in oviductal and endometrial membranes of all pregnant pigs investigated. A biologically intact EGF-R could be detected in all samples by means of autophosphorylation assay. Weak EGF mRNA signals were found in the expanded and filamentous blastocysts. No TGFalpha transcripts could be amplified during the embryonic stages. Only low amounts of EGF-R mRNA could be detected in zygotes and in filamentous blastocysts.

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.

Regulation of the expression and bioactivation of the epidermal growth factor receptor system by estradiol in pig oviduct and endometrium.

Growth factors, such as epidermal growth factor (EGF), have been suggested to mediate local effects of steroid hormones within female reproductive tissue. In the present study, the influence of estrogen on the expression and bioactivity of the EGF receptor (EGF-R) system was investigated in pigs. Oviducal and endometrial tissue from gilts was analysed either at two different time points after ovulation (Day 12 and Day 20), or from ovariectomized animals, with or without steroid-replacement treatment. Estrogen receptor protein concentrations were significantly down-regulated both in oviducal and endometrial tissue under estrogen-influence, in contrast to increased progesterone receptor concentrations. Transcript levels of EGF and transforming growth factor alpha remained unchanged in both the oviduct and endometrium during treatment. Oviducal EGF-R mRNA was found to be increased after estradiol treatment with concurrent increases in EGF-R protein. However, in endometrial tissue of estradiol-substituted ovariectomized pigs, the receptor transcript was significantly reduced, indicating a different regulation of EGF-R transcription within the endometrium. The bioactivity of the EGF-R, analysed by tyrosine kinase assays, was preserved throughout experiments in the porcine oviduct and endometrium without obvious changes caused by the steroids. In conclusion, estradiol may play a key role during the proliferation and differentiation of porcine oviducal tissue by activating the important paracrine or autocrine EGF system through its receptor. The cell-specific influence of progesterone during regulation of the EGF-R expression in the endometrium requires further investigation.

Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulosa cells.

Tris(4-chlorophenyl)methanol (TCPM) is a by-product in the manufacture of technical grade DDT, which is known to alter properties and functions of the female reproductive system. We investigated whether in vitro TCPM has an influence on the function of gap junction-mediated intercellular communication (GJIC) and gap junction protein expression of connexin 43 (Cx43) in cultured bovine granulosa cells. GJIC was assessed by fluorescent dye microinjection (dye-coupling). After a 1-h exposure to TCPM at a concentration of 32 microM, a significant (P<0.05) reduction in dye coupling occurred. The same result was obtained with o,p'-DDT. At a concentration of 32 microM both pesticides were cytotoxic as indicated by significant (P<0.05) increased propidium iodide staining of the cell nuclei. Little or no effect on the stainable pattern of connexons occurred after 1 h incubation time, while after 3 h treatment from 16 to 64 microM TCPM, a significant inhibition in the immunostaining resulted and the concentrations of 32 and 64 microM TCPM were cytotoxic for the granulosa cells. The freeze-fracture electron microscopy resulted in small differences in the morphology of gap junction plaques of cell cultures treated for 3 h with 8 or 16 microM TCPM in comparison to untreated cells. After treatment with 32 microM TCPM, gap junction plaques were very rarely detected and the lateral intramembraneous particles (IMP) distribution of many plasma membranes was strongly altered. Estimation of the cellular parameters may lead to an enhanced understanding of the mechanism of chemically induced toxicity by TCPM, that causes a general toxic effect on granulosa cells. We can conclude that TCPM is a toxic risk in the same manner as DDT.

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.

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.

[Control of implantation in rats and sows by peroral administration of prostaglandin synthetase inhibitors. 1. Theoretical aspects of control of implantation in swine]. / Untersuchungen zur Beeinflussung des Implantationsgeschehens bei Ratten und Sauen durch perorale Verabreichung von Prostaglandinsynthetasechemmern. 1. Mitteilung: Theoretische Aspekte der Beeinflussung des Implantationsgeschehens beim Schwein.

The reproduction situation of swine is considerably affected by embryonic foetal death. Reduction of loss of that origin, therefore, would open up palpable reserves. There is general agreement to the effect that the period close to implantation and the entire first month of gravidity make for the most critical phase of intra-uterine development. Proper mutual adaptation of progesterone and oestrogen levels are among the important prerequisites for favourable implantation conditions. However, fertility of swine was not always enhanceable by administration of progesterone and oestrogen. Tests of laboratory animals have shown that there are additional hormones and control mechanisms, on top of progesterone and oestrogen, which are involved in regulation of the implantation process. For example, higher prostaglandin F2 alpha concentrations in the blood and endometrial tissue of mammals in early gravidity seem to have adverse effects on implantation. The possibility is considered in this paper to eliminate such interference effect of prostaglandin by application of prostaglandin synthetase inhibitors.

[Control of implantation in rats and sows by peroral administration of prostaglandin synthetase inhibitors. 3. Effects of prostaglandin F2 alpha, progesterone/estrone, and acetylsalicylic acid on implantation and biochemical composition of amniotic fluid and fetuses of young sows]. / Untersuchungen zur Beeinflussung des Implantationsgeschehens bei Ratten und Sauen durch perorale Verabreichung von Prostaglandinsynthetasehemmern. 3. Mitteilung: Untersuchung des Einflusses von Prostaglandin F2 alpha, Progesteron/Ostron und Azetylsalizylsäure auf das Implantationsergebnis sowie die biochemische Zusammensetzung der Fruchtwässer und Feten von Jungsauen.

Low pregnancy rate (62.5 per cent), low implantation results (64.7 per cent), high percentage of dead foetuses (5.4 per cent), and low allantois fluid volume/foetus (130.7 ml) are likely to suggest adverse effect of prostaglandin administered in early gravidity since all these values deviate sizeably from control data (81.3, 68.0 and 1.1 per cent, 154.7 ml). On the other hand, those negative phenomena were not reflected in the results obtained from biochemical analysis of amniotic fluid and foetuses. The progesterone/oestron and acetylsalicylic acid doses used obviously had no impact upon pregnancy rates (81.3 and 75.0 per cent) and overall implantates (67.4 and 69.6 per cent), when compared to the controls (81.3 and 68.0 per cent). The lowest foetal loss occurred following application of acetylsalicylic acid (30.4 per cent, but 32.0 per cent for the controls). Increase in allantois fluid volume/foetus by progesterone/oestrone and acetylsalicylic acid was one of the findings of particular interest (progesterone/oestron: 174.4 ml, acetylsalicylic acid: 196.5 ml, control: 154.7 ml). The results obtained from amniotic fluid tests (rises in protein and progesterone concentrations) may, to some extent, suggest the possible existence of a relationship in action between the two substances. No information at all on the type of action of these substances was obtainable by biochemical analysis of foetal composition.

[Control of implantation in rats and sows by peroral administration of prostaglandin synthetase inhibitors. 2. Effects of prostaglandin F2 alpha, progesterone/estrone, and acetylsalicylic acid on implantation and various biochemical parameters of amniotic fluid in the rat]. / Untersuchungen zur Beeinflussung des Implantationsgeschehens bei Ratten und Sauen durch perorale Verabreichung von Prostaglandinsynthetasehemmern. 2. Mitteilung: Untersuchung des Einflusses von Prostaglandin F2 alpha, Progesteron/Ostron und Azetylsalizylsäure auf das Implantationsergehnis sowie einige biochemische Parameter der Amnionflüssigkeit der Ratte.

The highest pregnancy rate as well as most of all implantates and lowest foetal loss were recorded from the prostaglandin F2 alpha-(PGF2 alpha)-group (84, 84, and 16 per cent), while values following progesterone/oestrone and acetylsalicylic acid treatment were below those obtained from the controls. The highest number of normally developed (97 per cent) and the lowest number of degenerated foetuses (three per cent) were recorded following acetylsalicylic acid treatment, as compared to the control group (91 and nine per cent). Application of prostaglandin had no adverse effect on foetal development. The overall protein concentration in the amniotic fluid, following injection of progesterone/oestrone and PGF2 alpha, was lower with significance than the control value. Acetylsalicylic acid caused slight but insignificant rise in protein concentrations. The behavior of glucose concentrations in the amniotic fluid seemed to be diametrically opposed to that of protein. The activity of acid phosphatase was low and highly variable in all four experimental groups. Values moderately increased over the controls were recorded from the acetylsalicylic acid group.

Characterization of the epidermal growth factor receptor in pig oviduct and endometrium.

The aim of this study was to determine whether the stage of the oestrous cycle (day 1, n = 5; day 6, n = 5; day 12, n = 3 gilts) has an influence on the expression and activity of epidermal growth factor receptor (EGF-R) in the pig oviduct and uterus. Histochemistry, cross-linking of 125I-labelled EGF to isolated oviductal and endometrial membranes or on cryostat sections, and EGF-R binding assay were used to demonstrate the presence of the EGF-R in a qualitative and quantitative manner. The bioactivity of the EGF-R in the oviduct was estimated by means of a protein tyrosine kinase activity assay. This study suggests that EGF-R is widely distributed both in glandular and stromal cells of the endometrium and in epithelial cells of the oviduct in pigs. The concentrations of EGF-R were higher in oviductal membranes on day 1 (22.4 +/- 8.7 fmol mg-1 protein) in comparison with day 6 (11.0 +/- 0.42 fmol mg-1 protein; P < 0.05), but not on day 12 (16.0 +/- 2.9 fmol mg-1 protein). The concentrations dropped similarly in the endometrium (day 1: 66.8 +/- 16.4 fmol mg-1 protein; day 6: 39.1 +/- 3.4 fmol mg-1 protein (P < 0.05); day 12: 38.0 +/- 14.6 fmol mg-1 protein). The dissociation constant (Kd) showed the same pattern. These data were supported by cross-linking of 125I-labelled EGF to a 170 kDa membrane protein representing the EGF-R. In contrast to day 6 and 12 of the cycle, a significantly (P < 0.05) higher endogenous protein tyrosine kinase activity was observed on day 1. In summary, changes in concentrations and functional status of the EGF-R may play a significant role in the cascade of cellular events in oviductal and endometrial tissues.

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.