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The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25-kDa, mRNA cap-binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E-BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E-BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E-, eIF4G-, and 4E-BP1-specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E-BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E-BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E-BP1 and binding dynamic of eIF4E/4E-BP1 in distinct forms of implantation., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

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.

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.

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.

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.

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.

Contents 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. [ABSTRACT FROM AUTHOR]