Karin Wollenhaupt

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.

Activation of indoleamine 2,3-dioxygenase by LPS in a porcine model.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway, and its increased activation is associated with immunologic disorders. Because the specific role of IDO activation is not yet completely clear, the aim of the present study was to establish a pig model of IDO activation for further research. The activation of IDO in pigs was induced experimentally by LPS stimulation in vivo and ex vivo. IDO activation was characterized by measuring Trp, Trp metabolites and IDO protein expression in blood, liver, lung, muscle and different brain areas. The results show that the in vivo LPS administration induced increased plasma concentrations of TNF-α and IL-10, a depletion of Trp and an increase of Kyn, indicating an elevated enzymatic activity of IDO. This was supported by an LPS-induced IDO protein expression in blood, liver and lung. The ex vivo LPS stimulation also resulted in increased TNF-α concentrations and an IDO activation, characterized by an increase of Trp metabolites and IDO protein expression. In conclusion, our data emphasize that the LPS stimulation is a suitable model for IDO activation in the domestic pig, which provides a basis for further research on immunoregulatory IDO functions.

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig.

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. Mol. Reprod. Dev. 78:895–905, 2011.

The Akt/mTor signaling cascade is modified during placentation in the porcine uterine tissue

Recently we showed that essential components for the initiation of protein synthesis, namely the eukaryotic initiation factor 4E (eIF4E, mRNA-cap-binding protein) and its repressors 4E-BP1 as well as 4E-BP2, are proteolytically processed in the porcine endometrium during implantation. Here, the situation during placentation was compared with ovariectomized (OVX) animals and animals on pregnancy day 1 (PD1). Furthermore, the research was extended to factors which phosphorylate eIF4E and 4E-BPs and regulate their activities. These are the protein kinase B/mammalian target of rapamycin kinase (Akt/mTor) with the regulators Raptor and Rictor as well as the mitogen activated protein kinases (MAPKs): extra cellular-signal regulated kinase 1 and 2 (ERK1 and ERK2). Striking differences in the placentation site (PS) and the areas aside from PS (peri-PS) were observed. EIF4E and 4E-BP2 truncation as well as 4E-BP1 degradation took place in the endometrium of the peri-PS on PD24. Accompanied by a fragmentation of Akt/mTor, no expression of Rictor was observed, whereas the abundance of Raptor was not altered. On the contrary, MAPKs expression and phosphorylation remained almost stable in the peri-PS. In conclusion, the results indicated that on PD24 the translational regulation was shifted to 4E-BP2 control. Furthermore, the Akt/mTor signaling cascade seemed to be down regulated which suggest reduced phosphorylation of 4E-BP2. Whereas Akt was proteolyzed, the observed mTor fragments represented most likely splicing variants. The results indicate that translational control of gene expression is an important feature in the porcine endometrium during early pregnancy.

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 eIF4E Repressor Protein 4E-BP2 Is Merely Truncated, Despite 4E-BP1 Degradation in the Porcine Uterine Tissue During Implantation

Recently, we identified an N‐terminally truncated form of the mRNA cap binding protein eIF4E in the porcine luminal epithelium during implantation. EIF4E truncation is accompanied by degradation of the eIF4E‐repressor protein 4E‐BP1. In this study, we investigated whether or not the other members of the eIF4E‐repressor family, namely 4E‐BP2 and 4E‐BP3, were also modified during early pregnancy. We did not detect 4E‐BP3 in the uterine tissue; however, 4E‐BP2 emerged in one or two stable fragments on pregnancy day 15. 4E‐BP2 truncation most likely occurs at the N‐terminus, and this calcium‐stimulated processing depends on progesterone and estradiol. The activities targeting eIF4E, 4E‐BP1, and 4E‐BP2 were found in different fractions after anionic exchange chromatography, indicating the action of different proteases. Detailed protein interaction studies with immobilized anti‐eIF4E and m7GTP‐Sepharose showed a differential binding of the 4E‐BP2 isoforms to the eIF4E variants and to the cap structure. In general, truncation of eIF4E reduces the inhibitory impact of 4E‐BP2, whereas truncation of 4E‐BP2 restores repression by binding the prototype eIF4E. In this context, we suggest long‐term translational repression by the truncated 4E‐BP2 is affected by the loss of the RAIP motif located at the N‐terminus, which is indispensable for phosphorylation and deactivation of the molecule. In conclusion, we propose a tightly balanced regulation of the truncation of the cap‐binding complex component eIF4F and degradation of 4E‐BP1 and/or truncation of 4E‐BP2 that together ensures correct translational control during the dynamic process of conceptus implantation. Mol. Reprod. Dev. 79: 767–776, 2012.