Expression of PTX3, HAS2 AND TNFAIP6 genes in relation to real-time proliferation of porcine endometrial luminal epithelial cells in primary cultivation model.

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.

The origin, in vitro differentiation, and stemness specificity of progenitor cells.

Since the successful collection of the first progenitor stem cells (SCs), there has been an increased interest in these cells as a model for undiscovered and unlimited potential of differentiation and development. Additionally, it was shown that SC populations display an ability to form pluripotent and/or totipotent cell populations. It was found that human ovarian granulosa cells (GCs) maintain a large capacity for differentiation into several other cell lineages, such as chondrogenic, osteogenic, neurogenic, and adipogenic, particularly during long-term, in vitro culture. In these cases, the specific media supplements that promote various pathways of differentiation, such as leukemia-inhibiting factor (LIF) and/or FSH, are well recognized. However, these are only some examples of the differentiation possibilities of human SCs in vitro and other pathways still require further investigation. Many SC populations, which are directed to differentiate into specific cell types, are also successfully used in several human disease therapies, e.g. leukemia. Moreover, SCs are used for tissue scaffold construction in patients with respiratory and cardiovascular diseases. In this review, the most recent knowledge about the in vitro growth and differentiation capacity of SCs is presented. Furthermore, we discuss the possible worldwide application of SCs in advanced cell and tissue bioengineering. In conclusion, it is suggested that, in the future, SCs will be a basic strategy in human therapy, and their use will open new gates in regenerative and reconstructive medicine in the 21st century.

Molecular basis of growth, proliferation, and differentiation of mammalian follicular granulosa cells.

For normal folliculogenesis and oogenesis to occur many intrinsic and extrinsic factors are needed, i.e. positive feedback of hormone secretion and local ovarian-follicular growth factors distribution. During follicle formation, granulosa cells (GCs) change their morphology and physiological properties. The factors needed for GCs to differentiate within each layer are transforming growth factor beta (TGFB) and insulin-like growth factor (IGF), as well as the activation and modification of biochemical pathways involved in folliculogenesis. Physiological alterations occur when GC genes are characterized by several differences in their gene expression profile. Studies in recent years indicate a variety of processes involved in follicle morphology and biochemical remodeling during growth and development. It was demonstrated that IGFs play a central role in the differentiation of GCs both in vivo and in vitro. Moreover, the primary role of FSH and LH in the formation of the ovarian follicle, was also described. Our review article characterizes the most important pathways involved in the differentiation of GCs and the effect of various factors on gene expression in GCs during folliculogenesis.

Epithelialization and stromalization of porcine follicular granulosa cells during real-time proliferation - a primary cell culture approach.

The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation is also highly associated with porcine ovarian follicular granulosa cell differentiation in vitro.

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.

Association between the expression of LHR, FSHR and CYP19 genes, cellular distribution of encoded proteins and proliferation of porcine granulosa cells in real-time.

The process of granulosa cell luteinization is part of the main process determining growth, differentiation and proliferation of these cells. Although the mechanisms underlying the regulation of luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR) and cytochrome P450 aromatase expression in mammalian granulosa cells is well understood, still little is known about the expression of mRNA and encoded proteins in relation to cell proliferation and luteinization in vitro. Porcine granulosa cells were observed in vitro at a168-h period while undergoing real-time proliferation using an RTCA system. Furthermore, LHR, FSHR and CYP19 mRNA expression were detected using RQ-PCR after 168 h of in vitro culture (IVC) at 24-h intervals, and LHR, FSHR and P450arom were examined by confocal microscopic observation at 0 h, 24 h, 48 h, 96 h, and 168 h of IVC. We found increased expression of LHR and CYP19 mRNA at 24 h and 48 h of IVC compared to the other stages (P less than 0.01, P less than 0.001), whereas FSHR mRNA was higher only at 0 h (P less than 0.001). In contrast, LHR, FSHR and P450arom protein expression was significantly higher at the end of the 168-h IVC period compared to 0 h, 24 h, 48 h and 96 h (P less than 0.001). LHR, FSHR and P450arom were distributed in the cytoplasm of porcine GCs at each time point of IVC. When analyzing cell proliferation, differences in cell index were observed (at least P less than 0.05) between the first (0-24 h) and the last period (144-168 h) of IVC; however, soon after 24 h of IVC a logarithmic increase in proliferation was also seen. We assume that the expression of LHR, FSHR and CYP19 mRNAs depends on the period of in vitro cultivation and may be linked with the luteinization process of porcine GCs. Furthermore, the patterns of mRNA and protein expression suggest a post-transcriptional regulation of LHR, FSHR and P450arom. In summary, it can be presumed that mRNA and protein expression and in vitro luteinization and proliferation of porcine GCs are regulated by different mechanisms, because not all of these processes are correlated.

The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol.

The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 µg/mL, 1.0 µg/mL, and 2.0 µg/mL of P4 (experiment 1), or with (2) 2.0 µg/mL E2, and with (3) a combination of E2 (2.0 µg/mL) and P4 (0.5, 1.0, or 2.0 µg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 µg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 µg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 µg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.

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.

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.

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.

Luteinizing hormone release after administration of the gonadotropin-releasing hormone agonist Fertilan (goserelin) for synchronization of ovulation in pigs.

The generic GnRH agonist, Fertilan (goserelin), was tested for the ability to induce an LH surge and ovulation in estrus-synchronized gilts. Three experiments were performed to 1) examine the effect of various doses of Fertilan on secretion of LH in barrows, to select doses to investigate in gilts (Exp. 1); 2) determine doses of Fertilan that would induce a preovulatory-like rise of LH in gilts (Exp. 2); and 3) determine the time of ovulation after Fertilan treatment (Exp. 3). In Exp. 1, 10 barrows were injected on d 1, 4, 7, 10, and 13 with 10, 20, or 40 microg of Fertilan; 50 microg of Gonavet (depherelin; GnRH control) or saline (negative control); and sequential blood samples were collected for 480 min. There was a dose-dependent stimulation (P < 0.05) of LH release. Maximal plasma concentrations of LH (LH(MAX)) were 2.1 +/- 0.2, 4.1 +/- 0.3, 2.6 +/- 0.4, and 3.4 +/- 0.3 ng/mL after 10, 20, and 40 microg of Fertilan and 50 microg of Gonavet, respectively, and duration of release was 78 +/- 9, 177 +/- 12, 138 +/- 7, and 180 +/- 11 min, respectively. Fertilan doses of 10 and 20 microg were deemed to be the most suitable for testing in gilts. In Exp. 2, 12 gilts received (after estrus synchronization with Regumate and eCG) injections of 10 or 20 microg of Fertilan or 50 microg of Gonavet 80 h after eCG to stimulate a preovulatory-like LH surge and ovulation. An LH surge was induced in 3 of the 4 gilts in both of the Fertilan groups and in all of the Gonavet-treated gilts. Characteristics of induced release of LH did not differ among groups: LH(MAX), 5.0 +/- 0.9 vs. 4.6 +/- 1.8 vs. 6.6 +/- 1.1 ng/mL; duration, 11.7 +/- 2.0 vs. 12.3 +/- 2.2 vs. 14.3 +/- 0.5 h; interval from GnRH injection to LH(MAX), 4.0 +/- 2.0 vs. 6.7 +/- 1.3 vs. 5.8 +/- 1.6 h. In Exp. 3, estrus-synchronized gilts were injected with 20 microg of Fertilan (n = 8) or 50 microg of Gonavet (n = 4), and the time of ovulation was determined by repeated endoscopic examination. Time of ovulation ranged from 34 to 42 h postGnRH; however, ovulation occurred earlier in the Gonavet compared with the other groups (P < 0.05). Results of these experiments indicate that 1) barrows are an appropriate model for determining GnRH doses that can be effective in inducing a preovulatory-like LH surge in females; 2) the generic GnRH agonist Fertilan, at doses of 10 to 20 microg, can stimulate an LH surge in gilts, with subsequent ovulation; and 3) Fertilan at doses of 10 and 20 microg should be examined further for use in fixed-time insemination protocols.

Effects of a preovulatory administered depot gonadotrophin-releasing hormone agonist on reproductive hormone levels and pregnancy outcome in gilts.

The present study aimed to explore the influence of a preovulatory administered depot gonadotrophin-releasing hormone (GnRH) agonist (GnRHa; Decapeptyl Depot) on the endocrine parameters and pregnancy outcome of gilts (n = 6). A GnRHa-supported preovulatory luteinising hormone (LH) surge was detected in all treated gilts. LH pulses were abolished completely by depot GnRHa on Day 7 and partly on Day 21 of pregnancy. In this treatment group (n = 6) four gilts were pregnant at slaughter on Day 28. In the control group receiving Gonavet, a non-formulated GnRHa (n = 6), all pigs showed LH pulses and were pregnant at slaughter on Day 28 of gestation. Mean progesterone concentrations were elevated in controls during the early luteal phase and were similar for both groups during the implantation period. Mean concentration of unoccupied progesterone receptor was significantly higher in uterine myometrium than in endometrium, but without treatment effects. Peripheral estrone sulfate concentrations showed a similar increase in all pregnant gilts on Days 17 and 18, and remained elevated. In summary, treatment with a depot GnRHa for synchronisation of ovulation alters pulsatile LH secretion during early pregnancy in pigs. In general, this alteration seems not to exert an injurious influence on luteal function and, therefore, on embryo and early fetal development.

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.

Lamprey GnRH-III stimulates FSH secretion in barrows.

Although studies have indicated that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release can be dissociated in the pig, the underlying mechanisms are still to be answered. Since it was demonstrated that lamprey gonadotropin-releasing hormone (l-GnRH-III) has preferential FSH-releasing potency in several mammalian species, we have investigated the gonadotropin-releasing activity of l-GnRH-III in barrows. Each of nine barrows (body weight: 85-90 kg; age: 207 days) received 2 ml saline (S-barrow), followed by 150 microg l-GnRH-III (1.6-1.7 microg/kg body weight) dissolved in 2 ml saline intramuscularly 7 days later. Three pre-treatment and 13 post-treatment blood samples were taken at intervals of 30 min to 8 h to assess basal and treatment-associated concentrations of FSH and LH, respectively, by radioimmunoassay. Animals were defined as having responded to treatment if, 2 h post-treatment, plasma FSH and/or LH levels were >3 SD of the respective basal concentrations. There was no treatment-associated FSH response after saline treatment, but a clear FSH response in all l-GnRH-III-injected barrows. On average, the maximum FSH level (205% of the basal concentration) was observed at 1 h post-treatment. Mean FSH values were elevated until 10 h post-treatment. There was no LH response either to saline or to l-GnRH-III. In conclusion, this study demonstrates a selective FSH-releasing activity of 150 microg l-GnRH-III in barrows. Further studies are needed to investigate whether this effect is ubiquitous in the pig and what the physiological relevance is.

Maternal reproductive hormone levels after repeated ACTH application to pregnant gilts.

Prenatal stress has been seen as a reason for reproductive failures in pig offspring mostly originated or mediated by changed maternal functions. In pregnant gilts, three experiments (EXP I-III) were conducted to characterize the effects of repeated ACTH on maternal cortisol concentrations (EXP I) and on the secretion of maternal reproductive hormones (LH, progesterone, estrone sulfate; EXP II + III). Exogenous ACTH was given six times every alternate day beginning either on day 49 (EXP I + II) or day 28 (EXP III) of pregnancy. As a result of treatment, elevated cortisol levels were observed for more than 6 h (EXP I). Plasma concentrations of LH were at low basal level (0.1-0.2 ng/ml), but showed a pulsatory release pattern both during first and second trimester of pregnancy. The number of LH pulses/6 h (L.S.M. +/- S.E.) of saline treated controls increased with ongoing pregnancy (1.4 +/- 0.1 versus 2.0 +/- 0.2 in EXP III and EXP II, respectively). After ACTH treatment the number of LH pulses did not differ between the two gestational stages (1.3 +/- 0.2 and 1.4 +/- 0.2 in EXP III and EXP II, respectively). However, differences ( P < 0.05) were obtained comparing the LH pulse number of ACTH and saline treated sows during the second trimester of pregnancy. Moreover, areas under the curve (AUC) of each LH pulse and of all LH values over the baseline were significantly reduced by treatment. The levels of progesterone increased (P < 0.05) for 150-170 min after each ACTH application both in EXP II and EXP III. The concentrations of 17alpha-hydroxy-progesterone revealed likewise a significant elevation after each ACTH injection. Throughout EXP III, estrone sulfate concentrations were found to decrease (from 2.8-16.9 ng/ml on day 28 to 0.02-0.04 ng/ml on day 38) but without differences between ACTH-and saline-treated gilts. Further data of EXP II and EXP III, e.g. number of piglets born alive, confirmed the absence of detrimental treatment effects. Thus, repeated ACTH administration with subsequent release of cortisol is able to influence the release pattern of maternal reproductive hormones. However, these findings demonstrate that stress-related effects are dependent on the stage of pregnancy. The detected changes may affect feto-maternal interactions and, as a result, fetal reproductive development.

Administration of recombinant porcine somatotropin (rpST) changes hormone and metabolic status during early pregnancy.

The objective of this study was to examine the effects of somatotropin (ST) on porcine reproductive and metabolic statuses during early pregnancy. Four pregnant crossbred gilts received 6 mg of recombinant porcine somatotropin (rpST) daily from days 10 to 27 after artificial insemination while six pregnant gilts served as controls. Blood samples were taken on days 8, 10, 12, 14, 18, 22, and 27 prior to rpST injections (8:00 h) and subsequently at 9:00, 10:00, 12:00, 14:00, 16:00, 18:00, and 20:00 h. On all remaining days of treatment, samples were taken once daily before injections (8:00 h). The samples were assayed for the metabolic hormones: ST, insulin-like growth factor I (IGF-I), insulin, thyroxine (T(4)), triiodothyronine (T(3)), and cortisol; for metabolites: free fatty acids (FFA) and glucose; and for the reproductive hormones: luteinizing hormone (LH), progesterone, estradiol-17beta, estrone sulfate, and prostaglandin F(2alpha). Delivery of rpST daily induced a 20- to 40-fold increase in plasma ST concentrations. Moreover, repeated administration of rpST resulted in a continuous increase in plasma IGF-I concentration (P < 0.001), from 191.0 +/- 22.3-340.0 +/- 15.3 ng/mL 24 h after initial injection to 591.3 +/- 46.8 ng/mL after final injections. Mean serum insulin tended to be greater in rpST-treated gilts. Blood concentrations of T(4) were reduced (P < 0.05) from day 14 of gestation in treated gilts while T(3) concentrations remained unchanged. Concentrations of both glucose and FFA were greater (P < 0.01) and cortisol concentrations were unchanged in treated gilts. Changes in reproductive steroid hormones were minimally affected. Circulating progesterone (P = 0.078), and estradiol-17beta (P = 0.087) concentrations tended to be lower in treated animals. These data show that treatment of pregnant gilts with rpST during early gestation mainly impacts metabolic rather than reproductive status.

Follicular and oocyte development in gilts of different age.

The aim of the present study was to estimate follicular and oocyte development of the same gilts in three phases of their reproductive life--prepuberal gilt (6 months old), cycling gilt (9.5 months old) and primiparous sow. Follicular development was induced by injections of 1000 IU PMSG followed by 500 IU hCG 72 h later. Cumulus-oocyte-complexes (COCs) were recovered from preovulatory follicles of the left ovary, and follicular fluid (FF) from the right ovary always 34 h after hCG by endoscopy. Altogether, 19 gilts were used in the prepuberal (P) and cycling (C) trials and 12 of them in the primiparous trial (S). Altogether 168, 190 and 82 follicles were aspirated from the left ovary and 106, 125 and 42 COCs recovered (recovery rate 60.5 +/- 26.9, 62.7 +/- 20.9 and 52.9 +/- 21.8%). The average number of follicles was higher in C compared to P (19.7 +/- 6.8 vs. 15.7 +/- 6.8, p = 0.06) and to S (14.2 +/- 4.0, p < 0.05), respectively. More uniform expanded COCs were aspirated from prepuberal and cycling gilts as compared to sows (89.7 and 78.4% vs. 46.3%, p < 0.05). Furthermore, the meiotic configuration in oocytes differed (p < 0.05) between these groups (55.5 and 61.7% vs. 0% Telo 1/Meta 2). Concentrations of progesterone in FF decreased (p < 0.05) from 590.0 +/- 333.6 (P) to 249.1 +/- 72.6 (C) and 161.4 +/- 75.2 ng/ml (S). FF concentrations of oestradiol-17 beta were different between gilts and sows (9.3 +/- 2.9, 21.9 +/- 10.6 and 94.0 +/- 15.9 pg/ml, p < 0.05). The progesterone/oestradiol ratio was 72.1, 15.2 and 4.7. Results indicate a different follicular and oocyte development during the investigated lifetime periods. Cycling gilts should preferably be used in IVF and breeding programs. The lower reproductive potential of primiparous sows is taken into consideration at breeding. Prediction of lifetime performance based on individual ovarian reaction of prepuberal gilts is unsuitable.

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.