WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

BACKGROUND: The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). OBJECTIVE: The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. METHODS: WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). RESULTS: WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. CONCLUSIONS: There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

WNT5A gene and protein expression in endometrial cancer.

INTRODUCTION: WNT5A (Wnt family member 5A) belongs to the WNT family of secreted signaling glycoproteins that play essential role in developmental, physiological and pathological processes. WNT5A was shown to take part in carcinogenesis process playing both oncogenic and suppressor functions in various types of human malignancies. This study aimed to assess the expression of the WNT5A gene at the mRNA and protein levels in the specimens derived from endometrial cancer (EC) or unchanged control endometrium. The associations between the WNT5A expression levels and clinicopathological characteristics and survival of EC patients were evaluated. MATERIALS AND METHODS: Total RNA was isolated in order to assess the relative amounts of WNT5A mRNA by quantitative polymerase chain reaction (QPCR) in samples of unchanged endometrial control (n = 8) and tumor samples of EC patients (n = 28). Immunohistochemistry (IHC) was used to determine the presence of WNT5A protein in the sections of formalin-fixed, paraffin-embedded tissue specimens derived from unchanged endome-trial controls (n = 6) and EC tumors (n = 19). Significance of differences in WNT5A expression levels between the studied groups of EC patients and correlations between the WNT5A and demographic data, pathological features, hematological parameters and overall survival of the patients were evaluated by statistical analysis. RESULTS: The level of WNT5A mRNA was decreased in EC in comparison to unchanged endometrium. WNT5A expression was associated with primary tumor invasion status exhibiting reduced level of transcripts in EC that involved organs beyond the uterus when compared to the uterus-confined cancers. WNT5A immunoreactivity was visualized in the cytoplasm and nuclei of EC cells as well as in the luminal and glandular epithelial cells of unchanged endometrium. WNT5A mRNA expression levels correlated negatively with cytoplasmic, and positively with nuclear immunoreactivity of the WNT5A protein in the EC cells. In addition, the relationships between blood leucocyte count (in particular granulocytes and lymphocytes) of patients with EC and their WNT5A mRNA and protein expression levels were established. A positive correlation between the nuclear immunoexpression of WNT5A protein in the cancer cells in cell nuclei and mean platelet volume in blood was also found. CONCLUSIONS: The results of the first study of WNT5A expression at the transcript and protein levels indicate that it could be considered as a potential marker of molecular changes that take place during EC development.

Altered Expression of DDR1 in Clear Cell Renal Cell Carcinoma Correlates With miR-199a/b-5p and Patients' Outcome.

BACKGROUND/AIM: Accumulating evidence suggests that discoidin domain receptor tyrosine kinase 1 (DDR1) has an oncogenic role. Therefore, the aim of this study was to evaluate the potential utility of DDR1 and its post-transcriptional repressors, miR-199a-5p and miR-199b-5p, as prognostic factors in clear cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: The expression of DDR1 in tumor and normal renal tissues of 56 patients with ccRCC was assessed by reverse transcription quantitative polymerase chain reaction, western blotting and immunohistochemistry. Renal cancer cells were transfected with specific RNA sequences to validate DDR1 as a putative miR-199a/b-5p target. RESULTS: Decreased DDR1 mRNA and protein, as well as miR-199a/b-5p levels were found in ccRCC. Low DDR1 protein was associated with higher nuclear grade and shorter overall survival. DDR1 immunoreactivity was elevated in the nuclei and unchanged in the membrane/cytoplasmic compartment of tumor cells. DDR1 levels correlated with those of miR-199a/b-5p. In addition, we validated DDR1 as a target gene for miR-199a/b-5p in renal cancer cell lines. CONCLUSION: DDR1 expression is altered in ccRCC, but our findings do not support its oncogenic role. In-depth investigation will be necessary to elucidate the exact role and potential utility of miR-199a/b-5p in ccRCC.

IKBKB expression in clear cell renal cell carcinoma is associated with tumor grade and patient outcomes.

Inhibitor of nuclear factor kappa B kinase subunit B (IKBKB or IKKß) is a key activator of the nuclear factor κB transcription factor pathway. Increased expression and/or aberrant activity of IKBKB have been observed in various types of human cancer. Three independent techniques, reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemistry, were used to demonstrate that IKBKB expression is decreased in clear cell renal cell carcinoma (ccRCC). Notably, the patients with upregulated IKBKB protein expression were characterized by higher nuclear grade tumors and significantly shorter survival. The findings indicate that IKBKB protein may be of clinical relevance in ccRCC, serving as a marker of poor prognosis and as potential target for adjuvant chemotherapies. Further studies are required to validate the prognostic and predictive value of IKBKB.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon.

The anti-apoptotic protein survivin is one of the most promising cancer biomarkers owing to its high expression in human cancers and rare occurrence in normal adult tissues. In this work, we have investigated the role of supramolecular interactions between a graphene oxide (GO) nanosheet nanocarrier and a survivin molecular beacon (SurMB), functionalized by attaching fluorophore Joe and quencher Dabcyl (SurMB-Joe). Molecular dynamics simulations revealed hydrogen bonding of Joe moiety and Dabcyl to GO carriers that considerably increase the SurMB-GO bonding strength. This was confirmed in experimental work by the reduced fluorescence background in the OFF state, thereby increasing the useful analytical signal range for mRNA detection. A new mechanism of hairpin⁻hairpin interaction of GO@SurMB with target oligonucleotides has been proposed. A low limit of detection, LOD = 16 nM (S/N = 3), has been achieved for complementary tDNA using GO@SurMB-Joe nanocarriers. We have demonstrated an efficient internalization of SurMB-Joe-loaded GO nanocarriers in malignant SW480 cells. The proposed tunability of the bonding strength in the attached motifs for MBs immobilized on nanocarriers, via structural modifications, should be useful in gene delivery systems to enhance the efficacy of gene retention, cell transfection and genomic material survivability in the cellular environment.

Hairpin-Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells.

Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed hairpin-hairpin interaction method. The single nucleotide polymorphism sensitivity and a low detection limit of 26 nM (S/N = 3σ) for complementary targets have been achieved.

Decreased Expression of SATB2 Associates with Tumor Growth and Predicts Worse Outcome in Patients with Clear Cell Renal Cell Carcinoma.

BACKGROUND/AIM: SATB2 (special AT-rich sequence-binding protein 2) is a DNA-binding protein that is involved in transcriptional regulation and chromatin remodeling. SATB2 protein has been described as a promising novel marker in several human cancers. PATIENTS AND METHODS: This study compared SATB2 expression in tumor and matched unchanged renal tissues collected from 57 patients with clear cell renal cell carcinoma (ccRCC). SATB2 mRNA levels were determined by quantitative polymerase chain reaction, while SATB2 protein expression was estimated by immunohistochemistry. Moreover, the associations between SATB2 expression in ccRCC samples and clinicopathological and survival data of the patients were investigated. RESULTS: The mRNA level of SATB2 was lower in tumor tissues than in samples of corresponding unchanged kidney. Although the average immunoreactivity of SATB2 protein did not differ significantly between cancer cells and epithelial cells of proximal convoluted tubules, the decreased SATB2 expression in tumor specimens inversely correlated with the size of primary tumor and predicted worse patients' outcome. CONCLUSION: The results of the presented study suggest the tumor-suppressing function of SATB2 and that the expression level of this protein can be considered a potential prognostic factor in ccRCC.

Expression of the EP300, TP53 and BAX genes in colorectal cancer: Correlations with clinicopathological parameters and survival.

E1A binding protein P300 (EP300), tumor protein P53 (TP53) and BCL2 associated X, apoptosis regulator (BAX) genes encode proteins which cooperate to regulate important cellular processes. The present study aimed to determine the expression levels of EP300, TP53 and BAX in colorectal cancer (CRC) and to investigate their prognostic value and association with the progression of CRC. Tumor and matched unchanged colorectal tissues were collected from 121 CRC patients. Quantitative polymerase chain reaction and immunohistochemistry were used to assess the mRNA and protein levels of the studied genes. Altered expression of the studied genes in CRC tissues was observed at both the mRNA and protein levels. The depth of invasion was associated with TP53 mRNA levels and was correlated negatively with BAX mRNA expression. Moreover, a relationship between tumor location and BAX mRNA content was noted. BAX immunoreactivity was correlated positively with the intensity of p300 immunostaining and was associated with lymph node involvement and tumor-node-metastasis (TNM) disease stage. Univariate regression analysis revealed that overexpression of p53 and BAX in CRC tissues was associated with poor patient outcome. In conclusion, dysregulation of the expression of the studied genes was found to contribute to CRC pathogenesis. The association between p300 and BAX levels suggests the existence of an interdependent regulatory mechanism of their expression. Moreover, BAX expression may be regulated alternatively, in a p53-independent manner, since the lack of correlations between expression of these factors was observed.

Expression and Prognostic Significance of EP300, TP53 and BAX in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Histone acetyltransferase E1A-binding protein p300 (EP300), tumor protein p53 (TP53) and B-cell lymphoma-2-associated X protein (BAX) contribute to the regulation of the cell cycle and apoptosis, cellular processes that are often impaired in cancer cells. The aim of this study was to determine the expression levels of EP300, TP53 and BAX genes and their respective proteins in clear cell renal cell carcinoma (ccRCC) and evaluate the value of these factors as prognostic factors. MATERIALS AND METHODS: EP300, TP53 and BAX expression at the transcript and protein levels were determined by quantitative polymerase-chain reaction (QPCR) and immunohistochemistry (IHC) in paired tumor and kidney specimens from 31 patients with ccRCC. RESULTS: Levels of EP300, TP53 and BAX transcripts were found increased in tumor tissues. Immunoreactivity for TP53 was elevated in cancer cells when compared to unchanged kidney, while EP300 and BAX immunoexpression in ccRCC did not differ from that of normal renal tissue. Immunoreactivity for TP53 was positively associated with larger tumor size. In contrast, stronger BAX immunoexpression correlated with smaller tumor diameters. The average immunoreactivity for BAX was higher in localized, kidney-confined tumor than in advanced/recurrent tumors. None of the analyzed transcripts or proteins correlated with the overall survival of patients. CONCLUSION: Although TP53 and BAX immunoreactivity levels were associated with some clinicopathological parameters of the patients, the expression of EP300, TP53 and BAX did not reveal any prognostic significance in ccRCC.

SATB1 is Down-regulated in Clear Cell Renal Cell Carcinoma and Correlates with miR-21-5p Overexpression and Poor Prognosis.

BACKGROUND: Altered expression of special AT-rich sequence binding protein 1 (SATB1) was reported in several types of human cancers. This study aimed to determine the expression levels of SATB1, as well as miR-21-5p -the post-transcriptional repressor of SATB1 expression- in clear cell renal cell carcinoma (ccRCC) and to investigate their association with the progression of ccRCC. MATERIALS AND METHODS: Immunohistochemistry and quantitative polymerase chain reaction were used to assess the expression of SATB1 protein and mRNA as well as miR-21-5p in tumor and matched normal kidney tissues collected from 56 ccRCC patients. RESULTS: Nuclear SATB1 immunoreactivity was elevated in ccRCC cells while its cytoplasmic expression was decreased. SATB1 mRNA level was down-regulated in ccRCC tissue and inversely correlated with the content of miR-21-5p. Down-regulation of SATB1 mRNA and up-regulation of miR-21-5p were associated with shorter patient survival. CONCLUSION: Decreased expression of SATB1 in ccRCC may result from over-expressed miR-21-5p. Our data suggest that SATB1 may have a potential value as a prognostic marker in ccRCC.

PLAGL1 (ZAC1/LOT1) Expression in Clear Cell Renal Cell Carcinoma: Correlations with Disease Progression and Unfavorable Prognosis.

BACKGROUND: Pleiomorphic adenoma gene-like 1 (PLAGL1) protein was originally shown to induce cell-cycle arrest and promote apoptosis in several types of human tumors and therefore it was considered a candidate tumor suppressor. The involvement of PLAGL1 gene in the etiology and pathogenesis of clear cell renal cell carcinoma (ccRCC) has not been evaluated. The purpose of the present study was to determine the expression level of PLAGL1 in ccRCC and to investigate its potential utility as a prognostic factor. MATERIALS AND METHODS: We applied quantitative real-time polymerase chain reaction (QPCR), western blotting and immunohistochemistry to measure PLAGL1 mRNA/protein contents in paired tumor and kidney specimens of 40 patients with ccRCC. RESULTS: PLAGL1 mRNA and protein levels were increased in tumor tissues as determined by QPCR and immunohistochemistry, respectively. The average content of PLAGL1 protein measured by western blotting did not differ between tumor and non-cancerous kidney tissues. However, increased PLAGL1 protein level in ccRCC tissue positively correlated with the presence of distant metastases and worse patient outcome. CONCLUSION: These results suggest an oncogenic role of PLAGL1 in the progression of ccRCC and its potential value as a prognostic marker.

The interactions between SATB1 and F-actin are important for mechanisms of active cell death.

INTRODUCTION: The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). MATERIAL AND METHODS: The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. RESULTS: Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. CONCLUSION: We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur.

Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters.

Pleomorphic adenoma gene-like 1 gene (PLAGL1) encodes a zinc-finger nuclear transcription factor which promotes apoptosis and cell cycle arrest. Loss or downregulation of its expression has been observed in various human neoplasms. This study compared PLAGL1 expression in colorectal cancer (CRC) tissue and colon mucosa of healthy subjects at the mRNA and protein levels, and estimated its prognostic value. The PLAGL1 mRNA levels were also determined in CRC cell lines. We collected paired tumor tissue and unchanged mucosa of the large intestine from 121 CRC patients as well as 72 colon biopsies of healthy subjects obtained during screening colonoscopy. PLAGL1 mRNA levels were determined by quantitative PCR, while PLAGL1 protein expression was estimated by western blotting and immunohistochemistry. PLAGL1 mRNA level in tumor tissue was ~2-fold lower than in samples of corresponding unchanged tissues and biopsies of healthy colon mucosa. Downregulated expression of PLAGL1 mRNA was also observed in all tested CRC cell lines. Although the average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or colon mucosa of healthy individuals, the decreased PLAGL1 protein levels in tumor specimens correlated with lymph node involvement, the presence of metastases and higher TNM disease stage. The PLAGL1 expression level did not correlate significantly with patient overall survival; however, the hazard ratio for patients whose tumor tissues showed reduced PLAGL1 immunohistochemical staining was twice higher than in patients with increased PLAGL1 immunoreactivity. In conclusion, these results suggest that dysregulation of PLAGL1 expression may be involved to some extent in the progression of CRC, but the so far collected patient survival data do not confirm applicability of the PLAGL1 expression level as a prognostic factor in CRC.

Divergent expression patterns of SATB1 mRNA and SATB1 protein in colorectal cancer and normal tissues.

Special AT-rich sequence-binding protein 1 (SATB1) is a 'genome organizer,' and it has been proposed as a factor that affects the development and progression of various human neoplasms, including colorectal cancer (CRC). This study aimed to compare SATB1 expression in a group of CRC patients and healthy subjects at the mRNA and protein levels. We collected paired tumor tissue and unchanged mucosa of the large intestine from 102 CRC patients as well as 53 biopsies of normal colon mucosa obtained from healthy patients during screening colonoscopy. Tissue samples were quantified for SATB1 mRNA by quantitative PCR, while SATB1 protein expression was determined by Western blotting and immunohistochemistry. SATB1 mRNA level in tumor tissues was over twofolds lower than in samples of corresponding unchanged tissues and fourfolds lower than in biopsies of healthy colon mucosa. Western blotting analysis revealed that SATB1 protein content in tumor and unchanged tissues of CRC patients was over sixfold and fivefolds higher than in biopsies of healthy colon mucosa, respectively. Immunohistochemical staining demonstrated higher nuclear and cytoplasmic SATB1 reactivity in the tumor tissue compared to unchanged mucosa of CRC patients. Despite these differences, SATB1 mRNA, protein, and immunoreactivity levels did not correlate with patients' clinicopathological data and their overall survival, but the latter analysis was limited by a relatively short period of follow-up. In conclusion, we suggest that some as yet unidentified posttranscriptional mechanisms that regulate SATB1 expression may be altered in the CRC tissue.

Immunohistochemical visualization of pro-inflammatory cytokines and enzymes in ovarian tumors.

Epithelial ovarian cancer represents one of the most deadly gynaecological neoplasms in developed countries and is a highly heterogeneous disease. Epidemiological studies show that anti-inflammatory drugs reduce the incidence and mortality of several types of cancer, indicating the potential role of pro-inflammatory factors in carcinogenesis. The expression of pro-inflammatory factors in various cancer types, including ovarian cancer, was assessed in many studies, yielding in consistent results, often due to the histological heterogeneity of various cancers. The aim of the study was to investigate the expression of IL-1, IL-6, TGF-ß, TNF-α, COX-2, iNOS, and NF-kB in serous and mucinous ovarian cancers. Ninety cases of ovarian tumors classified into mucous and serous type (45 patients in each group) were selected. Each group was classified into subgroups according to the three stages of tumor differentiation, i.e. into (i) benign, (ii) borderline and (iii) malignant tumors. The presence of proteins of interest in paraffin sections was analysed by immunohistochemistry. The expression of most of the studied factors depended on the histological tumor subtype and the degree of malignancy. Expression of NF-κB appears to be related to the level of the neoplastic differentiation only in the group of serous tumors, while the presence of IL-6 in the mucinous tumor subtype was observed only in the case of benign lesions. Expression of IL-1, TNF-α and COX-2 increased with the stage of the disease in both serous and mucinous tumors. The highest level of TGF-ß expression was observed in serous borderline tumors. The different levels of iNOS immunoreactivity between the groups of serous and mucinous tumors were observed only in borderline tumors. The results of our study may be helpful in designing therapeutic strategies depending on the type of ovarian cancer.

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells.

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.

Expression of the vascular endothelial growth factor-receptor system in the porcine endometrium throughout the estrous cycle and early pregnancy.

The objective of this study was to investigate the protein and mRNA expression of vascular endothelial growth factor (VEGF), VEGFR-1 (fms-like tyrosine kinase, Flt-1) and VEGFR-2 (fetal liver kinase-1/kinase insert domain-containing receptor, Flk-1/KDR) in the endometrium during the estrous cycle and early pregnancy in pigs. The VEGF-receptor system was localized in epithelial and stromal cells, blood vessels, and myometrium. Western blot analysis showed higher levels of VEGF protein during the periovulatory and periimplantation periods (P < 0.001, and P < 0.05, respectively). Constant expression of VEGF mRNA during the cycle and significant upregulation on Days 22-25 of gestation (vs. Days 9-17; P < 0.001) was observed. Stable levels of VEGFR-1 mRNA and protein were detected in the endometrium of cyclic animals. However, higher VEGFR-1 protein expression was found on Days 16-17 of the estrous cycle (P < 0.01) and Days 13-15 of gestation (P < 0.05). Protein expression of VEGFR-2 was elevated on Days 2-4 of the estrous cycle (P < 0.001), but mRNA levels were constant during the cycle. In pregnancy, VEGFR-2 protein expression started to increase after Day 15 (vs. Days 9-12; P < 0.05), but induction of VEGFR-2 mRNA expression occurred earlier on Days 13-15. It appears from the present study that the VEGF-receptor system is regulated in a temporal and spatial manner during the estrous cycle and early pregnancy in pigs. The results suggest that VEGF-A family members are probably involved in appropriate preparation of endometrium for implantation and in vascular events during implantation in pigs.

Novel biological and possible applicable roles of LH/hCG receptor.

Luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors are widely expressed in gonadal cells, however, the presence of these receptors has also been demonstrated in several other non-gonadal female and male tissues. The expression level of non-gonadal LH/hCG receptors is much lower than in gonads, although their expression is regulated by similar mechanisms and they also exert biological effects using similar signaling pathways. Hormonally regulated LH/hCG receptor expression in the oviduct suggests that LH could be involved in the regulation of its contraction, gametes/embryos transport and synchronization of the fertilization. One of the major roles of the myometrial LH/hCG receptors may also be the stimulation of growth and maintenance of the uterine relaxation during pregnancy. In pigs, LH seems to be one of the pleiotropic factors which influence the endometrial prostaglandin F(2alpha) synthesis and initiation of the luteolysis. The LH/hCG receptor expression in several cancer cells provides new possibilities for developing new strategies for targeted cancer therapy based on lytic LH/hCG conjugates.

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy.

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig.

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.