Jolanta Kiewisz

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig.

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15-16 of gestation (P<0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15-16 of pregnancy (P<0.01 and P<0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL (P<0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) beta1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72h later. Gilts in the control group (n=23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P<0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P<0.05) and 15 (P<0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFbeta, LIF, and PGHS-2 on day 12 of pregnancy (P<0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

Seminal plasma affects prostaglandin synthesis in the porcine oviduct

Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF(2α) synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE(2) to PGF(2α), expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE(2) to PGF(2α) and PGFM to PGF(2α) ratios in the porcine oviduct.

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs.

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE(2), PGF(2alpha), and PGFM (PGF(2alpha) metabolite) content, as well as PGE(2) synthase (mPGES-1) and PGF(2alpha) synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10-11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE(2) in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE(2) content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE(2) accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF(2alpha) content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.

Effect of conceptus on transforming growth factor (TGF) β1 mRNA expression and protein concentration in the porcine endometrium--in vivo and in vitro studies.

Abstract Transforming growth factor (TGF) β and its receptors are expressed at the conceptus-maternal interface during early pregnancy in the pig. The present studies were conducted to examine: (1) the effect of conceptus products on TGFβ1 mRNA expression and protein concentration in the porcine endometrium using in vivo and in vitro models, and (2) the effect of TGFβ1 on proliferation of porcine trophoblast cells in vitro. During in vivo experiments, gilts with one surgically detached uterine horn were slaughtered on days 11 or 14 of the estrous cycle and pregnancy. For in vitro studies, endometrial explants and luminal epithelial (LE) cells co-cultured with stromal (ST) cells were treated with conceptus-exposed medium (CEM). Moreover, porcine trophoblast cells were treated with TGFβ1, and the number of viable cells was measured. On day 11, the presence of conceptuses had no effect on TGFβ1 mRNA expression, but decreased the TGFβ1 protein concentration in the connected uterine horn compared with the detached uterine horn. In contrast to day 11, on day 14 after estrus, TGFβ1 mRNA expression and protein content in the endometrium collected from the gravid uterine horn were greater when compared with the contralateral uterine horn. The treatment of endometrial slices with CEM resulted in greater TGFβ1 mRNA expression and protein secretion. LE cells responded to CEM with an increased TGFβ1 mRNA level. Moreover, TGFβ1 stimulated the proliferation of day 14 trophoblast cells. In summary, porcine conceptuses may regulate TGFβ1 synthesis in the endometrium at the time of implantation. TGFβ1, in turn, may promote conceptus development by increasing the proliferation of trophoblast cells.

Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs - involvement of steroid hormones

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E(2)) and progesterone (P(4)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

Global gene expression profiling of porcine endometria on Days 12 and 16 of the estrous cycle and pregnancy

The objective of the study was to investigate transcriptomic profile of pig endometrium on Days 12 and 16 of pregnancy in comparison with the respective days of the estrous cycle. Labeled complementary DNA was hybridized to Porcine Long Oligo microarray containing 13,297 oligonucleotide probes, which represented complementary DNA and expressed sequence tags. Statistical analysis revealed 110 differentially expressed genes (DEGs) on Day 12 of pregnancy and 179 DEGs on Day 16 of pregnancy. In silico analysis of gene function and functionality networks revealed links between genes implicated in cell death and survival, protein synthesis, lipid metabolism, cellular movement, tissue development, and cell-to-cell signaling. On Day 12 of pregnancy, estrogen, transforming growth factor (TGF) β1, and fibroblast growth factor (FGF) 2, and on Day 16 of pregnancy, epidermal growth factor (EGF), insulin, interleukin 11 (IL-11), and FGF family members were indicated as possible upstream regulators of several DEGs. Obtained results showed changes in global endometrial gene expression at the time of maternal recognition of pregnancy and embryo implantation. Additionally, these data revealed signaling molecules, which together with E2, may evoke molecular changes in the uterus, leading to successful pregnancy establishment.

Estrus synchronization affects WNT signaling in the porcine reproductive tract and embryos

The purpose of the study was to investigate an effect of estrus synchronization with prostaglandin (PG) F(2α) and PMSG/hCG on WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression. The weight of the uterus, morphometrical parameters of the endometrium and the number of CL were recorded. The analysis of estradiol (E(2)), prostaglandin (PG) F(2α) and E(2) content in the uterine luminal flushings (ULFs) and progesterone (P(4)) level in the blood serum were conducted. RNA was isolated from endometrial, luteal and embryonic tissue of pregnant non-synchronized (Control; n = 15) and pregnant synchronized (PGF(2α)/PMSG/hCG; n = 15) pigs. Whereas there was no change in uterine weight, differences in height of endometrial surface and glandular epithelium were found. However, height of the endometrium, number of the glands and capillaries were unaffected. The total number of the CLs was higher (P < 0.05) in animals treated with PGF(2α)/PMSG/hCG. The amount of E(2) and P(4) was lower (P < 0.05, P < 0.001, respectively) in pregnant gilts administrated with PGF(2α)/PMSG/hCG. The concentration of PGF(2α) in ULFs was not affected by hormonal management, while PGE(2) was higher (P < 0.01) in hormonally in comparison to non-hormonally treated pigs. The content of WNT4 mRNA in conceptuses increased on particular Days studied in Control and PGF(2α)/PMSG/hCG administered animals. WNT7A and CTNNB1 were affected by PGF(2α)/PMSG/hCG treatment in both conceptuses (P < 0.001, P < 0.05) and endometrial tissue (P < 0.001, P < 0.01). The PGF(2α)/PMSG/hCG treatment resulted in elevated expression of WNT4 (P < 0.001) and CTNNB1 (P < 0.05) in luteal tissue in comparison to the Control gilts. Moreover, luteal amount of WNT5A mRNA was higher in PGF(2α)/PMSG/hCG animals in comparison to the Control group (P < 0.05). Presented data show that exogenous hormones administration can affect gene expression in the porcine reproductive tract and embryo.

Participation of WNT and β-Catenin in Physiological and Pathological Endometrial Changes: Association with Angiogenesis

WNT proteins are involved in embryonic development, sex determination, stem cell recruitment, angiogenesis, and cancer. They take part in morphological changes in the endometrium during development, regulate processes of endometrial proliferation and differentiation. This review presents current knowledge about implication of WNT proteins and β-catenin in physiological endometrial functions as well as their involvement in uterine carcinogenesis. Influence of WNT proteins on the formation of blood vessel, taking place both under healthy and pathological conditions, is also considered. Participation of WNT proteins, β-catenin, and inhibitors and inducers of WNT signaling in the process of endometrial angiogenesis is largely unknown. Thus, confirmation of their local and systemic participation in the process of endometrial angiogenesis may in the long term help to establish new diagnostic and therapeutic approaches in conditions associated with the pathology of the female reproductive system.

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.