A. Andronowska

Mechanisms for the Establishment of Pregnancy in the Pig

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle.

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.

The effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 on chorioamnion secretion of prostaglandins (PG)F2α and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.

Influence of estradiol-17β and progesterone on nitric oxide (NO) production in the porcine endometrium during first half of pregnancy

The purpose of the study was to examine: 1/ endometrial concentrations of nitrate/nitrite (NOx) in pregnant pigs, and 2/ the influence of estradiol-17beta (E(2)) and/or progesterone (P(4)) on NOx production by porcine endometrium during the first half of pregnancy. Total NOx concentrations were determined using a microplate assay method based on the Griess reaction. Evident fluctuations of endometrial NOx content were found during the examined time of pregnancy (days 5, 10, 15, 20, 25, 30, 35, 40 and 60 of pregnancy). The NOx concentration was highest on days 10 and 15, and then lowered until day 60 of pregnancy. In addition, we demonstrated the stimulatory effect of E(2) and/or P(4) on NO in vitro production by porcine endometrial slices. The medium content of NOx depended on the steroid type, treatment dose and day of pregnancy. It is possible that the observed differences in the strength of the stimulatory action of E(2) and/or P(4) on endometrial NOx production are associated with activation of different isoforms of NOS.

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.

Semen biology of vendace (Coregonus albula L.)

The objective of this study was to describe the morphometry and motility parameters of vendace (Coregonus albula) spermatozoa. Morphometric parameters of vendace sperm head and tail were of values similar to rainbow trout. The effects of pH, sodium, potassium and calcium ion concentrations on computer-assisted sperm analysis (CASA) sperm motility characteristics were tested. Vendace sperm was motile in a wide pH range of 6.0–10.5 with the optimum pH established at 9.0. Increases in potassium and calcium ions caused decreases in the percentage of motile sperm. The CASA parameters and erratic sperm movement pattern of vendace spermatozoa were similar to whitefish (C. lavaretus) sperm motility, suggesting that there is a coregonid-specific sperm motility pattern.

Endothelin-1 and Endothelial Nitric Oxide Synthase Immunoreactivity in Lymphatic Vessels of the Uterine Broad Ligament during the Estrous Cycle in the Pig

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in precollector and collector lymphangions of lymphatic vessels leaving the ovary and were found in the vascular subovarian plexus (mesovarium) as well as in those emanating from the oviductal isthmus and uterine horn (mesosalpinx and mesometrium, respectively) forming the paraovarian lymphatic plexus in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and the monoclonal antibody for eNOS isoform were used for studies on the light-microscopic level. Immunoreactivities to both ET-1 and eNOS were observed in the endothelial cell cytoplasm of precollector and collector lymphangions and were not demonstrated in smooth muscle cells of the lymphatics examined. In the endothelium, the intensity of immunostaining for ET-1 and eNOS was found to be estrous phase-dependent and differed between precollector and collector lymphangions. In general, immunoreactivity to ET-1 was more intense in the endothelium of shrunken lymphangions, whereas that for eNOS was more intense in lymphangions with the large lumen. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the vascular contractile activity promoting lymph flow during the estrous cycle in the porcine broad ligament.

Immortalization of Swine Umbilical Vein Endothelial Cells (SUVECs) With the Simian Virus 40 Large-T Antigen

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G‐1410, using Simian virus 40 T‐antigen (SV40 T‐ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T‐ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G‐1410 cells did not differ morphologically from SUVECs. The G‐1410 cells exhibited positive staining for vascular endothelial (VE)‐cadherin and von Willebrand factor (vWF), and formed capillary‐like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T‐ag, these transformed G‐1410 cells have remained karyotypically normal and non‐tumorigenic. G‐1410 cells also responded to stimulation with VEGF, FGF‐2, and newborn calf serum. Moreover, G‐1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF‐A), and FGF‐2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G‐1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis. Mol. Reprod. Dev. 78:597–610, 2011.

Expression of the Vascular Endothelial Growth Factor (VEGF‐A) and its Receptors in the Umbilical Cord in the Course of Pregnancy in the Pig

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF-VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF-A, VEGFR-1 and VEGFR-2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT-PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR-1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR-2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF-A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR-1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR-2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF-VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF-A and VEGFR-1, but not VEGFR-2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF-VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.

mRNA and protein expression of FGF-1, FGF-2 and their receptors in the porcine umbilical cord during pregnancy.

The fibroblast growth factors (FGFs) are multifunctional proteins that, among other roles, regulate structural reorganization of uterine and placental vascular bed during pregnancy. Thus, we analyzed mRNA and protein expression and immunohistochemical localization of FGF-1 and FGF-2, and their receptors (FGFR-1 and FGFR-2) in the developing umbilical cord (UC) on days 40, 60, 75 and 90 of pregnancy and after the physiological delivery in the pig (day 114). qPCR analysis demonstrated an increase in FGF-1 and FGF-2 mRNA levels beginning on day 75 and on day 114 of pregnancy, respectively. In addition, significantly increased FGFR-1IIIc mRNA expression was also found on day 114. On the other hand, no significant changes in FGFR-2IIIb mRNA expression were observed. Western Blot analysis revealed a decrease in FGF-1 and FGFR-2 protein expression after day 40. Beside an increased protein expression of FGF-2 on day 60, no significant changes in FGFR-1 protein expression were detected. Immunohistochemical staining enabled detection of FGF-FGFR system, with different intensity of immunoreaction in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium as well as in myofibroblasts. In conclusion, our results show that members of FGF-FGFR system are expressed specifically in UC structures. Furthermore their day of pregnancy-related expression suggest that they may be an important players during UC formation and development.