Modulatory effect of chemokines on porcine endometrial stromal and endothelial cells.

The endometrium undergoes cyclical changes during the estrous cycle and pregnancy. These alterations are controlled by various factors, including cytokines. The present study aimed to screen the effect of several chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL9, CXCL10, and CXCL12) on endometrial stromal and endothelial cells. Real-time PCR analysis revealed mRNA expression of all examined chemokines and their receptors in primary stromal cells and undetectable levels of CXCL9, CXCL10, and CXCR3 in endothelial cells. Immunocytochemical staining showed variable distribution of chemokine receptors in stromal and endothelial cells. All examined chemokines enhanced stromal cell proliferation, and CCL2 and CXCL12 also increased the migratory potential of these cells. The evaluation of a possible indirect effect of chemokines on angiogenesis and lymphangiogenesis demonstrated that CXCL12 may potentially negatively affect lymphatic vessel creation. Downregulation of VEGFC mRNA and protein expression was noticed after CXCL12 stimulation. Among all examined chemokines, CCL4 and CCL8 positively affected the proliferation and migration of endothelial cells. The number of capillary-like structures was significantly reduced after CXCL8, CXCL10, and CXCL12 stimulation. In conclusion, among all examined chemokines, CCL2 is thought to act as the modulator of stromal cell functions, whereas CCL4 and CCL8 are suggested to be potent factors directly stimulating blood vessel creation.

Author Correction: Chemokines as the modulators of endometrial epithelial cells remodelling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chemokines as the modulators of endometrial epithelial cells remodelling.

Previous studies highlighted chemokines as potential factors regulating changes in the endometrium during early pregnancy. The current study aimed to screen the effects of a broad range of chemokines and indicate those that are involved in porcine luminal epithelial (LE) cell remodelling. Messenger RNA expression of chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL10 and CXCL12) and both the mRNA and protein expression of their receptors (CCR1, CCR2, CCR3, CCR5, CXCR2, CXCR3, CXCR4) were detected in LE cells. Exogenous CCL8 enhanced the proliferative and migration potential of LE cells and their motility in the environment with its stable concentration. The adhesive properties of LE cells were negatively affected by CCL8. However, CXCL12 positively affected the proliferation, motility and adhesion of LE cells as well as caused a decrease in MUC1 mRNA expression. To conclude, our studies determined that exogenous chemokines affected critical endometrial epithelial cell functions in the context of embryo implantation. We suggest that of all the examined factors, chemokine CCL8 participates in the establishment of a proper environment for embryo implantation, whereas CXCL12, apart from participation in endometrial receptivity, promotes embryo attachment.

Presence of trophoblast in the uterine lumen affects VEGF-C expression in porcine endometrium.

Early pregnancy is associated with morphological and functional changes within the uterus, accompanied by angiogenesis, increased vascular permeability and activation of immune tolerance. Intensive angiogenesis leads to accelerated vascular leakage and accumulation of interstitial fluid in endometrium. To protect the trophoblast from the harmful effect of extracellular fluid, process known as lymphangiogenesis is crucial. These studies are focused on VEGF-C, factor responsible of lymphatic vessels creating, and its receptors: Flk1 (VEGFR2) and Flt4 (VEGFR3) during the time of implantation as well as the effect of trophoblast signals (IFNG and E2) on VEGF-C production. Endometrial samples were collected from mature gilts from days 8, 10, 12, 14 of estrous cycle and pregnancy. Real-Time PCR analysis revealed increased mRNA expression of VEGF-C on days 10, 12, 14 of pregnancy compared to corresponding days of estrous cycle. The highest VEGF-C mRNA expression was observed on 14 day of pregnancy (p < 0.05). Increased mRNA expression of Flk1 and Flt4 was noticed on day 14 of pregnancy in comparison to day 10. Enhanced Flk1 mRNA expression during 14 day of pregnancy was observed compared to corresponding day of estrous cycle (p < 0.05). No significant difference on the protein level was revealed. VEGF-C and its receptors were localized mainly in luminal and glandular epithelial cells, but their presence were confirmed also in endothelial cells of blood and lymphatic vessels and 14 d trophoblasts. In vitro studies revealed positive effect of IFNG on VEGF-C mRNA expression in stromal cells and protein content in medium after stromal cells culture (p < 0.05). Our studies demonstrated the presence of VEGF-C system in porcine endometrium and indicated its possible important role during the time of implantation.

Lipopolysaccharide and cytokines modulate leukotriene (LT)B4 and LTC4 production by porcine endometrial endothelial cells.

Uterine inflammatory response is mediated by inflammatory mediators including eicosanoids and cytokines produced by immune and endometrial cells. Interactions between lipopolysaccharide (LPS) and cytokines, and leukotrienes (LTs) in endothelium, important for the host defence during the inflammation, are unknown. We studied the effect of LPS, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4 and IL-10 on 5-lipooxygenase (5-LO), LTA4  hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA and protein expression, LTB4 and LTC4 release from porcine endometrial endothelial cells, and cell viability. For 24 hr, cells were exposed to LPS (10 or 100 ng/ml of medium) and cytokines (each 1 or 10 ng/ml). 5-LO mRNA/protein expression augmented after incubation with larger doses of LPS, TNF-α, IL-4 and IL-10 and smaller dose of IL-1ß. Larger dose of TNF-α, smaller doses of LPS and IL-1ß and both doses of IL-10 increased LTAH mRNA/protein expression. LTAH protein content was up-regulated by larger dose of LPS, but it was reduced in response to both doses of IL-4. LTCS mRNA expression was elevated by larger doses of LPS, IL-4 and IL-10 or both doses of TNF-α and IL-1ß. LTCS protein level increased after treatment with both doses of IL-1ß, IL-4 and IL-10, smaller dose of LPS and larger dose of TNF-α. Both doses of LPS and larger doses of TNF-α and IL-10 increased LTB4 release. LPS, IL-1ß and IL-10 at smaller doses, or TNF-α and IL-4 at larger doses stimulated LTC4 release. Smaller doses of TNF-α and IL-1ß or both doses of IL-4 enhanced the cell viability. This work provides new insight on the participation of LPS, TNF-α, IL-1ß, IL-4 and IL-10 in LTB4 and LTC4 production/release from porcine endometrial endothelial cells, and the effect of above factors on these cells viability. The used cellular model gives the possibility to further establish the interactions between inflammatory mediators.

Downregulation of LH and FSH receptors after hCG and eCG treatments in the porcine oviduct.

The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor-positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct.

Prostaglandins are well-known mediators of crucial events in the female reproductive tract, eg, early embryo development and implantation. Prostacyclin (PGI2) is the most synthesized prostaglandin in the human oviduct during the postovulatory period, indicating its important role in supporting and regulating the oviductal environment. The present study was undertaken to determine the influence of insemination and ovarian stimulation with human chorionic gonadotropin (hCG)/equine chorionic gonadotropin (eCG) on PGI2 synthesis in the porcine oviduct on day 3 post coitus. Mature gilts (n = 25) were assigned into 2 experiments. In experiment I, gilts were divided into cyclic (control; n = 5) and inseminated (control; n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5), and superovulated/inseminated (1,500 IU eCG, 1,000 IU hCG; n = 5) gilts. Parts of oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (cyclic gilts of experiment I) or insemination (all other groups). Expression of messenger RNA for PGI2 synthase (PGIS) and its receptor (IP) was measured by real-time reverse transcription polymerase chain reaction (real-time RT PCR) and protein levels using Western blots. Concentrations of the PGI2 metabolite 6-keto PGF1α were evaluated by enzyme immunoassay and localization of PGIS and IP in the oviductal tissues using immunohistochemical staining. Insemination by itself increased PGIS protein levels in the oviductal isthmus (P < 0.05) and IP protein expression in the ampulla (P < 0.05). The concentration of 6-keto PGF1α increased significantly in the oviductal ampulla after insemination (P < 0.05). Induction of ovulation decreased IP protein levels in the oviductal ampulla (P < 0.05), whereas superovulation reduced IP levels in both parts of the oviduct (P < 0.01). Synthesis of 6-keto PGF1α was reduced by induction of ovulation and by superovulation in the oviductal ampulla (P < 0.05). Immunohistochemical staining confirmed the presence of PGIS in the muscular layer of the isthmus and both mucosa and muscular layers of the ampulla. IP-positive cells were observed in both mucosal and muscular layers of the isthmus and ampulla. This study showed for the first time that PGI2 synthesis and IP expression are insemination dependent. Moreover, ovarian stimulation with hCG/eCG decreases IP expression and 6-keto PGF1α concentrations in porcine oviducts. Therefore, disturbances in PGI2/IP expression and synthesis may lead to disruption of the oviductal environment and, in turn, perturbed development of embryos and their transport to the uterus.

Expression of factors associated with apoptosis in the porcine corpus luteum throughout the luteal phase of the estrous cycle and early pregnancy: their possible involvement in acquisition of luteolytic sensitivity.

The studies on the acquisition of luteolytic sensitivity have been focused mainly on molecular changes induced in the luteal tissue after treatment with exogenous PGF2α or on physiological changes occurring during the estrous cycle. The comparison of changes leading to the acquisition of luteolytic sensitivity after Day 12 of the estrous cycle and corresponding days of pregnancy has not been investigated in the pig. The present study was undertaken to evaluate (1) apoptosis measured as the proportions of early apoptotic, late apoptotic, and viable cells; (2) expression of factors involved in the extrinsic (TNFA/TNFα, TNFRSF1A/TNFR1, TNFRSF1B/TNFR2, FAS/Fas, and FASLG/FasL) and intrinsic (CASP3/Casp3, TP53/p-53, BAX/Bax, and BCL2/Bcl-2) apoptotic pathways, with two components of the activating protein-1 complex, i.e., FOS/Fos and JUN/Jun and IFNG/IFNγ; and (3) concentrations of luteal and blood plasma progesterone (P4) throughout the luteal phase of the estrous cycle and early pregnancy. Corpora lutea (CL) were collected postmortem on Days 8, 10, 12, and 14 of the estrous cycle and the corresponding days of pregnancy. The luteal tissue was subjected to RNA and/or protein isolation and disaggregation of CL cells followed by flow cytometry analysis aimed to determine apoptotic changes. Luteal and blood plasma P4 concentrations decreased on Day 14 of the estrous cycle versus pregnancy (P < 0.05 and P < 0.001, respectively). A significant increase in the number of early apoptotic cells and a decrease in the number of viable cells were observed on Day 14 of the estrous cycle (P < 0.001 and P < 0.05, respectively). Increase (P < 0.05) of TNFA messenger RNA (mRNA) level coincided with that of IFNG on Day 12 of the estrous cycle but not on the corresponding day of pregnancy. The content of FAS mRNA and protein increased on Day 14 of the estrous cycle versus pregnancy (P < 0.05). The mRNA expression of CASP3, BCL-2 and BAX was unchanged in cyclic and pregnant CL, while level of TP53 increased (P < 0.05) on Day 12 of the estrous cycle versus Day 8. The level of FOS and JUN mRNA increased (P < 0.05) on Day 14 of the estrous cycle versus the remaining days. The level of FOS and JUN mRNA was significantly higher (P < 0.001 and P < 0.05, respectively) on Day 14 of the estrous cycle than that on the corresponding day of pregnancy. In summary, the simultaneous increase of TNFA and IFNG transcript in cyclic CL suggests the crucial role of both cytokines in sensitization of porcine CL to further luteolytic action of PGF2α. The upregulated expression of FAS, FOS, and JUN mRNA in the late luteal phase in cyclic CL can indicate their involvement in structural luteolysis. The increased viability of luteal cells and elevated P4 concentrations in pregnant CL confirm the protective role of luteal P4 against apoptosis.

Expression of the vascular endothelial growth factor receptor system in porcine oviducts after induction of ovulation and superovulation.

This study was performed to determine the influence of insemination as well as treatment with human chorionic gonadotropin (hCG) and equine chorionic gonadotropin (eCG) on expression of the vascular endothelial growth factor (VEGF) system in porcine oviducts. In the first experiment, 10 gilts were assigned to 2 groups: cyclic (treated with phosphate-buffered saline; n = 5) and inseminated (n = 5). In experiment II, 15 gilts were assigned to 3 groups: inseminated (control; n = 5), induced ovulation and inseminated (750 IU eCG, 500 IU hCG; n = 5), and superovulated and inseminated (1500 IU eCG, 1000 IU hCG; n = 5). Oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (experiment I) or insemination. Blood samples were collected during slaughter for E2 (estradiol) and P4 (progesterone) analysis. Levels of messenger RNA (mRNA) of the VEGF system were analyzed by real-time polymerase chain reaction and protein by Western blot and E2 and P4 using radioimmunoassays. Insemination by itself decreased VEGF120 mRNA expression and VEGF-A protein level in the oviductal isthmus (P < 0.05) but did not alter VEGF164 mRNA. Expression of Flt-1 (c-fms-like tyrosine kinase VEGFR-1) mRNA increased in the isthmus of inseminated relative to cyclic gilts (P < 0.05), whereas KDR (fetal liver kinase-1 VEGFR-2) mRNA levels decreased in both the oviductal isthmus (P < 0.05) and ampulla (P < 0.001). Superovulation decreased VEGF120 and VEGF164 mRNA expression in the isthmus compared with the inseminated group (P < 0.05), and lowered protein levels of VEGF-A in the isthmus of both stimulated groups (P < 0.001). Expression of Flt-1 mRNA was affected by hCG and eCG treatment in both gonadotropin-stimulated groups in the isthmus as well as in the ampulla (P < 0.001) and protein levels in the ampulla of superovulated gilts (P < 0.05). Protein levels of KDR were reduced in the oviductal ampulla of gilts in both the induced ovulation and superovulated groups (P < 0.05). The concentrations of both E2 and P4 increased significantly in superovulated group of gilts (P < 0.01 and P < 0.05 for E2 and P4, respectively). Our study showed that insemination alone as well as ovarian stimulation affected the mRNA and protein profiles of the VEGF system in the porcine oviduct. Disrupted VEGF system expression may be crucial to many events occurring during the periovulatory period and consequently could lead to deprivation of VEGF-dependent factors that are necessary for proper fertilization, gamete transport, and embryo development.

Pro- and anti-inflammatory mediators change leukotriene B4 and leukotriene C4 synthesis and secretion in an inflamed porcine endometrium.

We studied the effect of lipopolysaccharide (LPS), proinflammatory cytokines (tumor necrosis factor α [TNF-α] and interleukin [IL]-1ß), and anti-inflammatory cytokines (IL-4 and IL-10) on leukotriene (LT) A4 hydrolase and LTC4 synthase (LTCS) protein expression in, and LTB4 and LTC4 secretion from, an inflamed porcine endometrium. On day 3 of the estrous cycle (day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) CFU/mL) was injected into each uterine horn of gilts (n = 12 per group). Endometrial explants, obtained 8 and 16 days later, were incubated for 24 h with LPS (10 or 100 ng/mL of medium), TNF-α, IL-1ß, IL-4, and IL-10 (each cytokine: 1 or 10 ng/mL of medium). Although acute endometritis developed in all bacteria-inoculated gilts, a severe form of acute endometritis was diagnosed more often on day 8 of the study than on day 16. The amount of the LTA4 hydrolase (LTAH) protein in the inflamed endometrium on day 8 was greater after applying the lower dose of TNF-α (P < 0.001) and both doses of IL-1ß (P < 0.001) and IL-4 (1 ng, P < 0.01 and 10 ng, P < 0.001) than in the saline-treated uteri. A similar situation was observed in the case of the inflamed tissue on day 16 in response to LPS (100 ng, P < 0.01), TNF-α (10 ng, P < 0.05), and IL-4 (1 ng, P < 0.001). The content of LTC4 synthase in the inflamed endometrium on day 8 was reduced by LPS (100 ng, P < 0.05), IL-1ß (10 ng, P < 0.05), IL-4 (1 and 10 ng, P < 0.05), and IL-10 (1 ng, P < 0.01) but increased after the application of LPS (100 ng, P < 0.05) and TNF-α (1 and 10 ng, P < 0.001), IL-1ß, and IL-4 (1 ng, P < 0.05 and 10 ng, P < 0.001) on day 16. On day 8, endometrial secretion of LTB4 from the saline-injected and E coli-injected organs was similar in response to all of the used mediators. On the other hand, the contents of LTB4 in the medium decreased after incubating the inflamed tissues from day 16 with TNF-α (1 ng, P < 0.05 and 10 ng, P < 0.01), IL-1ß (1 ng, P < 0.01), and IL-10 (10 ng, P < 0.05) compared with the saline-treated ones. Secretion of LTC4 from the inflamed uteri on day 8 was elevated by the lower doses of TNF-α (P < 0.01) and IL-10 (P < 0.05), whereas on day 16, such an effect occurred in response to the higher doses of IL-4 (P < 0.01) and IL-10 (P < 0.05). The obtained results show that pro- and anti-inflammatory mediators participate in the synthesis/secretion of LTs from an inflamed porcine endometrium. Our data suggest that inflammatory mediators may indirectly affect the processes regulated by LTs by influencing LT production.

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology, regulation of sperm motility and interpopulation diversity.

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species-specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl2 , respectively). Sperm maintained a high motility rate over a wide pH range (5.5-10.5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.

Effect of vaginal administration of prostaglandin E2 and/or 17beta-estradiol on luteal function and histological characteristics of the cervix in cyclic pigs.

The overall objective of this study was to examine the effect of vaginal administration of prostaglandin E2 (PGE2) and/or 17beta-estradiol (E2) on luteal function maintenance and histological properties of the porcine cervix. For this purpose, crossbred gilts were divided into three groups (n=5 per group) supplied on days 11-16 of the estrous cycle with suppositories containing: (1) placebo (Group I, Control); (2) 0.4 mg of E2 (Group II); (3) 0.4 mg of E2 and 2 mg of PGE2 (Group III). Blood samples were collected on days 11-19 of the estrous cycle to determine the concentration of progesterone (P4). Additionally, to examine local effects of the hormones applied, segments from the uterine and vaginal parts of the cervix and from the ovaries were collected post-mortem. Prolonged luteal function and extended synthesis of P4 were observed in 2 of 5 gilts receiving PGE2 and E2 simultaneously (Group III). Then, these gilts were subdivided into Group IIIA (n = 2; presence of corpora lutea on the ovaries) and Group IIIB (n = 3; lack of corpora lutea). Increased levels of plasma P4 were observed in Group IIIA on days 15-19 compared to Group IIIB and on days 16-19 compared to Group I and Group II (P < 0.05; P < 0.01; P < 0.001, respectively). In the cervix of gilts in Groups II and III, enlarged blood vessels in the lamina propria of both parts of the cervix were observed. Furthermore, in Group II the epithelium of the uterine part of the cervix was thicker (P < 0.001). Our study confirmed the proposed luteotrophic/antiluteolytic actions of E2 and PGE2 applied intravaginally. These results are significant considering that very low doses of E2 were used when compared to previous attempts. Despite the inadequate response to treatments in some of the gilts, the local effects of these hormones on the histological properties of the porcine cervix suggest that further improvements in the vaginal administration route might help to elaborate new methods for enhancing the luteal function in the pig.

The novel effect of hCG administration on luteal function maintenance during the estrous cycle/pregnancy and early embryo development in the pig.

Two independent experiments were performed on cyclic (Experiment I) and pregnant (Experiment II) gilts to examine the effect of human Chorionic Gonadotropin (hCG) administration on day 12 of the estrous cycle/pregnancy on ovarian and endometrial secretory function. Animals were divided into hCG Group (injection of 750 IU hCG) and Control Group (injection of saline). In Experiment I, the prolonged lifespan of the corpus luteum (CL), extended progesterone (P4) production (P < 0.05) and delayed luteolysis were found. In hCG Group increased ratio of PGE2:PGFM during 12 hrs period on day 15 (P < 0.05) of the estrous cycle was observed. In both experiments, higher concentrations of E2 in hCG treated gilts (P < 0.05) on days 14-15 of the estrous cycle/pregnancy were found. In Experiment II, hCG injection did not affect P4, PGE2 and PGFM concentrations in blood plasma, but reduced the number of resorbed embryos on day 30 of pregnancy. In the pregnant hCG treated gilts the immunostaining against von Willebrand Factor (vWF) demonstrated an enhanced (P < 0.05) angiogenesis in CLs and endometrium. Furthermore, the flow cytometry revealed an increased (P < 0.05) viability of cells in CLs of hCG Group. An augmented expression of Steroidogenic Acute Regulatory Protein (STAR; P < 0.05) and LH/hCG receptor mRNA (P < 0.05) in CLs of hCG Group were observed, but an elevated concentration of protein was confirmed only for STAR (P < 0.05). Our studies revealed, for the first time, that administration of hCG affects PGE2:PGFM ratio during the estrous cycle as well as the development of conceptuses through enhanced angiogenesis and decreased luteal apoptosis in early pregnant pigs.

Effect of hCG and eCG treatments on prostaglandins synthesis in the porcine oviduct.

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross-bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p < 0.05, p < 0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p < 0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES-1 mRNA levels in Groups III and IV, in both the isthmus (p < 0.01 in III, p < 0.001 in IV) and ampulla (p < 0.001). The prostaglandin E2 content of oviductal tissues was significantly lower in Groups III (p < 0.05) and IV (p < 0.01 in isthmus, p < 0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p < 0.01) compared with the non-treated Group II. In effect, the amount of prostaglandin F2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p < 0.001 in isthmus of Groups III and IV; p < 0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.

Expression of nerve growth factor and its receptors in the uterus of gilts with endometritis induced by infection with Escherichia coli.

Nerve growth factor (NGF) is a neurotrophin implicated in the pathophysiology of allergy, diseases of the immune system and inflammation. Expression of NGF and its receptors (TrkA and p75) has not been examined in inflamed uterine tissue. The aim of the present study was to evaluate the patterns of NGF, TrkA and p75 expression in normal and inflamed porcine uteri using real-time reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry. On day 3 of the oestrous cycle, 50 ml of saline or 50 ml of Escherichia coli suspension containing 10(9) colony forming units/ml, was injected into both uterine horns of control (n = 10) and experimental (n = 10) gilts, respectively. Infected animals developed moderate or severe acute endometritis after 8 days and moderate acute or subacute endometritis after 16 days. On day 8 of the study, the expression of NGF and TrkA mRNA and protein was higher in the endometrium of gilts with endometritis than in normal tissue. NGF protein was also more prominently expressed in the endometrium of those animals with endometritis 16 days post infection. There was no difference in endometrial p75 mRNA and protein expression on either day. The myometrium of animals in both groups showed no difference in expression of any of the three molecules. Immunohistochemically, on day 8 there was greater expression of NGF and TrkA by the luminal epithelium and some glandular and endothelial cells as well as the myometrium of the uteri of gilts with endometritis compared with normal tissues. At day 16, there was greater expression of NGF by the luminal epithelium and of TrkA by some endothelial cells and the myometrium, compared with control tissue. There is therefore, up-regulation of NGF and TrkA mRNA and protein expression in the porcine uterus when affected by E. coli-induced inflammation. NGF and TrkA may have roles in this inflammatory process and/or in the innervation of the uterus.

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.

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.

Expression patterns of endothelial and inducible nitric oxide isoforms in the porcine umbilical cord.

Stable fetal-placental blood pressure and flow are extremely important in fetal growth and development. Uncontrolled and long-standing increased or decreased vascular blood pressure in the umbilical cord (UC) affects hyperaemia or ischaemia and consequently causes fetal death. Nitric oxide (NO) is one of the most active factors controlling blood flow through relaxation of the vascular smooth muscle. In this study, we investigated endothelial (eNOS) and inducible (iNOS) nitric oxide synthase expression and NADPH-diaphorase activity (NADPH-d) in the porcine UC at various stages of pregnancy. The UCs were collected from pigs on days 40, 60, 75 and 90 of pregnancy and postpartum. Western blot analysis as well as immunohistochemical staining revealed protein presence for eNOS and iNOS in the UC of the pig. The eNOS expression was maintained at a significantly higher level in all analysed days of pregnancy when compared with postnatal stage. Additionally, a significant protein increase for eNOS was observed in a periplacental part of UC on day 90. There were no obvious differences in iNOS protein level in UC samples derived from different stages of pregnancy. NADPH-diaphorase histochemical activity was correlated with NOS immunoreactivity during all analysed days of pregnancy. These results suggest that NOS isoforms are responsible for regulation of blood circulation in UC and immune responses.

The influence of E2 and P4 on in vitro NOx production in the porcine myometrium during first half of pregnancy.

The aim of the present study was to determine 1) concentrations of NOx in the myometrium of pregnant gilts, and 2) the influence of estradiol-17beta (E2) and/or progesterone (P4) on NOx production by the porcine myometrium on days 5, 10, 15, 20, 25, 30, 35, 40 and 60 of pregnancy (n = 5 per day). Total NOx concentrations were determined using a microplate assay method based on the Griess reaction. During the first 60 days of gestation, a triphasic pattern in the concentration of NOx in the porcine myometrium was observed with a peak on days 10-15, 30 and 60 of gestation. We also demonstrated the stimulatory effect of E2 and/or P4 on in vitro NO production by the porcine myometrium. The stimulatory effect of steroid hormones on NOx release depended on the treatment dose of steroids and day of pregnancy. These data suggest that locally produced NO may inhibit spontaneous uterine contraction and therefore is involved in the maintenance of myometrial quiescence during pregnancy.