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.

Expression of VEGF-A, Flt-1, and Flk-1 in the arterial endothelial cells of the uterine broad ligament throughout the estrous cycle.

The aim of the present study was to determine the immunoreactivity of vascular endothelial growth factor (VEGF-A) and its two receptors, viz., Flt-1 (fms-like tyrosine kinase) and Flk-1 (fetal liver kinase), on the surface of endothelial cells of the uterine artery and its branches and of the arcuate arteries in the area of the uterine broad ligament during various phases of the estrous cycle in the pig. We also investigated their expression to determine whether this was phase-related. The highest immunoreactivity for VEGF-A was observed in the uterine artery and arcuate arteries at the early luteal phase and in the branches of the uterine artery during the follicular phase of the estrous cycle. The strongest immunostaining intensity of Flt-1 was found in the uterine artery and its branches at the follicular phase and in arcuate arteries at the mid-luteal phase, whereas Flk-1 immunostaining was at its highest in the uterine artery at the mid-luteal phase and in the branches of the uterine artery and arcuate arteries at the follicular phase. Additionally, VEGF-A expression was assessed by semi-quantitative Western blot analysis, which revealed significantly higher levels of VEGF-A protein during the early luteal and the follicular phase of the estrous cycle (P < 0.001). The phase-related differences in the immunoreactivity and expression of VEGF-A and VEGF receptors suggest that these factors are hormone-dependent during the estrous cycle in the pig.

Epidermal growth factor and epidermal growth factor receptor immunoreactivity in the endothelial cells of the uterine artery and its branches during different stages of the estrous cycle in the pig.

The uterine artery and its branches are the most important vessels that supply the uterus with blood, nutrients and active substances. Epidermal growth factor (EGF) and its receptor (EGFR) are expressed in many tissues, including reproductive organs, and is involved in angiogenesis, embryo implantation and development as well as in proliferation and differentiation of various cells. The aim of our study was to determine EGF and EGFR immunoexpression in the uterine artery and its branches during the estrous cycle in the pig. The experiment was performed on cryostat sections of the uterine artery and its branches stained immunohistochemically by ABC method. Light microscopic observations revealed the phase-related immunoreactivity of EGF and EGFR in the endothelial cells of the uterine artery and its branches. The highest intensity of EGF and EGFR immunoreaction in endothelial cells of the uterine artery was observed in the follicular phase. A significant decrease in the intensity of EGF and EGFR immunoreactivity was found in the middle luteal phase. Similar results of the immunostaining were found with regard to EGFR. In the endothelium of the uterine arterial branches, a significant increase in the intensity of EGF and EGFR-immunoreactivity was observed in the middle luteal phase. A decrease in the intensity of EGF immunostaining was observed in the late luteal phase. The phase-related expression of EGF and EGFR in the endothelium of the uterine artery and its branches suggest the modulatory effect of EGF and its receptor on the uterine artery and the region supplying these vessels.

VEGF, VEGFR-1 and VEGFR-2 immunoreactivity in the porcine arteries of vascular subovarian plexus (VSP) during the estrous cycle.

Abstract: Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the female reproductive tract. It binds to cell surface through ligand-stimulatable tyrosine kinase receptors, the most important being VEGFR-1 (flt-1) and VEGFR-2 (flk-1). The broad ligament of the uterus is a dynamic organ consisting of specialized complexes of blood vessels connected functionally to the uterus, oviduct and ovary. Endothelial cells form an inner coating of the vessel walls and thus they stay under the influence of various modulators circulating in blood including ovarian steriods involved in developmental changes in the female reproductive system. The aim of the present study was to immunolocalize VEGF and its two receptors: VEGFR-1 and VEGFR-2 in the broad ligament of the uterus in the area of vascular subovarian plexus during different phases of the estrous cycle in pig and to determine the correlation between immunoreactivity of the investigated factors and phases of the estrous cycle. The study was performed on cryostat sections of vascular subovarian plexus stained immunohistochemically by ABC method. Specific polyclonal antibodies: anti-VEGF, anti-VEGFR-1 and anti-VEGFR-2 were used. Data were subjected to one-way analysis of variance. Our study revealed the presence of VEGF and its receptors in endothelial and smooth muscle cells of VSP arteries. All agents displayed phase-related differences in immunoreactivity suggesting the modulatory effect of VEGF, VEGFR-1 and VEGFR-2 on the arteries of the VSP in the porcine broad ligament of the uterus.

Localization and macrostructure of subuterine lymphatic vessel networks in the bovine uterine broad ligament.

Localization and morphological features of lymphatic vessels leaving tissues of uterine horns and running through the broad ligament area were studied in mature cows after filling the lumen of the each lymphangion with varicoloured masses, mainly Microfil. The study has revealed that each branch of the lymphatic vessels emerged from both sides of the uterus and then formed double-layered networks, dorsal and ventral in the area of the mesometrium. The lymphatic branches under the uterus are very numerous and consist of enlarged precollector lymphangions. At the level of the ovary, the branches of vessels in both layers interweave each other forming the common pathway suggesting that lymph leaving the bovine uterus can be mixed in branches which are formed by elongated lymphangions running to lymphatic nodes.

The localization and connections of suboviductal lymphatic vessels in the bovine uterine broad ligament.

The distribution of oviductal lymphatic vessels in the bovine mesosalpinx and bursa ovarica, and their communications with other lymphatics in the uterine broad ligament were examined after exposition of all lymphatic pathways with varicoloured microfil and/or ink-gelatin mixture. However, filling of the lumen of the oviductal infundibulum lymphatics was difficult or impossible because of its slender walls. Lymphatics of the isthmus and ampulla with short precollectors formed branches 1.5-2.0 cm long on both sides: dorsal and ventral of the uterine broad ligament. Collectors with slender and long lymphangions, visible after filling their lumen, encircled the alymphatic area in the parainfundibullar mesosalpinx. The greatest number of lymphatic branches, which originated from the paraisthmal part of the oviductal ampulla were observed in the subovarian area. The characteristic feature was their immediate proximity with uterine and oviductal arterial vessels. Subsequent studies have provided convincing evidence that there are direct connections of lymphatics leaving the oviduct and ovarian sac with lymphatics emerging from the uterus and ovary (after bilateral filling of lymphatics pathways in the whole broad liagment of the uterus). The performed investigations showed particularly characteristic feature in the cow--mixing lymph leaving various reproductive organs in the area of the right and left ligament--before two long collector branches reach the nearest lymphatic node/-s.