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.

The patency of the utero-oviductal tract during the estrous cycle in the pig.

The study of corrosion casts after filling the utero-oviductal lumen with heat-hardened resin Mercox, and scanning electron microscopy (SEM) observations of the tissue cross-sections were carried out in 63 pigs during the estrous cycle. Investigations of the corrosion casts showed that the barrier for the transportation through the utero-oviductal canal may occur in the oviductal isthmus, because this pathway was closed for Mercox resin between days 6-21 of the estrous cycle. SEM observations of the oviductal tissues confirmed that the smallest cross-sections of the porcine oviduct were found in the isthmus on 1-1.5 cm from the uterine horn, although with open lumen throughout the estrous cycle. However, in the intermediate parauterine part of the isthmus, with thick muscular layer (2850-3800 microns in a diameter) and with short strongly tighten apical mucous folds, main pathways for the transportation can be parabasal fissures, particularly on days 6-21 of the estrous cycle.

Immunoreactivities of eNOS, ET-1 and ETB-R in blood vessels of the uterine mesometrium during the estrous cycle in the pig.

Cryostat sections of arterial and venous vessels from various size branches of the uterine artery and utero-ovarian vein of the pig mesometrium in different phases of the estrous cycle were stained immunohistochemically for endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1) and endothelin B receptor (ETB-R) using ABC method. Immunoreactivity was evaluated according to 6-point scale under light microscope. The differences in immunostaining intensity in the endothelium of the vessels studied at various levels of mesometrium suggest a correlation of eNOS, ET-1 and ETB-R expression with the estrous cycle. In the follicular phase, the highest eNOS immunoreactivity was noticed in arcuate arteries and veins, while immunoreaction of ET-1 was much lower, just as ETB-R. On the other hand, the highest ET-1 immunoreactivity was observed during first 2 days afterovulation, while ETB-R showed low immunoreactivity level during the whole luteal phase. Vessels from the middle part of mesometrium (I degrees and II degrees branches) and large vascular trunks revealed similar staining for eNOS during the cycle as compared to arcuate arteries. Those vessels showed very high immunoreactivity levels for ET-I and ETB-R during first 2 days after ovulation. Our results suggest that during the estrus eNOS, ET-I and ETB-R play a significant role in the regulatory process of blood flow through the mesometrial vessels, that are connected to the uterine horn.

The connections of lymphatic vessels draining female reproductive organs with lymphnodes in the pig.

The connections of lymphatic vessels leaving the reproductive organs (ovary, oviduct, uterine horn, corpus and cervix, and vagina) with lymphnodes (normal and hemal) located in the uterine broad ligament and paraaortal regions were examined in the cyclic and immature pigs. To visualize the lymph pathways, the lymphatic lumen was filling with varicoloured microfil and/or ink-gelatin mass introduced all the way down to appearance in several nodes. This study revealed that lymphatics emanating from the uterine horn, corpus and the anterior part of the cervix demarcated large a lymphatic area in the porcine broad ligament. From the caudal portion of the cervix and vagina 2-3 lymphatics were found to emerge running near walls of these organs and connecting with large complex nodes, and 2-3 lymphatics reaching the plica urogenitalis. Generally, the lymphatic vessels leaving the porcine reproductive organs were connected by collector lymphangions with 3-5 normal-, 0-3 hemal- and one large composed nodes all found between the distal vascular subovarian plexus and the uterine artery as well as with 9-27 normal and 3-9 hemal nodes in the paraaortal region.

Immunohistochemical localisation of ET-1 and eNOS in lymphatic stomata of the porcine broad ligament of the uterus.

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in lymphatic stomata in areas of their special accumulation in the porcine broad ligament during the estrous cycle. The study was performed using polyclonal antibody for ET-1 and monoclonal antibody for eNOS. ET-1 and eNOS immunoreactivities were demonstrated in some thin endothelial lymphatic lacuna walls throughout the estrous cycle. In the mesothelial cell layer, ET-1 and eNOS were detected only in stomata-related cuboidal mesothelial cells, however, the intensity of the immunostaining and distribution of the positive cells varied during the cycle. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the tone of lymphatic stomata during the absorption and passage of fluids, particles and cells from the peritoneal cavity to lymphatic vessels in the porcine broad ligament.

The morphometric parameters of adrenal cortex in sows: in normal condition and after prolactin infusion.

The aim of this study was to examine the effect of experimental hyperprolactinemia on the stereological parameters of porcine adrenal cortex. In cyclic sows, after preovulatory luteinizing hormone (LH) peak, porcine prolactin (PRL, 0.3 mg) or saline were administered i.v. for 48 h at 2 h intervals. Next sows were slaughtered and adrenal glands were dissected. Stereological analysis of the left adrenal gland did not reveal any significant differences between control and PRL-treated sows. Experimental hyperprolactinemia did not affect the volume of particular cortical zones, the number and the volume of adrenocortical cells or the average volume of their cell nuclei. Moreover, we present for the first time a detailed stereological description of adrenal cortex in sows.

The cytoarchitectonic and neuronal structure of the red nucleus in guinea pig: Nissl and Golgi studies.

The present studies were carried out on the brains of adult guinea pigs, Dunkin-Hartley strain. On the basis of preparations, they were stained according to the Nissl and the Klüver-Barrera method's; a short description of the cytoarchitectonics and the characteristics of the rubral cells were written. The red nucleus (RN) of the guinea pig is 1.2 mm in length. Three cellular parts in RN, and three classes (A, B, C) of the rubral cells were distinguished. Taking into consideration the predominant cell size, RN was divided into magnocellular part (RNm), parvocellular part (RNp) and intermediate part (RNi). On the basis of Golgi impregnated preparations four neuronal types (I, II, III, IV) were distinguished. To sum up, in the guinea pig were observed: the large, mainly multipolar (type I) and bipolar (type II) spiny being coarse (class A) in Nissl material; the medium-sized, triangular, aspiny (type III) corresponding to the fine cells (class B); and the small, both spiny and aspiny neurons (type IV), which are the fine or achromatic cells (classes B or C) in Nissl stained slices. The highest degree of dendritic branching was observed in type I, whereas the lowest in cells of types III and IV.

Oviductal lymphatics and their relations with the paraovarian lymphatic plexus in the pig.

The lymphatic vessels emanating from the oviductal infundibulum, ampulla and isthmus were examined in the pig paraovarian sac and broad ligament walls to determine their relations with paraovarian lymphatic plexus. To differentiate the oviductal, ovarian and uterine lymphatic pathways injections of the three-coloured microfil were used. The precollector lymphatics in the paraovarian sac mesosalpinx created two networks running independently pathways towards the lymph nodes. A large multimesh network from the oviductal isthmus, especially in the late follicular and early luteal phases, together with uterine precollectors made the lymphatic plexus in subovarian areas. Both of these lymphatic networks did not possess direct connections for the lymph flow. The lymphatic system in the supraovarian sac was not evident scanty.

Evidence for the presence of luteinizing hormone-chorionic gonadotrophin receptors in the pig umbilical cord.

Pig umbilical cord, like that of humans, contains two arteries and a vein surrounded by Wharton's jelly with amnion covering the exterior surface. The aim of the present study was to investigate whether LH-hCG receptors are present in the pig umbilical cord, using light microscope immunohistochemistry, semiquantitative autoradiography, western blotting and reverse transcription-polymerase chain reaction. Umbilical cords were collected on days 48, 71 and 103 of fetal life (n = 6). Monoclonal and polyclonal anti-LH receptor antibodies were used to study receptor distribution. Immunoreactivity was observed in the umbilical blood vessels, the epithelium of umbilical amnion and cells in the Wharton's jelly. No differences in LH-hCG receptor distribution related to the sex of the fetus, period of fetal life or section of the umbilical cord were observed. Strong immunostaining was observed in umbilical vein and in umbilical arteries. However, in the arteries, the tunica media expressed weaker receptor immunostaining than did the tunica intima and tunica adventitia. No immunoactivity was detected in non-target tissue (skeletal muscle) but LH receptors were immunostained in the pig ovary. Topical autoradiography showed that vein and arteries in the umbilical cord bind 125I-labelled hCG, which was highly diminished after co-incubation with an excess of unlabelled hCG. The binding of 125I-labelled hCG to the Wharton's jelly and epithelial amnion was less intense than it was to vessels. Gonadotrophin binding sites were not present in the skeletal muscle. The pig umbilical arteries, vein and Wharton's jelly contained a 75 kDa immunoreactive LH-hCG receptor protein similar to that found in corpora lutea. Southern blot analysis of reverse transcription-polymerase chain reaction products, performed to enhance the sensitivity and specificity of LH receptor transcripts determination in umbilical cord tissues, revealed that the expected fragments of 740 and 470 bp were present in the arteries, vein, Wharton's jelly and corpora lutea (positive control). An additional product of 670 bp was found in the corpora lutea and arteries of umbilical cord, but not in the vein and Wharton's jelly. This is probably the first reported evidence of the presence of LH-hCG receptors in the umbilical cord of a non-human female mammal.

Morphological features of lymphatic and mesothelial communications in the broad ligament of the pig.

The broad ligament containing uterine, paraovarian, and oviduct lymphatics was examined in the pig in various phases of the estrous cycle using light, scanning and transmission electron microscopy. The architecture of these regions differed and was independent of the lymphangions of the precollector and collector lymphatic vessels. Lymphangions were separated from mesothelium by connective tissue and/or muscle layers; however, in the vicinity of the thin walled paraovarian sac, large lymphangions were often compressed between two epithelial layers. Numerous lymphatic lacunae were in direct contact with the peritoneal and paraovarian sac cavities. The mesothelial lining of the broad ligament and the external and internal epithelium of the pig paraovarian sac displayed two distinct cell types. Only smaller cuboidal cells with prominent microvilli extended above the lymphatic endothelium. The surfaces of these cells were discontinuous and showed: 1) lymphatic stomata, 2) small pores or fenestrae, 3) a superficial network of epithelial-free communications with underlying connective tissue to the paraovarian sac in the postovulatory period independent of the lymphatic vasculature, and 4) endothelial (instead of epithelial) cells with crevice-like discontinuities in large portions of the internal sac surface during the follicular phase of estrus. Numerous lymphatic stomata had orifices composed of flattened cuboidal cells while lymphatic endothelial cells were characterized by macula or zonula adherent connections formed within rims of various sizes (up to 50 microns in diameter). During estrus, there were circular (0.5-2.0 microns) and irregular (to 10 microns) interendothelial openings in stomatal orifices with migrating cells. These morphologic findings suggest that absorption and passage of fluid, particles and cells between cavities and the lymphatic lumen in areas of the paraovarian lymphatic plexus in the pig is feasible.

Differentiation of lymphatics from blood vessels in the broad ligament of the swine using S-100 protein immunohistochemical localization in the vascular endothelium.

We examined the immunohistochemical staining characteristics of S-100 protein in the vessels of the broad ligament of the swine uterus. The endothelial cells of arterial vessels, lymphatics and blood capillaries as well as nerve fiber bundles showed S-100 protein positivity. In contrast, the endothelial cells of veins did not react for the S-100 antiserum. Immunoreactivity for S-100 protein in the endothelial cells of lymphatics did not consistently demonstrate strong staining intensity. Accordingly, we filled lymphatics with colored gelatin before immunohistochemical staining to facilitate identification of lymphatics under light microscopy. Numerous arterioles and capillaries (of which the endothelial cells were immunopositive for S-100 protein) in the lymphatic walls, especially those in the paraovarian vascular plexus, support the existence of a microvascular arterio-arterial rete mirabile network.

Lymphatic vessels in the broad ligament of the uterus in swine.

The lymphatics of the broad ligament were depicted in 24 pigs by multiple interstitial injections of latex into the ovary and subserosa of the mesometrial margin of the uterine horn. Two morphologically different networks of lymphatics emanating from the uterus and ovary were established. Ovarian lymphatics leave the hilus, invariably enter the parovarian plexus, lie superficially under the perimetrium of the mesovarium, and run a parallel course closely intertwined with the blood vessels in this region. Some lymphatics are thus located close to the branches of the ovarian artery and the utero-ovarian vein. Whereas the ovarian and uterine lymphatics in the swine are not directly connected, the uterine lymphatics enter the mesovarium and lie in close proximity to both the ovarian lymphatics and nearby blood vessels.