Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term.

Low efficiency of somatic cell cloning by nuclear transfer has been associated with alterations of placental vascular architecture. Placental growth and function depend on the growth of blood vessels; VEGF-A and bFGF are the most important factors controlling neovascularization and vascular permeability in the placenta. We hypothesize that the VEGF-A and bFGF systems are disrupted in placentomes from cloned animals, contributing to the placental abnormalities that are common in these clones. We determined mRNA expression and protein tissue localization of VEGF-A, bFGF, and their receptors in placentomes from cloned and non-cloned bovine fetuses at term. Real-time RT-PCR revealed that VEGFR-2 mRNA was increased in cloned male-derived placentomes, while mRNA of bFGF and its receptors were decreased in placentomes of cloned females. VEGF-A system proteins were found to be located in placentomal endothelial, maternal and fetal epithelial and stromal cells; there was a variable pattern of cellular distribution of these proteins in both cloned and non-cloned animals. Alterations in the expression of VEGF-A and bFGF systems suggest that angiogenic factors are involved in abnormal placental development in cloned gestations, contributing to impaired fetal development and poor survival rates.

Morpho-physical recording of bovine conceptus (Bos indicus) and placenta from days 20 to 70 of pregnancy.

The study is based on 141 pregnant Bos indicus cows, from days 20 to 70 post-insemination. First, special attention was given to the macroscopically observable phenomena of attachment of the conceptus to the uterus, i.e. the implantation, from about days 20 to 30 post-insemination up to day 70, and placentome development by growth, vascularization and increase in the number of cotyledons opposite to the endometrial caruncles. Secondly, as for the conceptuses, semiquantitative, statistical analyses were performed of the lengths of chorio-allantois, amnion and yolk sac; and the different parts of the centre and two extremes of the yolk sacs were also analysed. Thirdly, the embryos/foetuses corresponding to their membranes were measured by their greatest length and by weight, and described by the appearance of external developmental phenomena during the investigated period like neurulation, somites, branchial arcs, brain vesicles, limb buds, C-form, pigmented eye and facial grooves. In conclusion, all the data collected in this study from days 20 to 70 of bovine pregnancy were compared extensively with corresponding data of the literature. This resulted in an 'embryo/foetal age-scale', which has extended the data in the literature by covering the first 8 to 70 days of pregnancy. This age-scale of early bovine intrauterine development provides model for studies, even when using slaughtered cows without distinct knowledge of insemination or fertilization time, through macroscopic techniques. This distinctly facilitates research into the cow, which is now being widely used as 'an experimental animal' for testing new techniques of reproduction like in vitro fertilization, embryo transfer and cloning.

Influence of the magnesium aspartate hydrochloride administration to the maternal circuit on the aspartate concentration of the fetal circuit under in vitro perfusion of human placenta.

OBJECTIVES: Magnesium aspartate hydrochloride (Magnesiocard, Mg-Asp-HCl) is proposed as a substitute of magnesium sulfate for the treatment of preeclampsia and premature labor. After an i.v. administration of a dose equivalent to that used in the treatment of preeclampsia to nonpregnant volunteers, a 10-fold increase of aspartic acid (Asp) over the physiological level was observed. Animal experiments have demonstrated that highly increased fetal levels of acidic amino acids such as Asp could be associated with neurotoxic damage in the fetal brain. The influence of such an elevation of Asp concentration in the maternal circuit on the fetal level, using the in vitro perfusion model of human placenta, was investigated. STUDY DESIGN: After a control phase (2h), a therapeutic dose of Mg combined with Asp (Magnesiocard, Mg-Asp-HCl) was applied to the maternal circuit approaching 10 times the physiological level of Asp. The administration was performed in two different phases simulating either a peak of maximum concentration (bolus application, 2h) or a steady state level (initially added, 4h). RESULTS: In four experiments, during experimental phases (6h) a slow increase in concentration in the fetal circuit was seen for Mg, AIB (alpha-aminoisobutyric acid, artificial amino acid) and creatinine confirming previous observations. In contrast, no net transfer of Asp across the placenta was seen. A continuous decrease in the concentration of Asp on both maternal and fetal side suggests active uptake and metabolization by the placenta. Viability control parameters remained stable indicating the absence of an effect on placental metabolism, permeability and morphology. CONCLUSION: Elevation of Asp concentration up to 10 times the physiological level by the administration of Mg-Asp-HCl to the maternal circuit under in vitro perfusion conditions of human placenta has no influence on the fetal level of Asp suggesting no transfer of Asp from the maternal to fetal compartment. Therefore, the administration of Mg-Asp-HCl to preeclamptic patients would be beneficial for the patients without any impact on placental or fetal physiology.

Immunohistochemical localization of oestrogen receptors alpha and beta, progesterone receptor and aromatase in the equine placenta.

The functions of placental oestrogens during equine pregnancy are still unclear. Yet, they may act predominantly as local regulators of growth and differentiation in the microplacentomes. Thus, expression patterns of oestrogen receptors (ERs) alpha and beta were investigated in the microcotyledonary placenta from pregnant mares at 110, 121, 179, 199 and 309 days of gestation by immunohistochemistry. In microplacentomes, both the ER isoforms were detected in trophoblast (T) cells, chorionic villous stroma (FS), microcaruncular epithelium (ME) and microcaruncular stroma (MS). Proportions of positive cells were 38-91% (T), 11-41% (FS), 55-89% (ME), 17-51% (MS) for ERalpha and 66-76% (T), 21-37% (FS), 41-68% (ME) and 24-55% (MS) for ERbeta. Between days 110 and 199, proportions of cells positive for progesterone receptor (PR) varied between 19% and 62% (T), 3% and 50% (CS), 15% and 46% (ME), and 4% and 33% (MS). At day 309, PR was virtually absent in T, CS and ME (percentages < 0.1), whereas in MS 14.3% of cells were still positive. The expression of ERs and PR in equine microplacentomes gives evidence for a role of placental steroids as regulators of placental growth, differentiation and function. The detection of ERalpha, ERbeta and PR in foetal and maternal vascular tissue suggests that placental steroids are also involved in the control of placental angiogenesis and /or vascular functions. The co-localization of ERs with aromatase in T suggests auto- or intracrine functions of oestrogens in this cell type.

Gross and microscopic anatomy of the pineal gland in Nasua nasua--coati (Linnaeus, 1766).

Nasua nasua, coati, is a mammal of the Carnivora order and Procyonidae family. It lives in bands composed of females and young males. The pineal gland or epiphysis of brain is endocrine, producing the melatonin. Its function is the control of the cycle of light environment, characteristic of day and night. For this research, five adult coatis were used, originating from CECRIMPAS-UNIfeob (Proc. IBAMA 02027.003731/04-76), Brazil. The animals were killed and perfusion-fixed in 10% formaldehyde. Pineals were measured and a medium size was found to be 2.3-mm-long and 1.3-mm-wide. Pineal gland was located in the habenular commissure in the most caudal portion of the third ventricular roof, lying in a dorso-caudal position from the base to the apex. Pinealocytes were predominantly found in the glandular parenchyma. Distinct and heterogeneous arrangements of these cells throughout the three pineal portions were observed as follows: linear cords at the apex, circular cords at the base of the gland, whereas at the body a transition arrangement was found. Calcareous concretions could be observed in the apex. The pineal gland was classified as subcallosal type [Rec. Méd. Vét.1, 36 (1956)] and as AB type [Prog. Brain Res. 42, 25 (1979); The Pineal Organ, Berlin/Heidelberg: Springer-Verlag (1981)].

Histological changes of the feline cervix, endometrium and placenta after mid-gestational termination of pregnancy with aglepristone.

This study was conducted to investigate endometrial and placental structural changes that occurred in response to mid-gestational termination of pregnancy in queens using aglepristone, a progesterone receptor antagonist. Thirteen European Shorthair pregnant queens were either treated with aglepristone (10 mg/kg body weight; subcutaneously) twice on days 25 and 26 after first mating (am; group I; n = 9), or remained untreated and served as control (group II; n = 4). Queens of group I were ovariohysterectomized between days 30 and 41 am, either at the onset (n = 3) or during (n = 1) abortion and 12 h (n = 1), 24 h (n = 3) or 10 days after abortion (n = 1). Queens of group II were ovariohysterectomized on day 30 am. Tissue was collected from the cervix, and the interplacental zone as well as the paraplacenta/placental girdle of the uterus, subjected to standard histological procedures and evaluated using light microscopy. During abortion, gaps appeared within the paraplacenta and the placental girdle which were filled with blood, leading to an embryo-maternal disconnection. Blood was also observed within the uterine lumen as well as the interstitial mucosal stroma of the cervix and the placental girdle zone and probably originated from damaged venules, whilst arterioles remained intact. As the interval between abortion and surgery increased, the interstitial and luminal haemorrhages became less pronounced and completely disappeared except interstitial remnants 10 days after abortion. The endometrial regeneration was not fully completed on day 10 after abortion and a few cystically dilated glands were evident. In conclusion, abortion of queens through aglepristone given during mid-gestation is assumed to be the result of damage of uterine venules. This leads to an interstitial haemorrhages and bleeding into the uterine lumen, subsequently resulting in utero-placental detachment.

Light and scanning electron microscopic study on the blood vascular system of the donkey placenta.

The donkey placenta is diffuse and epitheliochorial with numerous microplacentomes consisting of a fetal microcotyledonary and a maternal microcaruncular part. The microplacentomal vasculature during the last third of pregnancy has been investigated by light microscopy in comparison to scanning electron microscopy of the materno-fetal contact surface and corrosion casts of blood vessels after plastic instillation from either the microcotyledonary or the microcaruncular side, and, for the first time in a perissodactyle, from both sides. Morphological data were semiquantitatively evaluated. The supplying parts of both, the microcotyledonary and the microcaruncular vascular system are strictly proximo-distally oriented, thus reaching the capillary systems or working parts in the shortest way possible. The straight course of the vasculature, particularly on the fetal side, suggests the occurrence of venulo-arteriolar back diffusion. The fetal capillary system consists of convolutes confronting the maternal septal capillary complexes in a countercurrent way. This materno-fetal blood flow interrelationship is highly efficient in terms of placental exchange, which is further supported (1) by dilations and increasing coiling of the fetal venular capillary limbs in particular and (2) by a decrease in the interhaemal distance from 12.5 to 7.2 microm between the two capillary systems. Besides the countercurrent blood flow interrelationship, some maternal branch arterioles reach the septal capillary system from the maternally oriented pole of the microplacentome or microcaruncle, respectively, resulting in the less efficient crosscurrent blood flow. Hence, in the donkey placenta fetal and maternal blood vessels meet in a mix of countercurrent and crosscurrent flow patterns.

Bovine caruncular epithelial cell line (BCEC-1) isolated from the placenta forms a functional epithelial barrier in a polarised cell culture model.

In the bovine synepitheliochorial placenta key sites of fetal-maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither alpha-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (+/-1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal-maternal communication, transport and infection.

Development of an in vitro model for bovine placentation: a comparison of the in vivo and in vitro expression of integrins and components of extracellular matrix in bovine placental cells.

BACKGROUND/AIMS: Interaction of trophoblastic integrins with the extracellular matrix plays a role in embryo implantation and trophoblast invasion. The phenomenon of restricted trophoblast invasion, observed in the bovine epitheliochorial placenta offers intriguing conditions to study invasive processes. The migration of bovine trophoblast giant cells is accompanied by the expression of specific integrins and corresponding extracellular matrix ligands. METHODS: Primary cultures of different cell populations from cow placentomes were established and characterized, and in vitro phenotypes were compared with in vivo conditions by immunofluorescence. RESULTS: Propagated epithelial cells were positive for cytokeratin and vimentin, while fibroblasts contained alpha-smooth muscle actin, desmin and vimentin. Epithelial cells coexpressed integrin subunits alpha(6) and beta(1) with laminin, and fibroblast cells were positive for alpha(v), beta(3), fibronectin and laminin. In contrast to cells in vivo, cultured epithelial cells secreted fibronectin, while collagen IV was not detected. The occurrence of integrin subunits was confirmed at mRNA level by RT-PCR. CONCLUSION: We have established cell cultures isolated from maternal and fetal components of bovine placentomes expressing typical cytoskeletal filaments and integrin receptors also present in their in vivo counterparts. These bovine placentomal cells provide a suitable in vitro model for the study of cell-cell interactions.

Placentation in cloned cattle: structure and microvascular architecture.

To elucidate the morphological differences between placentas from normal and cloned cattle pregnancies reaching term, the umbilical cord, placentomes and interplacentomal region of the fetal membranes were examined macroscopically as well as by light and scanning electron microscopy. In pregnancies established by somatic nucleus transfer (NT), the umbilical cord and fetal membranes were edematous. Placentomal fusion was common, resulting in increased size and a decreased number of placentomes. Extensive areas of the chorioallantoic membrane were devoid of placentomes. An increased number of functional or accessory microcotyledons (<1 cm) were present at the maternally oriented surface of fetal membranes. Extensive areas of extravasated maternal blood were present within the placentomes and in the interplacentomal region. The crypts on the caruncular surface were dilated and accommodated complexes of more than one primary villus, as opposed to a single villus in non-cloned placentae. Scanning electron microscopy of blood vessel casts revealed that there was also more than one stem artery per villous tree and that the ramification of the vessels failed to form dense complexes of capillary loops and sinusoidal dilations as in normal pregnancies. At the materno-fetal interface, however, the trophoblast and uterine epithelium had normal histology. In conclusion, the NT placentas had a range of pathomorphological changes; this was likely associated with the poor clinical outcome of NT pregnancies.

Validation of primary epitheloid cell cultures isolated from bovine placental caruncles and cotyledons.

In order to study feto-maternal interactions in the bovine synepitheliochorial placenta primary cell cultures of both placentomal components throughout pregnancy, namely caruncular epithelial cells and trophoblast cells were developed. The aim of this study was to validate and improve a method to culture caruncular epithelial cells and fetal trophoblast from manually separated placentomes. Prior to seeding the presence of fetal cells in caruncular samples and vice-versa could be demonstrated by the detection of the Y-chromosome via fluorescence in situ hybridization (FISH) provided the fetus was male. Epitheloid shaped cells present in both cultures (cotyledon and caruncle) were characterized on a morphological basis as well as by immunofluorescence and Western blot thereby detecting cytokeratin, zonula occludens-1 and vimentin but not alpha-smooth muscle actin and desmin. The absence of the Y-chromosome demonstrated the caruncular origin of epitheloid cells. In addition, a population of polygonally shaped cells derived from the cotyledon was propagated and displayed the same cytoskeletal characteristics as described above. The presence of the Y-chromosome confirmed the fetal origin of these cells and the lacking uptake of fluorescence conjugated low density lipoprotein, specific for endothelial cells, identified polygonally shaped cells as fetal trophoblast cells. In conclusion, the cross-contamination of maternal and fetal cells in manually separated placentomes should be considered in future experiments as it may lead to false positive results dependent on the sensitivity of the method applied. This study highlights the importance of an appropriate cell characterization and identification, especially when isolating primary cells.

Microvascular architecture of the near-term uterine caruncles in water buffaloes (Bubalus bubalis).

The present investigation was carried out on five near-term pregnant water buffaloes for studying the microvascular architecture of the uterine caruncles. The vascular casts were obtained by injection of 4:1 mixture of mercox and methylmethacrylate through the branches of the uterine arteries. After complete polymerization of the plastic, corrosion was conducted in 20% potassium hydroxide, then the vessel casts were immersed in distilled water, cut into small pieces, sputter coated with gold, and examined by using a scanning electron microscope. The buffalo uterine caruncle is highly vascularized through two slightly convoluted arteries and a single less tortuous vein. The arteries branch into several stem arteries at the base of the uterine caruncle, which follow nearly straight course in the primary septa towards the fetal side. During the courses of these stem arteries arterioles of variable diameters arise. The arterioles run in the secondary and tertiary septae and at this location arterioles and venules are connected through a voluminous capillary complex. The latter consists of capillaries of greatly variable diameters with vigorous coiling and sinusoidally dilated zones. From the capillary complexes the blood is driven through postcapillary venules back to the tertiary, secondary and primary septa, respectively, and then converge into stem veins which leave the caruncles through the branches of the uterine vein.

VEGF system expression in different stages of estrous cycle in the corpus luteum of non-treated and superovulated water buffalo.

Water buffaloes are easily adaptable animals, whose raising and economical exploitation have been growing in the last three decades all over the world. Hyperstimulation of ovarian function in this species is a common technique aiming to improve reproductive performance. Superovulatory treatment affects corpus luteum (CL) function, which is highly correlated to angiogenic process. The aim of this study was therefore to assess the temporal protein and mRNA expression of VEGF and its receptors in the CL of non-treated and superovulated buffaloes. For that purpose blood samples and CL from 36 healthy (30 untreated, groups 1-5, and 6 superovulated, group 6) non-pregnant buffaloes were collected and the samples were divided into 6 groups according to the age of CL. Plasma samples were submitted to RIA to measure progesterone concentration and CL were subjected to immunohistochemistry and real time PCR for VEGF (vascular endothelial growth factor), Flt-1 (fms-like tyrosine kinase receptor 1) and KDR (kinase insert domain containing region). The VEGF system protein and mRNA expression during CL life span of untreated animals showed a specific time-dependent profile, although protein did not always reflect mRNA concentrations. VEGF expression in luteal cells was high correlated to plasma progesterone levels. Superovulated CL showed a significant increase of the VEGF-system protein and a significant decrease of mRNA expression compared to untreated animals in the same stage of the oestrous cycle. We conclude that VEGF, Flt-1 and KDR protein and mRNA expression in buffalo CL is dependent of estrous cycle stage and superovulatory treatment is able to increase the translation rate of this system.

The glycosylation of pregnancy-associated glycoproteins and prolactin-related protein-I in bovine binucleate trophoblast giant cells changes before parturition.

Binucleate trophoblast giant cells (BNC) in the bovine placenta produce glycoproteins, which are delivered into the mother after fusion of BNC with uterine epithelial cells. During most time of pregnancy, BNC produce pregnancy-associated glycoproteins (PAGs) and prolactin-related protein-I (PRP-I) with asparagine-linked lactosamine-type glycans terminating with N-acetyl-galactosamine. We show by lectin histochemistry that terminal N-acetyl-galactosamine (detected by Dolichos biflorus agglutinin, DBA) in placentomal BNC is greatly reduced prior to parturition, while lactosamine-type N-glycans (detected by Phaseolus vulgaris leucoagglutinin, PHA-L) remain unaltered. The change in DBA-staining showed no statistically significant differences between placentomes of cows with and without retention of fetal membranes. Western blots revealed that, at parturition the apparent molecular mass of PAGs and PRP-I is 1-2 kDa lower than in late pregnancy. These changes are due to alterations of asparagine-linked glycans, since the molecular weight of the peptide backbones after enzymatical release of asparagine-linked glycans is identical at late pregnancy and parturition. Lectin western blots showed a reduction of terminal N-acetyl-galactosamine on PAGs at parturition. A lectin sandwich-ELISAwas used to differentiate DBA- and PHA-L-binding PAGs in sera of pregnant and non-pregnant cows. The values for DBA-binding PAGs at parturition were not significantly different from non-pregnancy, while the values for PHA-L-binding PAGs were significantly higher at parturition. The peripartal changes of PAG- and PRP-I-glycosylation could alter functional properties of these proteins and might therefore be considered for functional studies. The differentiation of PAG glycoforms in maternal serum could be valuable for a further optimization of PAG-based pregnancy diagnosis in cattle.

Structural and haemovascular aspects of placental growth throughout gestation in young and aged mares.

This investigation was carried out to study the equine placenta from early gestation to near term, with special reference to morphological changes associated with the development of the vasculature of the fetal component of the microcotyledons. Pregnant uteri were removed post mortem from five Thoroughbred mares between 110 and 309 days of gestation, two of which were aged, multiparous animals suffering age-related degenerative changes in their endometrium (endometrosis), while the other three were young, and had primigravid normal healthy uteri. Pieces of endometrium with placenta attached were fixed for light microscopy and fetal vascular casts were made by injecting the placental arteries with a mixture of Mercox and methylmethacrylate. The casts were examined under the scanning electron microscope. In an aged, endometrotic mare at 110 days of gestation, most of the microplacentomes were irregular in shape with a mean+/-sem diameter of 399+/-30.53 microm. Capillaries with variable diameters made up widely meshed network villi with pointed ends (Type 1 terminal villi), and narrow-meshed networks with finger-like ends (Type 2 terminal villi). In the "paired" young healthy mare at day 121 of gestation, most of the microplacentomes were globular in shape and appeared smaller in diameter than those in the 110-day "pair". The narrow-meshed capillary networks formed villi with stems that consisted of both intermediate and terminal parts, the latter of which represented more the Type 2 than the Type 1 terminal villus. In another aged endometrotic mare at 179 days of gestation, the microplacentomes were typically globular in shape and they showed a mean diameter of 534+/-36.07 microm. The villi were short and thick and they were distinctly differentiated into stem, intermediate and terminal parts. The density of the fetal capillaries had now greatly increased so that, three dimensionally, they constituted bulb-like capillary networks at the base of the stem of each villus. At 199 days in the young healthy "pair", the microplacentomes were again smaller in diameter (402+/-16.24 microm) than in the old mare at 179 days and the interhaemal distance had now reduced to 14.28+/-0.42 microm. The vascular density was lower than in the day 179 aged mare and the fetal villi were much longer and thinner. In the single late stage, healthy young mare at 309 days of gestation (term=336 days), the microplacentomes, each of around 2 mm diameter, exhibited maximal length villi. The capillaries were arranged simply, mostly in straight lines along the axis of the villus, and with communications visible at irregular intervals. Simple and slightly more complicated side capillary loops could be seen along the whole length of the villi and at the top of the terminal villi. Most of the capillaries were characterized by zones containing dilated sinusoids, which increased the surface area for materno-fetal exchange. Thus, the morphological development of the microplacentomes on the surface of the horse placenta during gestation was studied, with special reference to the growth and organisation of the fetal and maternal capillary beds within each microplacentome. The study also reinforced previous work showing the disadvantageous influence of age-related endometrial degenerative changes on microplacentome development and on both the extent and intimacy of physical and haematological contact at the fetomaternal interface, and hence upon fetal growth.

Fibroblast growth factor (FGF)-1, FGF2, FGF7 and FGF receptors are uniformly expressed in trophoblast giant cells during restricted trophoblast invasion in cows.

The bovine placenta is characterized by a limited invasion of trophoblast giant cells (TGC). In contrast to mononuclear trophoblast cells (MTC), TGC are non-polarized cells, which migrate and fuse with single uterine epithelial cells throughout gestation. Fibroblast growth factors (FGF) were shown to be associated with the migratory activity of cells, cell differentiation and angiogenesis, and due to its localization in trophoblast cells were proposed as important regulating factors in hemochorial placentae of rodents and humans, and the (syn)epitheliochorial placenta of pig and sheep. Since migrating bovine TGC are of epithelial origin, but exhibit similarities to mesenchymal cells we hypothesize that the restricted trophoblast invasion in cattle is characterized by a specific FGF expression pattern. Therefore, the spatiotemporal expression of specific FGF factor:receptor pairs, either acting on cells of mesenchymal origin or on epithelial cells was examined in bovine placental tissues throughout gestation and prepartum by immunohistochemistry, semiquantitative RT-PCR and in situ hybridization. FGF1 protein was found in trophoblast, caruncular epithelium (CE) and stroma (CS), stroma of chorionic villi (SCV), and in fetal and maternal blood vessels. FGF2 signals dominated in maternal vascular endothelia (VE), immature TGC, and MTC, whereas staining in other cell types was clearly weaker. FGF7 protein was detected in fetal and maternal blood vessel as well as in immature TGC and MTC predominantly at the chorionic plate. FGFR immunoreaction was localized in immature TGC, MTC, and to a clearly lesser extent in CS, CE and fetal and maternal blood vessels. Mature TGC stained negatively for all examined factors and FGFR. The corresponding mRNAs specific for FGF1, -2, -7, total FGFR, and FGFR2 isoforms IIIb and IIIc were colocalized in immature TGC, whereas hybridization was substantially lower in CE and absent in CS, SCV and mature TGC throughout gestation, but switched to CS and VE immediately prepartum. Semiquantitative RT-PCR revealed higher mRNA levels for FGF1, FGFR, and FGFR2IIIc in cotyledons compared to caruncles (p<0.05), whereas it was the opposite with FGF2 (p<0.001). FGF7 and FGFR2IIIb mRNA levels did not differ between caruncles and cotyledons. Significant changes (p<0.05) of mRNA levels related to gestational age were found for FGF1 and FGFR2IIIc, but not for FGF2, -7, total FGFR, and FGFR2IIIb. The specific localization of all examined FGF family members in TGC suggests that TGC, apart from their classical function as producers of hormonal products, play other important roles in the regulation of bovine placentomal growth, differentiation and angiogenesis.

Expression of gap junctional connexins 26, 32 and 43 in bovine placentomes during pregnancy.

Gap junctional connexins (Cx) are induced in the endometrium during implantation in rodents, the human receptive window, and in the decidua Cx26 and Cx43 expression increases in response to trophoblast invasion. In contrast, this gap junctional response and decidualization is absent in non-invasive epitheliochorial placentae of pigs and horses. Bovine (syn)epitheliochorial placentation represents an intermediate type of trophoblast invasion, since it is characterized by the continuous migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells. Therefore the objective of the present study was to investigate the expression of Cx26, Cx32, and Cx43 in placental tissues during bovine pregnancy, to determine if Cx expression patterns correlate with the depth of trophoblast invasion. Cx26, Cx32, and Cx43 proteins were detected by immunohistochemistry and corresponding specific mRNAs were shown by RT-PCR and localized in tissue sections by in situ hybridization. Cx26 protein was detected at the feto-maternal contact interface and as cytoplasmic staining in TGC. Cx26 mRNA was located in maternal epithelium and in TGC. Cx32 protein expression was observed in the maternal epithelium exclusively on the tips of maternal septa, whereas Cx32 mRNA was detected in all maternal epithelial cells and single TGC. Cx43 protein and mRNA were coexpressed in TGC. Cx43 protein was present in maternal septal stroma and to a lesser extent in chorionic villous mesenchyme, while Cx43 mRNA was associated with the vasculature. In the course of gestation, expression of Cx26, Cx32, and Cx43 did not change. In conclusion, the intermediate invasive status of bovine trophoblast is supported by the fact that TGC coexpress Cx26, Cx32, and Cx43, which may be important for trophoblast migration (invasion), and fusion with maternal epithelial cells. Cx32 could be involved in the control of invasion.

Localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in bovine placentomes from implantation until term.

Interactions of vascular endothelial growth factor (VEGF) with its receptors VEGFR-1 and VEGFR-2 promoting angiogenesis have been described in placentation of human, mink and pig. The bovine placenta is multiplex, villous and synepitheliochorial due to migratory trophoblast giant cells (TGC). To determine the role of VEGF in bovine implantation and placentation, placentomes and interplacentomal areas from 33 cows from early implantation until near term were evaluated by immunohistochemistry. VEGF immunoreactivity was detected in fetal and maternal blood vessel tissues during implantation and throughout gestation, and in preimplantatory trophoblast cells and uterine epithelium. After implantation the immunoreaction was confined to TGC and uterine epithelium. An antibody against bovine VEGF revealed a strong reactivity in the stroma of maternal caruncular septa in early and mid-gestation, which distinctly decreased near term. In interplacentomal areas, VEGF was found in luminal and glandular epithelia as well as in trophoblast, with distinctly higher reactivity in giant cells. VEGFR-1 was observed in trophoblast and uterine epithelium around implantation. Later, in definite placentomes, VEGFR-1 was localized in TGC near the chorionic plate and in maternal endothelial cells in the center of the placentome. VEGFR-1 and VEGFR-2 were co-localized in uterine epithelium and trophoblast as well as in blood vessel tissue and uterine glands. The presence of VEGF, VEGFR-1 and VEGFR-2 at the feto-maternal interface and in vasculature indicates that in the bovine VEGF may have (1) classic functions in angiogenesis and vascular permeability, (2) growth factor properties, facilitating feto-maternal exchange via paracrine action, (3) chemotactic activity on capillary endothelium, and (4) an autocrine influence on TGC migratory activity.

Morphological and immunocytochemical investigations on mast cells in porcine ureter.

Morphological, morphometric, histochemical and immunocytochemical investigations on mast cells, located in the wall of ureter of 8 months aged pigs were performed. Mast cells were found in all three layers of ureteral wall, but their distribution was irregular and the number unequal. It was established that alcian blue (AB)-positive mast cells were significantly more than toluidine blue (TB)-positive mast cells. A statistically significant smaller number of both AB and TB-stained mast cells were observed in the tunica mucosa. The largest number of mast cells was found in the tunica muscularis. In the adventitia, mast cells were higher in number in the main connective tissue than in the connective tissue near the blood vessels. Mast cells stained with TB showed variably expressed gamma-metachromasia, which was best visible in those situated in the lamina propria of the mucosa. The prevailing parts of mast cells, however, were AB-positive after AB-safranin staining. This was mostly found in mast cells of the tunica muscularis and in mast cells of perivascular location in the tunica adventitia. Immunocytochemically, mast cells were found to be positive for histamine and vasoactive intestinal polypeptide in the muscle coat, and to histamine in the adventitia, as well. On the basis of obtained results it was presumed that the mast cells in porcine ureter most probably took part not only in keeping of local homeostasis, but played also an important role of mobility of smooth muscle cells in the middle layer of ureter on one hand, and, on the other, in the adventitial blood vessels.

Glial fibrillary acidic protein and myelin basic protein as markers for the immunochemical detection of bovine central nervous tissue in heat-treated meat products.

Because bovine central nervous tissue (CNT) is the main risk material in transmission of the infective agent of bovine spongiform encephalopathy, a suitable test is needed to enforce the ban on CNT in human foodstuffs in the United States and the European Union and to ensure that meat products are free of CNT To detect bovine CNT in heat-treated meat products, we used immunohistochemistry and Western blots with antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). Both antigens were resistant to processing methods used for meat products. The anti-GFAP antibody showed a high degree of tissue specificity, whereas the anti-MBP antibody had high species specificity, clearly differentiating between porcine and bovine CNT Therefore, immunochemistry performed with both proteins provides an effective means for detecting bovine CNT in meat products.