Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro.

INTRODUCTION: The feto-maternal interface during bovine implantation was studied in vivo and using three-dimensional bovine endometrial (BCECph) and trophoblast spheroids (CCS), each with underlying fibroblasts. METHODS: The expression of ezrin and cytokeratin 18 (CK18) was analyzed via immunohistochemistry (IHC), RT-PCR and western blotting in bovine endometrium (GD 18-44) with in vivo (VIVO) and in vitro-produced embryos (VITRO). BCECph were stimulated with cotyledon-conditioned media (CCM) and analyzed by TEM/SEM and IHC. CCS were stained (IHC) for TGC markers, to test if spheroidal trophoblast cells had differentiated into TGC. RESULTS: At GD 20, caruncular epithelium (CE) and uterine glands (UG) showed a loss of cytosolic ezrin and CK18 followed by a complete loss of both proteins. At GD 35 both reappeared in CE and UG. The endometrial expression pattern did not differ between VIVO and VITRO. RT-PCR and western blotting confirmed the presence of ezrin and CK18. All spheroids had an outer polarized, cytokeratin and ezrin positive epithelium (CE or trophoblast) with apical microvilli. Stimulation of BCECph with CCM induced similar changes in ezrin expression as observed in endometrial tissue. However, no ultrastructural alterations were found by transmission electron microscopy. Absence of TGC-specific glycoproteins in CCS indicated that TGC differentiation was not induced by three-dimensional culture conditions. DISCUSSION: Ezrin and CK18 are downregulated during implantation in cattle. The expression changes represent a temporal depolarization, which could be important for an establishment of bovine pregnancy. Our in vitro experiments demonstrate that the trophoblast could contribute to this change in vivo.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama.

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and ß-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcß1-4)Galß1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

The glycosylation pattern of secretory granules in binucleate trophoblast cells is highly conserved in ruminants.

The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of three ruminant families were thus investigated: greater malayan chevrotain (Tragulidae); fallow deer, red deer, chinese water deer (Cervidae); and domestic goat, springbok, impala (Bovidae). BNC granules in all species studied strongly expressed tri-/tetraantennary complex N-glycans and bisecting N-acetylglucosamine [GlcNAc] as shown by binding of leuco- and erythroagglutins of Phaseolus vulgaris respectively. The presence of terminal N-acetylgalactosamine [GalNAc]) in BNC granules is shown by intense staining with lectins from Dolichos biflorus, Vicia villosa and Wisteria floribunda. Terminal galactose or GalNAc was also present, bound by Glycine max agglutinin. Treatment of slides with neuraminidase strongly intensified staining of Erythrina cristagalli lectin (ECA) to terminal lactosamine in all species studied; this was otherwise absent except in goat. Sambucus nigra-1 lectin bound to BNC granules in all species except in Impala, indicating the presence of abundant alpha2,6 linked sialic acid. These results indicate that these unusual highly branched glycans, with bisecting GlcNAc and terminal GalNAc are a general feature of BNC granules in Ruminants, including the most basal Tragulid branch. It therefore appears that the specific glycosylation pattern of BNC granules evolved early in ruminant phylogenesis, together with the appearance of BNC. The conserved glycan structure in BNC secretory granules indicates that this pattern of glycosylation is likely to be of considerable functional importance for the secretory glycoproteins of ruminant BNC.

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

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.

Evolutionary differentiation of Cetartiodactyl placentae in the light of the viviparity-driven conflict hypothesis.

We analysed the evolution of placental traits in the novel mammalian clade Cetartiodactyla (Cetaceans and Artiodactyls) by a parsimony-based computer program (MacClade). A diffuse epitheliochorial placenta was identified as the stem species pattern of this clade. Trophoblast giant cells (TGCs) independently evolved in Camelids and Ruminants. The polycotyledonary placenta is an apomorphic character for Pecora (higher ruminants) and the oligocotyledonary placenta developed as a further step on the stem lineage of cervidae and moschidae. We interpret these findings by application of the "viviparity-driven conflict hypothesis", which states that divergent interests of mother and offspring lead to a rapid antagonistic coevolution, which might cause placental diversity. According to this hypothesis the evolution of camelid and ruminant TGCs can be interpreted as means to increase fetal endocrine influence on the maternal metabolism. The development of the cotyledonary placenta could be related to a diminished availability of glucose, which is associated with the evolution of forestomach fermentation in Pecora. An arms race, in which the mother tried to restrict and the fetus tried to increase transplacental glucose flow, might have promoted the evolution of the cotyledonary placenta, which has a high feto-maternal exchange area, but a low conductivity for glucose.

Glucose transporter 1 localisation throughout pregnancy in the carnivore placenta: light and electron microscope studies.

Glucose is one of the major fetal nutrients. Maternofetal transfer requires transport across the several placental membranes. This transfer is mediated by one or more of the fourteen known isoforms of glucose transporter. So far only Glucose Transporters 1 and 3 (GT1, GT3) have been shown to be located in placental membranes. GT1 may be the only one on the syncytiotrophoblast (human) or both may be present on the same membrane (rodents) or be required in sequence (ruminants, horses and elephant). This paper shows GT1 to be the only transporter demonstrable by immunocytochemistry in carnivore (cat, dog and mink) endotheliochorial placental membranes. GT1 is invariably present on both apical and basal surfaces of the cyto- and syncytiotrophoblast in all carnivore species examined and the pattern of development is described from implantation to term.

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.

Serotonin-containing cells in the gastrointestinal tract of newborn foals and adult horses.

Serotonin (5-hydroxytryptamine, 5-HT), a regulatory amine of mucosal enterochromaffin cells plays an important role in the control of gastrointestinal smooth muscle contraction and epithelial secretion. Serotonin has also been associated with gastric ulcers, diarrhoea and abdominal pain. In spite of the high incidence of these gastrointestinal disorders in newborn foals and adult horses, no data are available regarding 5-HT immunoreactive cells (i.c.) in the gastrointestinal tract (GIT) of foals, and for adult horses, data are incomplete and contradictory. In this study, the distribution and relative frequency of 5-HT i.c. in the GIT of newborn foals and adult horses were determined immunohistochemically. In foals as in adults, a relatively large number of 5-HT i.c. were detected in all portions of the GIT. In foals, a significantly higher amount of cells was found in the pyloric region and margo plicatus of the stomach, as well as in the caecum and colon ascendens compared with adults. Our results provided rationale for further research concerning the role of 5-HT i.c. during the milk diet or in the regulation of gastrointestinal growth/cell proliferation, and in the pathogenesis of gastric ulcers, especially in newborn foals.

Expression of cyclooxygenase-II (COX-II) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD)/prostaglandin F-synthase (PGFS) in bovine placentomes: implications for the initiation of parturition in cattle.

The chain of events leading to prepartal luteolysis in cattle is not yet fully understood. Prostaglandin F(2alpha) (PGF(2alpha)) seems to be a major factor involved. However, only little information is available about the underlying regulatory mechanisms. Consequently, the expression of cyclooxygenase-II (COX-II) and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), an enzyme recently shown to be most likely responsible for the production of luteolytic PGF(2alpha) in the endometrium of cyclic cows, was investigated in bovine placentomes. Immunohistochemical methods were applied to placentomes from 17 pregnant cows between days 100 and 284, from three cows during the prepartal progesterone decrease (days 273-282) and from five parturient cows. COX-II was found in uninucleated trophoblast cells (UTC) from day 100 until parturition. However, between days 100 and 235 expression was only weak to moderate, focal and mainly restricted to the chorionic plate and adjacent basal parts of chorionic stem villi. In placentomes from a 270 and a 284 day pregnant cow, in which the prepartal decline of progesterone levels had not started yet, staining had substantially increased and extended to secondary and tertiary chorionic villi. In prepartal and parturient cows strong to intense staining was present in UTC all over the villous tree. Real time RT-PCR confirmed an extensive pre- and intrapartal rise of COX-II expression in bovine placentomes with a 70-100-fold increase of COX-II-mRNA levels. 20alpha-HSD/PGFS was widely expressed in UTC of chorionic villi. Like COX-II it was down-regulated in UTC differentiating into trophoblast giant cells. Immunostaining pattern did not change remarkably during the period under investigation, and 20alpha-HSD/PGFS-mRNA levels increased only 2.6-fold in the prepartal phase. Thus, in UTC PGF(2alpha) may be produced via COX-II and 20alpha-HSD/PGFS, but only COX-II may be substantially involved in the control of a putative prepartal placentomal output of luteolytic PGs, whereas 20alpha-HSD/PGFS seems to be expressed in a more constitutive manner.

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta.

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.

Mitotic polyploidization in trophoblast giant cells of the alpaca.

Genome multiplication is a typical feature of trophoblast giant cell (TGC) development in many species. Elevated nuclear DNA contents can be achieved by modified cell cycles with a complete lack of mitosis (endoreduplication) or with incomplete mitoses. The aim of this study is to characterize genome multiplication in the alpaca TGC. Placental tissues of gestation days 150, 264 and 347 (near term) and term placentae were processed for light microscopy and for transmission electron microscopy. Each TGC showed many nuclear profiles. Observation of serial sections revealed that TGCs are truly multinucleate with several highly lobulated nuclei. Feulgen staining showed that TGC nuclei have a higher DNA content than nuclei of other trophoblast cells. The number of argyrophilic nucleolar organizer regions (AgNORs) in nuclear profiles of TGC was between 15 and 100, while other trophoblast cells showed 1 or 2 AgNORs. Large multipolar mitotic figures with maximal diameters of 80 mum were observed in the alpaca placentas on gestation days 264 and 347. No cytokinesis was seen in TGC. The results show that the mode of genome multiplication in the alpaca TGC is mitotic polyploidization. Subsequent acytokinetic mitoses may lead to an accumulation of chromosomes and centrioles in TGC. With increasing ploidy levels, the shape of these polyploidizing mitoses becomes more irregular. The restitution of nuclei after these complex multipolar mitoses is likely to result in the irregular nuclear shape in TGC.

Development of the areola in the early placenta of the one-humped camel (Camelus dromedarius): a light, scanning and transmission electron microscopical study.

This study aimed to elucidate development of the areola in the early dromedary placenta in comparison with that of the pig and mare. Placental tissues from 25 pregnant camels were obtained from Cairo abattoir and prepared for light, scanning and transmission electron microscopy by routine methods. Vascular casts were made by injection of 4 : 1 liquid plastic mixture of mercox and methylmethacrylate. Areolar formation was first observed at 4.5 cm curved-crown-rump CVR length, while by 5-9 cm CVR length, the endometrial surface was uneven and studded with numerous uterine gland openings, where corresponding foetal areolae were barely detectable and the foetal areolar cells were of variable appearance and covered with long microvilli. At 10-13 cm CVR length the uterine gland openings developed irregular folds and the maternal areolar cells showed numerous apical blebs. At 14-29 cm CVR length the foetal areolae showed a great increase in height at the expense of their width. At 30-34 cm (CVR) length the maternal areolae appeared discoid and sharply demarcated from the surrounding inter-areolar tissues and the foetal areolae were rounded to irregular in shape with well-developed areolar rims. The vascular casts showed a widely meshed capillary network on the maternal areola, connecting with the pre- and post-capillary vessels, whereas the foetal side showed a relatively dense capillary meshwork. These studies indicate that the areola in the placenta of the one-humped camel is of the regular type like in the pig, and is poorly vascularized.

The three-dimensional feto-maternal vascular interrelationship during early bovine placental development: a scanning electron microscopical study.

Both the fetal and maternal microvasculature of bovine placentomes was examined by scanning electron microscopy of vascular casts. So far the development of the vascular architecture of the bovine placentome in early gestation has only been studied 2-dimensionally due to technical difficulties arising from the fragility of the early placental blood vessels. Repeated experiments led to the selection of the microvascular corrosion casts presented here. The vasculature of the maternal compartment is supplied by large caruncular stalk or spiral arteries, which release short maternal stem arteries. In the 3rd month of gestation, these arteries branch into several arterioles at their base, thus providing the vascular framework for the lower part of the septal walls of the primary crypts. In the 4th month, due to progressive longitudinal growth of the stem arteries, branching into arterioles occurs not only at the base, but over the whole length of the stem arteries. These arterioles supply the capillary complexes of the septa which resemble the major part of the septal vasculature and face the secondary crypts. Further indentation results in the formation of tertiary crypt capillary complexes, encircling the earlier secondary unit. From the 6th month of gestation the architecture resembles the fully developed maternal placenta with stem arteries running directly to the fetal side to branch into 4 to 6 arterioles, which turn back to enter secondary and tertiary septa. Maternal venules, collecting the blood from the capillary bed of secondary and tertiary septa, converge onto stem veins leaving the caruncle via branches of the uterine vein. The fetal part of the placentome is supplied by the cotyledonary arteries, which branch into fetal stem arteries that are the tributary to single villous trees. Over their whole course towards the maternal side, these give off arterioles entering secondary villi. The tertiary or terminal villous vasculature consists of capillaries, which are organised in serial capillary loops. This system is progressively elaborated in the course of gestation. In the 4th month there are only finger-like loops, whereas from the 6th month large fan-like structures can be observed. In early gestation the maternal and fetal blood vessels meet predominantly in a countercurrent fashion, changing to the less efficient crosscurrent exchange when the tertiary unit develops. These results indicate the development of a highly elaborated fetomaternal villous-crypt exchange system, already established in the 1st half of gestation, thus meeting the increasing needs of the fetus.

Immunolocalization of progesterone receptors in bovine placentomes throughout mid and late gestation and at parturition.

The corpus luteum is the main source of progesterone (P(4)) responsible for maintenance of gestation in cattle. So far it has not been possible to assign any biological role to placental P(4), which contributes only marginally and temporarily to peripheral maternal blood levels. In order to identify possible P(4) target cells within the placenta, placentomes from 150-, 220-, 240-, and 270-day-pregnant cows and from parturient cows (3 animals per group) were screened immunohistochemically for expression of the progesterone receptor (PR). During gestation, PR-positive staining was found exclusively in the nuclei of caruncular stromal cells (CSC; maternal part of the placentome) and of caruncular vascular pericytes. In placentomes from parturient cows, occasional positive nuclear staining was also observed in the walls of small caruncular arteries. The percentage of PR-positive CSC increased slightly from 51.8 +/- 2.6% on Day 150 to 56.2 +/- 5.6% at Day 270 (p < 0.05) and was 58.9 +/- 1.8% at parturition. These results suggest that in pregnant cattle, CSC are under the control of P(4) of placental rather than luteal origin. Thus, whereas luteal P(4) may regulate "coarse" systemic progestational functions in the maternal compartment in the classical hormonal manner, placental P(4) may act as a paracrine factor involved in the local regulation of caruncular growth, differentiation, and functions.

DNA content and ploidy level of bovine placentomal trophoblast giant cells.

Cytophotometric measurement of the DNA content of Feulgen-stained nuclei in touch preparations of bovine placentomes (n =5) revealed that 8C nuclei occurred in all, 16C nuclei in two, and 32C nuclei in one specimen. The determination of ploidy level by in situ hybridization with a Y-chromosome specific DNA probe showed that the majority of the fetal nuclei in touch preparations of placentomes from male fetuses (n =5) are tetraploid. Generally two tetraploid nuclei lie close together. These findings indicate that polyploidization is a normal feature in the development of the mostly binucleate trophoblast giant cells (TGCs). A new model for the development of these cells is proposed: a primary acytokinetic mitosis leads to a binucleate cell with two diploid nuclei. This cell enters a second acytokinetic mitosis during which the chromosomes of both nuclei form a common metaphase plate. The resulting cell with two tetraploid nuclei undergoes an additional S-phase but does not enter a renewed mitosis. The functional significance of this genome multiplication may be an increased synthetic capacity of bovine TGCs, caused by an increased number of gene copies available for transcription. Since genome multiplication is a property of invasive trophoblast cells of different species, it may be advantageous for trophoblast invasion.

Tripolar acytokinetic mitosis and formation of feto-maternal syncytia in the bovine placentome: different modes of the generation of multinuclear cells.

The vast majority of trophoblast giant cells in the ruminant placenta are binuclear and are believed to derive from mononuclear trophoblastic cells by a single acytokinetic mitosis. There is no satisfactory explanation for the generation of the small proportion of trophoblast giant cells with one, three, or more nuclei. In this light-and electronmicroscopic study of bovine placentomal tissue from the second half of gestation, developmental stages of the trophoblast giant cells are investigated. Large mitotic figures indicate mitotic polyploidization, which is proposed to be due to two subsequent acytokinetic mitoses. Tripolar mitoses offer an explanation for the development of trinucleate trophoblast giant cells. Measurements of nuclear volumes in a series of semithin sections revealed that three size classes of trophoblast giant cells occur. The approximately doubling of nuclear volume between each class is thought to reflect different levels of DNA content that result from polyploidization in this cell type. Although trinuclear feto-maternal hybrid cells are the standard outcome of the fusion of binuclear trophoblast giant cells with uterine epithelial cells, some syncytia with at least five nuclei were observed in the uterine epithelium.

Fetal villosity and microvasculature of the bovine placentome in the second half of gestation.

The architecture of the fetal villous tree and its vasculature in the bovine placentome were studied in the second half of gestation using both conventional histology and histology of ink-filled blood vessels. These were compared with corrosion casts of plastic fillings of the vasculature, prepared for scanning electron microscopy. This combination of morphological methods allows perception of the villous tree throughout gestation from broad-conical to tall-conical form where branch ramification occurs mainly at right angles to the stem. The stem villus typically contains a single central artery and several peripheral veins arranged in parallel. The proximal branches to the stem, the intermediate villi, contain a central arteriole and accompanying venules. The distal branches, the terminal villi, enclose capillary convolutions which consist of an afferent arterial capillary limb, capillary loops and efferent venous capillary limbs. Vascular interconnections exist within the terminal villi, as capillaries or venules between the capillary convolutions, serially bridging them in up to 5 places, and as capillary anastomoses between the capillary loops. Coiling and sinusoidal dilatations of these loops develop near the end of gestation. The intraplacentomal rearrangement of villous trees with progressive gestation and their morphological vascular adaptations are discussed in relation to placental function, including the ever increasing need for transplacental substance exchange. This adaptation allows the blood to traverse the shortest possible arterioarteriolar route to the periphery of the trees where exchange takes place. The need for an increasing blood flow stimulates capillary growth and at the same time optimises the blood flow reaching the placental barrier represented by the vessel cast surface. The capillaries also carry the blood back into the very voluminous system of venules and veins where back diffusion may occur. The total volume of terminal villi of bovine placentome, the 'working part' of villous trees, hence distinctly increases with respect to the stem and intermediate villi, the 'supplying part' of the villous tree. In morphological terms the efficiency of the bovine transplacental diffusional exchange is higher than in the closely related 'co-ruminants' sheep and goats and distinctly higher when compared with the human placenta.