Placental steroids in cattle: hormones, placental growth factors or by-products of trophoblast giant cell differentiation?

The bovine placenta produces large amounts of steroids, mainly estrone (E1) and progesterone (P4). Specific features of bovine placental steroidogenesis are 1) the expression of all enzymes needed for the production of estrogens from cholesterol in the trophoblast 2) an only marginal and temporal contribution to peripheral maternal P4 levels restricted to a period between approx. days 150 - 240 of gestation 3) the predominance of sulfoconjugated over free E1 and 4) a complementary setting of steroidogenic enzymes in the two morphologically discriminable trophoblast cell types, the uninucleated trophoblast cells (UTC) and the trophoblast giant cells (TGC). In cattle so far no definite information is available on the specific biological roles of placental estrogens and P4. However, the detection of estrogen receptors and progesterone receptors in the placentomes suggests a role primarily as local regulators of caruncular growth, differentiation and functions. Inconsistent with a function as a caruncular growth factor is the strong evidence that in cattle placental estrogens enter the maternal compartment almost completely as estrone sulfate (E1S), which is not active at classical nuclear receptors. On the other hand, E1S may be converted locally to free active estrogens via the action of steroid sulfatase (StS), which has been detected in specific parts of the bovine caruncular epithelium. Alternatively or in addition, StS expression in the caruncular epithelium may serve the utilization of sulfated neutral steroid precursors (e.g. pregnenolone sulfate or cholesterol sulfate) supplied with maternal blood, thus providing free substrates for further metabolization in the adjacent trophoblast. The down-regulation of P450scc and P450c17 and the up-regulation of 3beta-HSD and aromatase during the differentiation of TGC from UTC in parallel with the up-regulation of ER beta and estrogen sulfotransferase in maturing TGC suggests a function of placental estrogens primarily as autoor intracrine regulators during this process and assigns to conjugated placental estrogens a role as inactivated by-products of TGC differentiation intended for excretion. Collectively, despite some evidence from recent studies for putative roles of placental steroids in cattle their exact functions in the bovine species remain still undefined.

Bovine placental steroid sulphatase: molecular cloning and expression pattern in placentomes during gestation and at parturition.

Apart from during the prepartal period, the main oestrogen produced by the bovine trophoblast is oestrone sulphate (E1S) which does not bind to nuclear oestrogen receptors (ER). High steroid sulphatase (StS) activities previously detected in the maternal part of bovine placentomes (caruncles) suggest the local activation of E1S ("sulphatase pathway"). Consequently, the expression pattern of StS in bovine placentomes was investigated by immunohistochemistry using an antiserum against human placental StS. Cross-reactivity for bovine StS was confirmed by Western blot yielding a single band of 62 kDa in both bovine and human placenta. Immunostaining for StS was detected in caruncular epithelial cells (CEC), which was clearly related to gestational age. In animals pregnant between 100 and 284 days (n=17), signals were restricted to CEC adjacent to the chorionic plate and basal primary and secondary chorionic villi. After the onset of prepartal luteolysis (days 273-282; n=3) and during active labour (n=5) overall staining intensity had increased substantially and signals occurred ubiquitously in the flattened and partially dismantled caruncular epithelium. A 2204 bp full-length mRNA transcript of the bovine StS exhibiting 74% and 77% sequence identity to human StS on the mRNA and protein levels, respectively, was cloned by RACE-PCR. Real-time RT-PCR detected a 2.5-fold increase of StS-mRNA in prepartal placentomes, which, however, was not statistically significant. The co-localisation of ERalpha and StS in CEC is consistent with a role of placental oestrogens as regulators of caruncular growth and differentiation, and the up-regulation of carunclar StS may be involved in the marked prepartal increase of free oestrogens.

Complex secretory products in the oviduct of the plethodontid salamander Bolitoglossa dofleini (Amphibia, Urodela).

The ultrastructure of the secretory granules in the cells of the subdivisions of the oviduct in the neotropical plethodontid salamander Bolitoglossa dofleini was studied by transmission electron microscopy. In addition, we applied the cationic dye Cuprolinic Blue (CB) at different electrolyte concentrations to demonstrate proteoglycans, and the pyrogallol red-copper (PR-C) method to stain proteins at the ultrastructural level. The entire oviduct is lined by a simple epithelium that contains ciliated and microvillous cells in the first subdivision, the aglandular pars recta; microvillous cells show a moderate secretory activity. The following pars convoluta is differentiated into five glandular subdivisions and the aglandular "uterine portion". Especially in the glandular parts, the epithelium is arranged in longitudinal folds. At their crests ciliated and microvillous cells similar to those in the pars recta occur. Gland cells are crowded with secretory granules that differ in their structural complexity (with and without electron-dense spheres or masses; elaborated, homogeneous or granular matrix; spherical; distorted) along the various subdivisions. Further, as suggested by the CB-technique, the cranial subdivisions contain large amounts of sulphated proteoglycans that decrease in the caudal direction. Carboxylated proteglycans appear to be present in all subdivisions examined. Electron-dense spheres of secretory granules are largely free of CB-precipitates, but stain more or less intensely with PR-C. The ultrastructure of the pars recta, and especially the "uterine portion" indicates transporting capability. The epithelial cells of the "uterus" have coated pits and a considerable amount of lysosome-like bodies.

Development of serous cutaneous glands in Scinax nasica (Anura, Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families.

We examined the development of serous (poison) cutaneous glands in larval and juvenile Scinax nasica (Hylidae) at the ultrastructural level. We describe the biosynthesis and maturation of the cutaneous poison in comparison with the corresponding processes in representatives of Discoglossidae, Leptodactylidae, Pelobatidae and Pipidae. Serous biosynthesis in S. nasica starts in discrete adenoblasts and continues in the syncytial secretory unit. Biosynthetic processes involve rough endoplasmic reticulum and the Golgi apparatus, that releases membrane-bounded material, varying from fine grained to flocculent. During the post-Golgian secretory phase, this material undergoes initial maturation, and two products are formed: dense granules and larger vesicles holding a thin substance that will later be structured into a three-dimensional, honeycomb-like net. Both the secretory granules and vesicles change into glomerular-like aggregates of bowed, rod-shaped subunits (modules). In adult frogs, formation of dense granules is bypassed. The modular granule substructure seems to be related to the merocrine release of small amounts of poison, involved in regulating skin homeostasis. Comparison with maturational changes in larval glands of species representing four anuran families discloses similar patterns in the Leptodactylidae, but production of opaque homogeneous granules occurs in the Discoglossidae, clear vesicles in the Pelobatidae and aggregates of dense bars in the Pipidae.

Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study.

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.

Structural and mechanical aspects of the skin of Bufo marinus (Anura, Amphibia).

Specific biomechanical characters and some structures possibly related to them were investigated in the skin of the toad Bufo marinus using tensile testing techniques (at constant strain till rupture) as well as morphological methods (histological, immunohistochemical and electronmicroscopical). Mechanical parameters of the native skin varied considerably according to sex, individual variability and/or site of specimen collection. In skin strips of males and females excised from different parts of the body thickness ranged from 0.45 to 0.87 mm, strain (epsilonf) from 96.52 to 211.03, tensile strength (sigmam) from 5.72 to 9.38 MPa, and stiffness (E-modulus) from 5.76 to 6.73. The dermis of B. marinus is provided with a collagenous stratum compactum of considerable thickness, a stratum spongiosum with loosely arranged fibres and a marked calcified layer (substantia amorpha). Collagen appears to be the main determinant of skin mechanics. However, the slope of the J-shaped static stress-strain curves indicates elastin to be responsible for the high values of strain. Contrary to van Gieson and orcein staining, immunostaining with a monoclonal antibody against elastin revealed very few elastic fibers between collagen bundles and in the vertical fiber tracts (perforating bundles), but a considerable amount in the tela subcutanea. This was partly confirmed at the ultrastructural level by tannic acid staining.

Survey of the oviduct of salamandrids with special reference to the viviparous species.

Urodeles include oviparous and a few obligately viviparous species that belong to one family, the Salamandridae. Oviducts of both groups have basically the same design, but some modifications became necessary as viviparous species evolved. The entire oviduct of urodeles is lined by a monolayered epithelium, which is regionally differentiated into large glands and smaller secretory cells rich in glycoproteins. Secretory products important for fertilization form the egg jelly, which also is present in viviparous species. In the latter species, however, there appear to be fewer oviductal convolutions and subdivisions of the glandular part of the organ, as well as fewer gland cells. Viscous, acidic secretory products predominate in viviparous species, whereas resistant neutral mucins predominate in oviparous species. In viviparous species, the caudal portion of the oviduct became altered to retain the developing offspring. This "uterus" lacks glands, but epithelial cells produce carbohydrate-rich material. Offspring remain in the uterus anywhere from several months to five years, depending on the species and climatic conditions. The Na(+)-K(+)-AT-Pase system, common in vertebrate epithelia, is used to regulate the intrauterine milieu. Subepithelial vessels mediate gas exchange and the removal of waste products, mainly urea. Secretions of the uterine epithelium may contribute to the fluid surrounding the young, but do not seem to support nutrition. Sources of nutrition for the young vary from one (sub)species to the next. Some feed on yolk reserves (Salamandra salamandra and certain of its subspecies), others on unfertilized eggs and siblings (other S. salamandra subspecies and Mertensiella luschani with subspecies), and yet others on degrading embryotrophic eggs as well as on cells derived from a specialized uterine trophic zone (S. atra and subspecies). Certain basic alterations in the uterine epithelium during pregnancy are most likely under endocrine control. Others, such as the flattening of cells, the discharge of secretory products, and the development of the trophic zone, may be induced by interactions with the offspring.

Proteoglycans (PGs) in the larval amphibian tooth as visualized by cuprolinic blue.

Proteoglycans (PGs) were localized in the predentine and dentine of young larvae from the urodelan species Salamandra salamandra. After cuprolinic blue (CB) staining at the critical electrolyte concentration of 0.1 M MgCl2, CB-positive, electron dense filaments with considerable variations in length and width were found in the collagen-free zone adjacent to the odontoblast processes (length up to 1.3 microns, width 21 nm), in predentine (660 nm/3.2 nm), in dentine around (20 nm/9 nm) and in the dentine tubules (35-150 nm/8 nm). Size classes very likely represent different PGs.

The development of the dentigerous bones and teeth in the hemiramphid fish Dermogenys pusillus (Atheriniformes, Teleostei).

The development of the dentition and dentigerous bones was studied in the hemiraphid Dermogenys pusillus using histological sections, scanning electron microscopy, and cleared and stained specimens. Five days after birth, the toothless tip of the lower jaw begins to grow longer than the tip of the upper jaw. The growth originates from small cartilaginous triangles connected with Meckel's cartilage. The peri- and enchondrial ossification of the growing cartilaginous bars advances rostrad. The pharyngeal tooth plates are formed by fusion of the slat-like dentigerous dermal bones with the bony fractions of the gill branches. Hence, the tooth plates are composite bones. The ventral tooth plate ist formed by the two ceratobranchials V and the basibranchial IV together with the respective dermal bones. The paired pharyngobranchials III and IV are fused with the dorsal tooth plate, and the pharyngobranchials II is fused with the two respective lateral tooth plates. Mineralization starts after birth in elements of the pharyngeal tooth plates and their teeth. There are no indications that the pedicel on which the tooth is established is formed by the enamel organ, which is covered by pulpal cells. The enamel organ originates from the stratum basale of the oropharyngeal epithelium and moves within the jaw from labial toward lingual, the site of the establishment of the tooth. The anlage of the tooth on the tooth plates of the pharynx lies at the level of the tooth base.

Dentigerous bones and dentition in the hemiramphid fish Dermogenys pusillus (Atheriniformes, Teleostei).

Structure and arrangement of the teeth were studied in the hemiramphid Dermogenys pusillus, using scanning electron microscopy as well as cleared and stained specimens. The teeth of the jaws are small, monocuspid, and tilted towards the esophagus. They are arranged along the lateral edges of the premaxillas and dentaries. Each premaxilla bears additional teeth on an osseous bar extending from rostro-lateral to medio-lingual. The dentition of both dentaries curves slightly within the cavity of the mouth and gently tapers off laterorostrad just beyond the tip of the upper jaw. The part of the lower jaw, which typically protrudes beyond the upper jaw, is without teeth. One dorsal and two lateral tooth-bearing bony plates (tooth plates) are found in the pharyngeal region. Their teeth are largely irregular in arrangement. The teeth on the two lateral plates are small and monocuspid, whereas the dorsal and the ventral tooth plate possess additional strong bi- and tricuspid teeth. The teeth of the jaws and of the pharyngeal region obviously have a bony pedicel ("attachment bone") which is asymmetric in all teeth. An elastic suture connects the cone of dentine with the bony pedicel. The special construction of the teeth and their arrangement on the various dentigerous bones will be discussed with respect to their function in catching prey.

Mechanical properties of the skin of Xenopus laevis (Anura, Amphibia).

The skin of the aquatic pipid frog, Xenopus laevis, was examined for specific biomechanical features: 1) thickness, 2) maximal strain at break (epsilon f), 3) tensile strength (sigma m), 4) modulus of elasticity (E, stiffness), and 5) the area under the stress-strain curve (W) (breaking energy, toughness). Skin freshly removed from dorsal, ventral, and lateral areas of the body was subjected to uniaxial tension. In both sexes, the dorsal skin is thicker than the ventral. The skin of male frogs was consistently thinner in all body regions than that of females. Most biomechanical parameters showed a considerable range of values in both males (epsilon f = 59-63%, sigma m = 15-16.5 MPa, E = 33.5-38.4 MPa, W = 3.8-4.5 MJ/m3) and females (epsilon f = 102-126%, sigma m = 11.5 MPa, E = 10.4-12 MPa, W = 5.2-6.7 MJ/m3). The disparate epsilon f values in males (low) and females (high) might reflect sexual dimorphism. Static stress-strain curves were typically J-shaped; with the exception of a "toe," the curves rose approximately linearly with increasing strain. The skin of X.laevis, although heterogeneous in structure, possesses features similar to those found in tissues with aligned collagen fibers such as tendons or fish skin. However, in anurans, the skin seems to play a more passive mechanical role during locomotion than in fish.

Cadmium-induced mRNA encoding a nonmetallothionein 33-kDa protein in Enchytraeus buchholzi (Oligochaeta).

Enchytraeus buchholzi (Oligochaeta) were exposed to various concentrations of CdCl2 in agar and aqueous solution. The Cd uptake was determined by atomic absorption spectroscopy as well as Cd effects on survival, reproduction, and mRNA synthesis by in vitro translation of total RNA in a rabbit reticulocyte lysate system. Although Cd was rapidly accumulated by the worms, any acute toxic Cd effects at concentrations below 4 mg Cd/liter were not detectable. However, such subtoxic Cd concentrations caused the induction of an mRNA species coding for a nonmetallothionein 33-kDa protein as revealed by 2D electrophoresis of in vitro translated in vitro translated proteins using [35S]methionine. The Cd-induced synthesis of this transcriptionally regulated protein might be a preindicator for a Cd intoxication in enchytraeids.

cDNA cloning of a cadmium-inducible mRNA encoding a novel cysteine-rich, non-metallothionein 25-kDa protein in an enchytraeid earthworm.

Cadmium accumulation and its effect on gene expression have been investigated at sublethal cadmium concentrations in the soil oligochaete Enchytraeus buchholzi. This worm is capable of accumulating cadmium to large amounts, which coincides with the induction of a mRNA isolated as a cDNA clone by differential screening of a cDNA library constructed from cadmium-treated enchytraeids. The cDNA clone designated CRP1 is 1474 base pairs in length and contains a 753-base pair open reading frame, encoding a novel Cys-rich non-metallothionein protein. In vitro translation of the in vitro transcribed CRP1 results in a protein with a molecular mass of 25 kDa and an pI of approximately 7.5. These values are consistent with those predicted from the deduced amino acid sequence. The CRP protein contains 27% Cys, most of them arranged in Cys-X-Cys and Cys-Cys segments. The sequence is also characterized by a 31-amino-acid motif, which is tandemly repeated along the sequence. Northern blot analysis reveals that the CRP gene is not constitutively expressed in untreated worms, but rather it is rapidly induced by cadmium. The CRP gene may be a promising candidate gene for monitoring bioavailable cadmium at subtoxic levels in terrestric environments.

CD15 in trophotaeniae of the viviparous goodeid fish Xenotoca eiseni (Cyprinodontiformes, Teleostei).

The antigen CD15, also known as 3-fucosyl-N-acetyl-lactosamine (FAL), or Stage Specific Embryonic Antigen (SSEA-1) is a cell membrane and cytoplasmic antigen, that is present in several mammalian tissues. Using immunohistochemistry the monoclonal antibodies 3B9 and B4,3 detected CD15 in whole mount preparations of trophotaeniae of embryos of the matotroph viviparous teleost Xenotoca eiseni. A specific localization is encountered in the ecto- and entodermal portion of the trophotaenial epithelium. A positive reaction in the fibrocytes of the inner mesenchymal core of trophotaeniae is doubtful since a relative high background is present in these cells. CD15 positivity has been observed in all developmental stages studied. The exact role of CD15 is unclear. An involvement in endocytic activity is postulated.

Ultrastructural localization of tyrosinase in the tardigrade cuticle.

Tyrosinase activity has been demonstrated ultra-structurally in the cuticle of the eutardigrade Macrobiotus hufelandi. The enzyme could be localized in the outer layer (=epicuticle) of the integument. A weak deposition of reaction product was also seen in the cytoplasm of storage cells, free floating in the haemolymph.

Protein-gold transport in the endocytic complex of trophotaenial absorptive cells in the embryos of a goodeid teleost.

This paper reports adsorptive endocytosis of exogenous proteins by the trophotaenial absorptive cells (TACs) in the viviparous goodeid teleost, Ameca splendens. In vitro incubations were performed with gold conjugated to bovine serum albumin (Au-BSA), human transferrin (Au-HTf), fetuin (Au-Fet), and asialofetuin (Au-ASFet). Localization of gold label on the TAC surface was nearly exclusive to patches of an amorphous coat associated with part of the intermicrovillous plasma membrane. On addition of excess BSA, HTf, Fet, or ASFet to incubation media containing, respectively, Au-BSA, Au-HTf, Au-Fet, or Au-ASFet, the density of gold particles adsorbed on the TAC surface decreased drastically. Moreover, attachment of the four protein-gold complexes to the same plasma membrane sites was suggested by reciprocal inhibitory effects. Further proteins such as hemoglobin, myoglobin, and cytochrome c were as well potent inhibitors of Au-BSA and Au-HTf binding and uptake. Binding of TACs of native BSA or HTf was visualized by immunogold labeling. The interactions between proteins and binding sites required both the presence of Ca2+ and appropriate pH greater than 6.6. Analyses of the concentration-dependent BSA and HTf binding curves, plotted from morphometric data, resulted in apparent dissociation constants, Kds, of approximately 5 x 10(-7) M and 4 x 10(-7) M, respectively. Following binding at the TAC surface and internalization via clathrin-coated pits and vesicles the several ligands were routed along the lysosomal pathway with transit through the endosomal compartment. Prolonged incubation periods led to massive intracellular accumulation of tracer proteins. The effects of NH4Cl (10 mM) treatment on TACs included enormous cytoplasmic vacuolation, a reversible loss of protein binding sites on the plasma membrane, and a block in the transport of protein-gold complexes to lysosomes.

Cell surface distribution and intracellular fate of human beta-very low density lipoprotein in cultured peritoneal mouse macrophages: a cytochemical and immunocytochemical study.

The receptor-mediated endocytosis pathway of colloidal gold labeled beta-very low density lipoprotein (beta-VLDL-Au) derived from patients with familial dysbetalipoproteinemia was analyzed at the ultrastructural level in macrophages. The results showed that beta-VLDL-Au complexes were specifically recognized by a cell surface receptor of the macrophages. beta-VLDL-Au particles once bound to the randomly distributed cell surface receptors clustered in coated pits and were taken up by coated vesicles. Subsequently, the beta-VLDL-Au particles passed through tubular structures and small endosomes before deposited into large electron lucent smooth surfaced endosomes. As revealed by ruthenium red and enzyme cytochemistry the endosomes appeared to be separated from the extracellular space and did not contain acid phosphatase. There were no clear signs of passage of beta-VLDL through the Golgi complex. The accumulation of many flocculated gold particles within Ac-Pase positive vesicles suggests that beta-VLDL once internalized by the macrophages is diverted into a degradative pathway. Incubation of beta-VLDL-loaded macrophages with the hydrophobic fluorescent dye nile red revealed numerous large fluorescent bodies within the cells indicating that the macrophages accumulate large amounts of lipid droplets with time. Additional studies large amounts of lipid droplets with time. Additional studies with native beta-VLDL in conjunction with postembedding immunocytochemical techniques were used to delineate further the intracellular pathway. Immunolabeling was carried out on thin sections of LR White embedded cells using affinity-purified polyclonal rabbit antibodies against apolipoprotein B with the protein A-gold or goat anti-rabbit IgG-gold technique. Indirect visualization of beta-VLDL by these immunocytochemical studies yielded results comparable to those with gold-labeled beta-VLDL. On the basis of both indirect immunocytochemical and direct cytochemical localization of beta-VLDL it is concluded that although colloidal gold labeling of beta-VLDL molecules unquestionably modifies their morphology, their function appears to be unaltered, at least with respect to the process of receptor-mediated endocytosis.