Structure and function of placental exchange surfaces in goodeid fishes (Teleostei: Atheriniformes)

The species of the family Goodeidae have evolved reproductive strategies involving intraovarian gestation, early evacuation of nearly yolk‐exhausted embryos from the ovigerous tissue into the ovarian cavity, placental matrotrophy during intraluminal gestation, and the birth of highly developed fry. The inner ovarian lining becomes hypervascularized during gestational periods and functions as the maternal component of the placental association. Embryotrophic liquid is secreted by the inner ovarian epithelium into the ovarian cavity. Comparative electrophoretic analyses of embryotrophe and maternal blood serum provide evidence for the transfer of maternal serum proteins into the embryotrophe. Trophotaeniae, proctodaeal processes of the embryos, provide a surface for nutrient absorption. Endocytic activity was demonstrated by ingestion of unspecific tracer proteins in various species. Moreover, the trophotaenial absorptive cells (TACs) in Ameca splendens ingest various proteins or random copolymers conjugated to colloidal gold as well as radioiodinated proteins in a way that satisfies the criteria of receptor‐mediated endocytosis. Several aminopeptidases (APs) on the surface of TACs were identified as protein binding sites as evidenced by inhibition of binding and uptake of marker proteins in the presence of AP substrates or AP inhibitors. Morphological adaptations of the embryonic circulatory system pertaining to nutrient and gas exchange were characterized. The embryonic epidermis comprises two layers of squamous cells closely underlain by a dense capillary net. Efficient gas exchange is facilitated by a thin embryotrophe‐blood barrier of both the embryonic skin and the intraovarian lining. J. Morphol. 276:991–1003, 2015. © 2014 Wiley Periodicals, Inc.     

The trophotaenial placenta of a viviparous goodeid fish. I. Ultrastructure of the internal ovarian epithelium, the maternal component

Embryos of goodeid fishes develop to term within the ovarian lumen, where they undergo considerable increase in weight due to transfer of maternal nutrients across a trophotaenial placenta. The placenta consists of an embryonic component, the trophotaeniae, and a maternal component, the ovarian lining. The latter was examined by transmission electron microscopy, scanning electron microscopy, and light microscopy in both gravid and nongravid ovaries of the viviparous goodeid fish, Ameca splendens. The single median ovary of A. splendens is a hollow structure whose lumen is divided into lateral chambers by a highly folded longitudinal ovarian septum. Germinal tissue occurs within folds of the ovarian lining that extend into each of the two lateral chambers. Matrotrophic embryonic development takes place within ovarian chambers. During gestation, the lining of the ovarian lumen is in direct apposition to body surfaces and trophotaenial epithelia of developing embryos. The ovarian lining consists of a simple cuboidal epithelium, termed the internal ovarian epithelium (IOE), overlying a well-vascularized bed of connective tissue. Cells of the IOE are apically convex. Well-developed granular and agranular endoplasmic reticula and numerous large membrane-bound vesicles with electron-dense content occupy the apical cytoplasm of IOE cells. Two functional states of the same cell type are distinguished within the IOE. Phase I cells contain few, if any, large apically situated vesicles; Phase II cells contain many. Secretory products of the IOE are presumed to be an important source of nutrients for embryonic development. Structural and functional relationships of the IOE to the trophotaenial epithelium of developing embryos are discussed in relation to maternal-embryonic nutrient transfer processes.     

The trophotaenial placenta of a viviparous goodeid fish. III: Protein uptake by trophotaeniae, the embryonic component

Protein uptake and degradation by trophotaenial cells of the viviparous goodeid fish Ameca splendens were studied colorimetrically and ultrastructurally using horseradish peroxidase (HRP) as a tracer and acid (ACPase) and alkaline (ALPase) phosphatase cytochemistry. Trophotaeniae are ribbon-like external projections of the embryonic gut that are equivalent to greatly hypertrophied intestinal villi. During gestation within the ovarian lumen, trophotaeniae are directly apposed to the internal ovarian epithelium (IOE) where they establish a placental association between the developing embryo and maternal organism. Trophotaenial absorptive cells possess an ALPase reactive brush border, an endocytotic apparatus, and ACPase reactive standing lysosomes. Ultrastructural studies of protein uptake indicate that cells of the trophotaenial epithelium take up HRP by micropinocytosis and degrade it within lysosomes. Initially (from 1.5-10 min), HRP is taken up in vitro at 22 degrees C at the apical cell surface and passes via endocytotic vesicles into an apical canalicular system. From 1.5 to 10 min exposure, HRP passes passes from the apical canalicular system to a series of small collecting vesicles. After 10 min, HRP is detected within large ACPase reactive supranuclear lysosomes. Three hours after an initial 1 h exposure to HRP, most peroxidase activity within supranuclear lysosomes is no longer detected. Presence of Golgi complexes, residual bodies, and secretory granules in the infranuclear cytoplasm suggest that products of protein uptake and hydrolysis are discharged across basal and lateral cell surfaces and into the trophotaenial circulation. Trophotaeniae of embryos incubated in vitro in HRP-saline take up HRP at an initial rate of 13.5 ng HRP/mg trophotaenial protein/min. The system becomes saturated after 3 h. Trophotaeniae incubated at 4 degrees C show little or no uptake. In trophotaeniae continuously pulsed with HRP for 1 h, then incubated in HRP-free saline, levels of absorbed peroxidase declined at a rate of 0.5 ng/mg trophotaenial protein/min. HRP does not appear to enter the embryo via extra-trophotaenial routes. These findings are consistent with the putative role of trophotaeniae as the embryonic component of the functional placenta of goodeid fishes. Trophotaenial uptake of maternal nutrients accounts for a massive (15,000%) increase in embryonic dry weight during gestation.     

Isolation of human nerve growth factor from placental tissue

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the supernatant was sequentially chromatographed, at neutral pH, on Sephadex G-100, DEAE-11, and Sephadex G-150. A high-molecular-weight protein fraction (150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1–3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse -NGF.To whom reprint requests should be addressed.     

Fine structure of pars neuro-intermedia of the loach,Misgurnus fossilis L.

Summary Two types of glandular cells are present in the pars intermedia of the loach, Misgurnus fossilis. Basophils are characterized by the presence in their cytoplasm of numerous secretory granules containing electron-dense and homogeneous material and by scarce endoplasmic reticulum. Weak acidophils contain in their cytoplasm abundant endoplasmic reticulum and numerous granules of different electron densities.The distal part of the neurohypophysis is composed of several types of neurosecretory axons, strongly branched pituicytes, numerous capillaries, and connective tissue elements. The axon terminals form the neuroglandular, neurovascular and neurointerstitial contacts. Some axon terminals are closely apposed to the basement membrane separating neurohypophysis from meta-adenohypophysis. At points of absence of continuity of this membrane, some neurosecretory axons become directly contiguous with cytoplasmic membranes of the intermedia cells.The investigation was partly supported by a research grant from the Zoological Committee of the Polish Academy of Sciences.     

Pi Mheerlen,a Pi M allele resulting in very low a 1-antitrypsin serum levels

Summary A patient with pulmonary emphysema is described, who had a very low ₁-antitrypsin serum concentration (2% of normal). After isoelectric focusing and staining, the patient's serum revealed no visible ₁-antitrypsin bands. Immunofixation, following isoelectric focusing, gave a banding pattern identical to that of a normal M type. The existence of this deficient M-allele was confirmed by family studies. Low ₁-antitrypsin concentrations, due to the presence of the deficient allele, were coupled with low serum antitrypsin activities.     

A comparative study of embryonic gene expression inDrosophila andEphestia

Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and -glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

The rôle of membrane-bound cytoplasmic inclusions during gametogenesis in Lilium longiflorum thunb.

In the prophase of both mega- and microsporogenesis, a sizeable proportion of the meiocyte cytoplasm becomes invested in double or multiple membrane-bround inclusions. This cytoplasm remains thus isolated from the rest of the cell until the completion of meiosis II in the female cells, or the young spore stage in those of the male. Significantly this encapsulation proceeds immediately the elimination of the major part of the ribosome population from the cytoplasm and, further, the electron microscope reveals that those ribosomes contained in these membranous inclusions remain unaffected by the lytic enzymes active elsewhere in the cytoplasm at this time. This encapsulated cytoplasm is proposed to fulfill two rôles; one, that it carries reserves necessary for postmeiotic development through from the diplophase to the haplophase environment and, two, that it permits the continuity of protein synthesis throughout meiosis I and II, a period when the major part of the protein synthetic apparatus is absent.Abbreviations MI membrane-bound inclusions - MMI multimembraned inclusions     

Expression of developmental genes during early embryogenesis of<Emphasis Type="Italic"> Hydra</Emphasis>

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.Edited by D. Tautz     

Effects of mutations at thestambh A locus ofDrosophila melanogaster

We report novel findings on the cytogenetic location, functional complexity and maternal and germline roles of thestambh A locus ofDrosophila melanogaster. stmA is localized to polytene bands 44D1.2 on 2R.stmA mutations are of two types: temperature-sensitive (ts) adult and larval paralytic or unconditional embryonic or larval lethal. Twelve alleles reported in this study fall into two intragenic complementing groups suggesting thatstmA is a complex locus with more than one functional domain. Some unconditional embryonic lethal alleles show a ‘neurogenic’ phenotype of cuticle loss accompanied by neural hypertrophy. It is shown that embryos of ts paralytic alleles also show mild neural hypertrophy at permissive temperatures while short exposure to heat induces severe cuticle loss in these embryos.stmA exerts a maternal influence over heat-induced cuticle loss. Unconditional embryonic lethal alleles ofstmA are also germline lethal.     

The nucleolar fibrillar centres in various cell types in vivo or in vitro

Summary The compound eye of male (haploid) Xyleborus ferrugineus beetles was examined with scanning and transmission electron microscopy. The eye externally consists of ca. 19 to 33 facets. Each ommatidium is composed of a thickly biconvex lenslet with about 50 electron dense and rare layers, but at the junction area between two lenslets there are only about 35 to 37 layers that can be distinguished. A very short (3.4–4.0 m) acone type crystalline cone is located directly beneath the lenslet. Each ommatidium is surrounded by pigment cells, and pigment granules also appear throughout the cytoplasm of the retinular cells. Some pigment granules are even present below the basement membrane. There are 8 retinular cells. The rhabdomeres of 2 centrally situated photoreceptor cells fuse into a rhabdom which is enveloped by the rhabdomeres of 6 peripheral retinular cells. The rhabdomeres of the 6 peripheral retinular cells join laterally to form a rhabdomeric ring around the central rhabdom. No tracheation was observed among the retinular cells. Virus-like particles are evident near the nucleus in each Semper cell of the crystalline cone.This research was supported by the Director of the Research Division, C.A.L.S., University of Wisconsin, Madison; and in part by research grant No. RR-00779 from the Division of Research Resources, National Institutes of Health and by funds from the Schoenleber Foundation, Milwaukee, WI to D.M.N.     

Role of α-galactosidase in cell wall metabolism of date palm (Phoenix dactylifera) endosperm

Summary The endosperm of developing date palm (Phoenix dactylifera) seeds was sampled at regular intervals from pollination to mature fruit. The galactose content of the cell wall mannans was assessed. Accumulation of -galactosidase, a cell wall hydrolase, during endosperm development was analyzed by isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with Western blotting and immunolocalization on tissue sections. N-terminal amino acid sequence of the first 15 amino acids showed homology with amino acids 71 to 85 of the sequence reported for the mature guar protein. Four forms of the enzyme with isoelectric points ranging from 4.4 to 5.2 appeared by 11 weeks after pollination, and all forms remained until maturity. A major band of 41 kDa and several lower M_r, lightly staining bands cross reacted with the anti--galactosidase antiserum. The major band remained until maturity while the lightly staining bands gradually disappeared. In the mobilizing endosperm of germinated seeds, two darkly staining bands were observed at 41 and 40 kDa. At 9 weeks after pollination, the endosperm was cellular and the silver enhanced gold label localizing -galactosidase occurred predominantly in the cell periphery. By 11 weeks, the label was present in the cytoplasm, but lacking on the thickening cell wall. -Galactosidase accumulated in the protein bodies along with the storage protein. At 13 to 17 weeks, the label accumulated and then was lost in a centrifugal pattern (from the middle lamella inward) from the cell walls as they matured and was lost in the cytoplasm. The mature endosperm cells had intense label present only over the protein bodies and over the inner cell wall. These observations suggest that -galactosidase is synthesized during endosperm development and unique forms of the enzyme are associated with cell wall maturation and cell wall mobilization in this species.     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Maternal-fetal transport kinetics of copper,selenium, magnesium and iron in perfused human placental lobule: in vitro study

Transport characteristics of certain inorganic elements such as copper, magnesium, selenium and iron have been studied in maternal-fetal direction in normal pregnancies, using in vitro perfusion of isolated placental lobules. Copper, selenium, magnesium and iron salts corresponding to twice physiological concentrations were injected as a 100 l bolus, into the maternal arterial perfusate. Serial perfusate samples were collected from venous outflows for a study period of 5 min. Concentrations of various inorganic elements and their transport kinetics were determined. Transport fractions of copper, selenium, magnesium and iron averaged 0.14, 0.19, 0.06 and 0.23% of maternal load respectively. The pharmacokinetic parameters such as area under the curve, clearance, elimination constant, and time for maximum response showed some significant differences between the various elements. We speculate that copper and selenium share the same transport pathway along a concentration gradient in maternal-fetal direction, while for iron and magnesium, active transport plays a predominant role for element transfer across the human placental membrane.     

Microtubules and cell shaping in the mesophyll ofNigella damascena L.

Summary Cell shaping in the mesophyll ofNigella damascena was investigated with the aim of determining the origin of the arm-like protrusions, which are characteristic of, e.g., arm-palisade cells. It was found that hoops of cell wall were deposited during the early stages of cell expansion. The hoops were interconnected, thus embracing the cells with a wide-meshed net of local wall reinforcement. The pattern of wall deposition in the extra-cellular matrix correlated with a pattern of bands of microtubules in the cortical cytoplasm of the cells. During lateral expansion bulges were forced through the comparatively thin walls of spaces between the meshes, giving rise to the arm-like protrusions. After establishing the cell shape the bands of microtubules disintegrated and cell wall was uniformly deposited. The results are discussed in the context of the mode of cell shaping observed in the mesophyll of other systems and of a previous, classical hypothesis on the origin of arms in mesophyll cells.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - PME phosphate-magnesium-EGTA-buffer