bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of cell volume determination for stereological studies of adrenocortical cells: Comparison of values from semifine sections with those of dissociated zona fasciculata cells
Authors: Taki Taki M. , Nickerson Peter A.
Published: February 2005

The average cell volume of rat adrenocortical zona fasciculata cells, determined using three different stains on semifine sections, was compared to that of dissociated, unfixed zona fasciculata cells. The maximum number of nuclei per area (NA) was obtained by counting nuclear profiles in semifine sections from nonosmicated adrenocortical tissue stained with toluidine blue. In osmicated adrenocortical tissue on the other hand, fewer nuclear profiles were seen and more of the small size classes were missing from the distribution of profiles. The skewness of the nuclear‐profile distribution was much greater in osmicated than in nonosmicated tissue, reflecting the missing small profiles. Although the average diameter of nuclei (D) in nonosmicated tissue was smaller than that for osmicated tissue, profiles were in all likelihood more readily identified because they stained quite intensely. Three different methods for correcting nuclear‐size profile distribution and determining nuclei per volume (NV) (Giger‐Riedwyl, Cruz‐Orive, and Weibel‐Gomez) were generally in agreement for nonsmicated tissue. Considerable shrinkage occurred during preparation for electron microscopy, with the greater change occurring in nonosmicated tissue. Cell volume in osmicated tissue corrected for shrinkage varied considerably among the different methods, producing significantly greater values for cell volume than those in nonosmicated tissue. The cell volume of nonosmicated tissue, corrected for shrinkage during processing and for missing small nuclear profiles, did not differ significantly from that of freshly dissociated zona fasciculata cells. The one exception was nonosmicated tissue stained by Feulgen‐methylene blue and corrected for missing small nuclear profiles by the method of Cruz‐Orive. Use of nonosmicated adrenocortical tissue in stereological studies with an appropriate correction for shrinkage for each experiment is recommended for determining cell volume, although osmicated tissue should also be included to facilitate identification of intracellular membranes when electron microscopy is required.
Reduction of density and anisotropic distribution of intermediate filaments occur during avian skeletal myogenesis
Authors: Price Maureen G. , Sanger Joseph W.
Published: February 2005

Chicken skeletal muscle taken from embryos in ovo was examined by thin‐section electron microscopy. Measurements of filament diameters reveal three nonoverlapping groups of filaments: thin (actin myofibrillar) filaments with mean diameters of 5.3 ± 0.6 nm (S.D.), thick (myosin myofibrillar) filaments with mean diameters of 15 ± 1.4 nm, and intermediate filaments with mean diameters of 9.3 ± 0.9 nm. During muscle development these diameters do not change. By counting the number of filaments observed in the sarcoplasm at different stages, we find that the spatial density of intermediate filaments decreases during avian myogenesis in ovo, from 91 intermediate filaments/μm2 at 6 days to 43 intermediate filaments/μm2 at 17 days in ovo. Initially randomly arranged, some intermediate filaments become associated with Z discs, sarcoplasmic reticulum, nuclear membrane, and the sarcolemma between 6 and 10 days in ovo. These associated intermediate filaments course both parallel and transverse to myofibrils, forming lateral connections between myofibrillar Z discs and longitudinal connections from Z disc to Z disc within myofibrils. Intermediate filaments also appear to connect Z discs with the nuclear membrane. The intermediate filament associations persist through day 17 of development, after which the presence of cytoskeletal filaments is obscured by the densely packed myofibrils and membranes. Intermediate filament distribution becomes anisotropic during development. A greater proportion of intermediate filaments in the immediate perimyofibrillar area are oriented parallel to myofibrils than in other areas, so that the majority of the intermediate filaments nearest the myofibrils course parallel to them. The longitudinal intramyofibrillar intermediate filaments persist throughout development, as shown by their existence in KI‐extracted adult myofibrils.
Ultrastructural changes in the oviductal epithelium of merino ewes during the estrous cycle
Authors: Hollis D. E. , Frith P. A. , Vaughan J. D. , Chapman R. E. , Nancarrow C. D.
Published: February 2005

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days ‐1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron‐lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.
Immunodissection of sperm surface modifications during epididymal maturation
Authors: Eddy E. M. , Vernon R. B. , Muller C. H. , Hahnel A. C. , Fenderson B. A.
Published: February 2005

Mammalian spermatozoa undergo changes in morphology, composition, and function during transit through the epididymis. These changes correlate with acquisition by sperm of the ability to fertilize ova. It has been found that sperm from the cauda epididymidis, but not those from the caput epididymidis, are able to bind to the zona pellucida. This would imply a modification in sperm surface characteristics. Biochemical and immunological studies have demonstrated changes in sperm surface composition during epididymal maturation. These changes involve addition of epididymal maturation. These changes involve addition of epididymal secretory products to the sperm surface, loss or alteration of existing sperm surface molecules, and possibly the unmasking of preexisting molecules or epitopes. Several laboratories have studied the epididymal secretory proteins in the rat, but a consensus has not been reached on the identification, characterization, source, and sperm surface association of these proteins. Monoclonal antibodies are beginning to be used to characterize sperm surface components and sperm maturation antigens. They are proving to be valuable tools for the dissection of epididymal maturation when used in conjunction with biochemical and physiological approaches.
Sperm surface changes during the acrosome reaction as observed by freeze‐fracture
Authors: Fléchon Jacques‐E.
Published: February 2005

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze‐fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle‐free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea‐pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.
Inner acrosomal membrane of mammalian spermatozoa: Its properties and possible functions in fertilization
Authors: Huang T. T. F. , Yanagimachi R.
Published: February 2005

The inner acrosomal membrane (IAM) develops during the spermatid stage of differentiation as that portion of the Golgi‐derived acrosome granule that tightly associates with the condensing sperm nucleus. In some mammalian species, an electron‐dense proteinaceous material accumulates between the IAM and the nuclear envelope, collectively comprising the “perforatorium.” Evidence, including its partial purification and its structural resistance to detergents and sonlcation, suggests that the IAM is an unusually resiliant membrane. Dense paracrystalline arrays of intramembranous particles, a lack of lectin‐mediated receptor modulation, and its lack of participation in sperm‐egg fusion suggest that the IAM lacks the same degree of fluidity as the egg surface plasmalemma. Observations using monoclonal antibodies, however, suggest that some specific antigenic modulations may be possible within the IAM. Its structural rigidity is of obvious mechanical value during sperm penetration through the zone pellucida. An additional role as a scaffold for putative zona lysin material remains controversial. Biochemical evidence suggests that acrosin, for example, is not entirely soluble and that some remains sperm‐associated, depending on the conditions of acrosome disruption. Nevertheless, morphological studies do not agree on acrosin's specific localization to the IAM. Currently there is only very limited information concerning the localization of the other acrosomal enzymes to the IAM. Another possible role for the IAM in some species may be in recognizing the zona pellucida. Evidence for this derives from the observation that fucoidin, a fucose heteropolysaccharide, inhibits guinea pig sperm‐zona binding, and bound fucoidin can be localized to the IAM and equatorial regions of the living acrosomereacted spermatozoa. Finally, the IAM may have a role in early recognition/adhesion with the colemma.
Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface
Authors: Meizel Stanley
Published: February 2005

This review deals with exogenous molecules that stimulate the acrosome reaction (AR) of mammalian sperm in vitro, presumably by acting at the sperm surface. Such molecules may exert their effect(s) by stimulating capacitation and/or by stimulation or initiation of the AR, and they are probably present at one of three putative in vivo sites (also discussed here) for the AR of a fertilizing sperm: the oviductal fluid, the cumulus oophorus matrix, and the zona pellucida. The molecules discussed include serum albumin, hydrolytic enzymes (particularly proteases); hormones including biogenic amines, estradiol, and arachiodonic acid metabolites; sulfur‐containing β‐amino acids; glycosaminoglycans such as hyaluronic acid; and a zona pellucida glycoprotein. Possible mechanisms to explain the effects of these molecules are also discussed. Several conclusions and suggestions are offered in this review: (1) There is more than one site for the AR of a fertilizing sperm in vitro, depending on experimental conditions and species, but the site(s) at which the AR of a fertilizing sperm occur(s) in vivo is/are still a matter of disagreement; (2) there are a number of molecules that can stimulate or initiate the AR in vitro, and such molecular duplication may also exist in vivo to ensure fertility; and (3) synergistic interaction between some of those exogenous molecules may occur in the stimulation of capacitation and the stimulation or initiation of the AR.
Fine structure of the mammalian egg cortex
Authors: Longo Frank J.
Published: February 2005

The cortices of a number of mammalian eggs are not strucurally homogeneous but are polarized. In mouse ova the plasma membrane is a mosaic; the cytoplasm overlying the meiotic spindle is devoid of cortical granules and consists of a filamentous layer containing actin. Functionally, this cortical polarity may be related to the restriction of sperm‐egg interaction and fusion to a specific region of the ovum cortex and to dynamic changes of the egg cortex during fertilization, including cortical granule exocytosis, polar body formation, and fertilization cone development. The origin of cortical polarity in mammalian oocytes and its possible relation to components of the cytoskeletal system and meiotic apparatus are discussed and compared with cortical features of eggs of other vertebrates and invertebrates.
Freeze‐fracture observations on mammalian oocytes
Authors: Koehler James K. , Clark Judy M. , Smith Dianne
Published: February 2005

Freeze‐fracture studies on mammalian oocytes have been hampered by the relatively small numbers of cells available at a given time as well as by difficulties encountered in effectively freezing these large, watery cells. We have nevertheless pursued this area because of the benefits of visualizing membrane faces involved in various fusion reaction by the freezefracture method. Our observations indicate no overall change in intramembranous particle (IMP) distribution before and after sperm penetration, although the question of possible alterations of these structures at the precise locus of sperm attachment remains open. Preliminary statistical analysis indicates that there is a much higher IMP density on the P face than on the E face of the plasma membrane and that the microvillar membranes bear more IMPs than those of the intermicrovillus regions. Probes of lipid subclasses were used to determine the distribution of cholesterol and anionic lipid in the egg plasma membrane. Filipin and tomatin showed extensive complex formation in microvillus as well as nonmicrovillus regions, whereas anionic lipids (using polymyxin B) have been difficult to detect on the oocyte surface. These results are discussed relative to current views of membrane fusion mechanisms.
Sperm penetration through oocyte investments in mammals
Authors: Talbot P.
Published: February 2005

Literature on the interactions between eutherian gametes is reviewed. The oocyte cumulus complex of the female is surrounded by a zona pellucida, corona radiata, and cumulus layer. Sperm undergo an acrosome reaction before penetrating the zona pellucida. The morphological consequences of this reaction and its possible role(s) in penetration of the oocyte cumulus complex are considered. The acrosomal enzyme, hyaluronidase, has been thought to aid sperm in penetrating the cumulus layer and corona radiata. Several recent investigations, including one that shows that motile cells lacking hyaluronidase can penetrate to the zona surface, do not support this idea. Other possible roles of this enzyme in fertilization are discussed. The development of in vitro fertilization systems that employ physiological numbers (1–100) of sperm will be valuable in studying the mechanisms used by sperm to penetrate the oocyte cumulus complex.
The basement‐membrane‐like matrix of the mouse EHS tumor: I. Ultrastructure
Authors: Inoue S. , Leblond C. P.
Published: February 2005

The fine structure of the extracellular matrix was examined in the Engelbreth‐Holm‐Swarm (EHS) tumor of the mouse. The matrix is composed of layers parallel to the surface of the associated cells; the layers are poorly defined close to the cells (proximal region) but quite distinct at a distance from the cells (distal region). In the proximal region of the matrix, the indistinct layers are composed of three types of structures: (1) a network of 3‐to 8‐nm‐thick „cords”︁ makes up the bulk of the tissue. (2) Within the network are scattered few 7‐ to 10‐nm‐wide, hollow rods of indefinite length, referred to as „basotubules”︁; their cross section has a dense, more‐or‐less‐circular or pentagonal wall and a light lumen containing a spherule. In addition, many plae profiles similar to these cross sections are present; they are interpreted as small, independent structures. (3) Minute structures composed of two parallel, 3.5‐nm rodlets are referred to as „double pegs.”︁ In the distal region of the matrix, the distinct layers include the same three types of structures, but basotubules are numerous and prominent; in each layer, they are arranged in picket‐fence fashion along two parallel planes. Cords are packed between them. Double pegs are scattered throughout the clear interlayer spaces.

Inasmuch as cord network, basotubules, and double pegs are present in the two regions of the tumor matrix, both regions resemble basement membrane. To explain the contrast between the paucity of basotubules in the proximal region and their abundance in the distal region, it is proposed that, as the production of newer matrix by the cells causes the older matrix to be displaced distally, the independent structures seen as pale profiles in the proximal region gradually assemble into basotubules.
Binding of lectin‐fluorescein conjugates to intracellular compartments of growth‐plate chondrocytes in situ
Authors: Farnum Cornelia E.
Published: February 2005

In this study, lectin‐binding techniques are applied to growthplate cartilage to analyze the intracellular localization of lectin‐binding glycoconjugates of chondrocytes in situ. The binding of ten fluorescein‐conjugated lectins is analyzed on 1‐μm‐thick Epon‐embedded, nondecalcified sections of growth plates from Yucatan swine. Comparisons are made to intracellular binding in chondrocytes of tracheal, articular, and auricular cartilage. Ear epithelium, tracheal epithelium, and kidney are used as positive control tissues for the specificity of lectin binding. Only the mannose‐binding lectins had affinity for the RER and nuclear envelope. Eight lectins reacted within the Golgi complex with characteristic patterns which ranged from localized fine linear strands to extensive vesicular accumulations. When cartilage slabs were exposed before embedment to the ionophore monensin to disrupt intracellular transport through the Golgi, it was possible to define differential subcompartments of the Golgi complex, based upon sites of sugar addition. Also, it was possible to characterize the cytoplasmic deposits of reserve‐zone chondrocytes which were positive with concanavalin A as glycogen, based upon their sensitivity to amylase. This method allows resolution at the light‐microscopic level of lectin‐binding glycoconjugates with localization to specific organelles. Patterns of intracellular binding were consistent with biochemical data relating to the subcellular localization of processing steps of complex carbohydrates prior to secretion.
Distribution of intramembranous particles and filipin‐sterol complexes in mouse sperm membranes: Polyene antibiotic filipin treatment
Authors: Toshimori Kiyotaka , Higashi Ruiko , Ōura Chikayoshi
Published: February 2005

The distribution of intramembranous particles (IMPs) and membrane filipin‐sterol complexes (FSC) was examined ultrastructurally in mouse spermatozoa from the male reproductive tract and ejaculates. IMPs were qualitatively analyzed on freeze‐fracture replicas of glutaraldehyde‐fixed tissue, while membrane FSC were quantitatively analyzed on replicas of filipin‐treated cells. The distribution pattern of IMPs of mouse spermatozoa was fundamentally similar to that of other mammalian spermatozoa. (1) In the head, the plasma membrane had a heterogeneous population density, e.g., few IMPs on the acrosomal region, particularly few on the marginal segment, and somewhat regularly arranged IMPs on the postacrosomal region. The acrosomal membrane had many IMPs in hexagonal arrays. The nuclear membrane had many IMPs on the P‐face, few IMPs on the variegated E‐face, and an intense population density on the P‐face of the basal plate. (2) In the neck, the plasma membrane had many IMPs with square arrangements of small IMPs in some areas on the P‐face; the redundant nuclear membrane had a few IMPs on both P‐ and E‐faces. (3) In the tail, the plasma membrane had diagonal rows of IMPs in some areas amongst larger IMPs on the middle piece, while it had „zippers”︁ composed of IMPs running parallel to the axis on the principal piece. The distribution of sperm membrane FSC may be summarized as follows: (1) In the head, the acrosomal plasma membrane, which was heavily labeled with filipin, had much more FSC in the equatorial segment than in the marginal segment throughout the study. The postacrosomal plasma membrane generally had no FSC, but some sperm in ejaculates were slightly positive to filipin. The acrosomal membranes (both outer and inner) had no FSC. The nuclear membrane in the main part of the head had less FSC in vas deferens and ejaculated sperm than in the epididymal sperm. The nuclear membrane on the basal plate had no FSC. (2) In the neck, the plasma membrane had little FSC. The redundant nuclear envelope had scattered FSC with a higher incidence in the epididymal sperm than in those from the vas deferens and ejaculates. The membrane scroll, which was elongated from the extreme caudal end of the redundant nuclear envelope, had abundant FSC in the vas deferens and ejaculated sperem. (3) The tail plasma membrane (both middle and principal piece), which was weakly labeled with filipin, had less FSC in sperm from the vas deferens and ejaculates than in those from the epididymis. The limiting membrane covering the mitochondria had no FSC.
The early development of the sheep trophoblast and the involvement of cell death
Authors: Carnegie J. A. , McCully M. E. , Robertson H. A.
Published: February 2005

During the period of rapid elongation prior to the initiation of placental attachment (days 12–16 of gestation), the ovine blastocyst consists of a single layer (primarily) of roughly cuboidal trophoblastic cells with an inner lining of flattened endodermal cells. Well‐developed spot desmosomes link the adjacent cell borders in both the trophoblastic and endodermal layers. The trophoblastic cells contain acid phosphatase‐positive, lysosomelike organelles, the mean diameter of which increases greatly between days 12 and 16 and whose contents vary during development. Also during the developmental period studied, trophoblast cells accumulate lipid; and periodic acid‐Schiff‐positive binucleate cells appear within the trophoblast layer. A consistent observation throughout the 5 days of rapid growth and differentiation of the blastocyst was the death and disintegration of some trophoblast cells. These disintegrating cells are usually singly dispersed within the trophoblast, although occasionally groups of four or five are observed. The cell death may indicate overall remodelling of the blastocyst, or the cells may represent genetically deficient cells which are unable to respond to the appropriate signal to differentiate.
An excursion through the ultrastructural world of quick‐frozen pancreatic islets
Authors: Dudek R. W. , Boyne A. F.
Published: February 2005

In this article we have presented a philosophical and historical perspective on quick freezing, freeze‐drying, freeze‐substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that (1) quick‐freezing does not produce any gross distortion of islet tissue; (2) the amount of usable islet tissue for ultrastructural analysis is approximately 13 μm deep from the frozen edge; (3) three different cell types can be identified in quick‐frozen tissue based on general morphological characteristics; (4) freeze‐substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; (5) with the use of protein A‐gold, insulin and glucagon can be localized immunocytochemically on silver‐gray (50‐nm‐thick) sections treated with 1% ovalbumin at room temperature overnight; (6) secretory granules of quick‐frozen alpha and beta cells may exist in either a swollen or condensed state; (7) swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; (8) insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; (9) our methods provide not only discrete immunocytochemical localization of hormone, but also well‐preserved cellular compartments; (10) energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and (11) the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.
Scanning electron microscopy of cell surface antigens labeled with colloidal gold
Authors: de Harven E. , Soligo D.
Published: February 2005

A method is described and discussed that permits the specific labeling of the surface of prefixed cells with the colloidal gold marker viewed with the scanning electron microscope. Its value depends exclusively on the use of backscattered electron imaging. Its advantages include (1) the possibility of preserving the surface features of the labeled cells, (2) the ease with which specificity can be established, (3) the possibility of making total counts of the labeled surface antigenic sites, and (4) the possibility of achieving distinct labeling for two different antigens expressed on the surface of the same cell.
Immunocytochemical studies of pituitary hormones with PAP, ABC, and immunogold techniques: Evolution of technology to best fit the antigen
Authors: Childs Gwen V. , Unabia Geda , Ellison Dayle
Published: February 2005

The immunocytochemical technology in our laboratory has evolved in response to specific needs for more efficient, refined stains for each antigen. The rationale for the application of each of the immunocytochemical techniques used today is described, and detailed methods are given. In the early 1970s, it was determined that the peroxidase‐antiperoxidase complex (PAP) stain provided the most sensitive means of detection of adrenocorticotropin (ACTH) at the electron microscope level even in tissues prepared with conventional fixation and embedding techniques that are considered rather harsh for the maintenance of antigenicity. Application of the same PAP complex technique to the larger glycoprotein antigens, like follicle‐stimulating hormone (FSH), however, proved far more difficult; and the problem was resolved partially when more gentle fixation‐embedding protocols were applied. The production of an efficient, reliable stain for FSH was achieved only with the avidin‐biotin peroxidase complex (ABC) stains were applied in the early 1980s. This technique also allowed more efficient reactions for all the antigens, and morphometric data could thereby be collected more rapidly. Thus, we concluded that the light microscope immunoperoxidase techniques were excellent for the morphometric analysis of pituitary cell types in both pre‐embedding and postembedding stains. However, the need for a more refined stain for its quantification at the electron microscope level on individual organelles led to the development of the colloidal gold stain in 1983–1984. This technique, which is new to our laboratory, is also described and illustrated in this report. Also included is a description of our studies of the effect of fixation and embedding processes on hormone antigenicity and techniques used to control background and nonspecific reactions. It is hoped that the novice will find the description of the rationale for the evolution of technology in our laboratory useful in making choices for his or her own immunocytochemical stains.
Dual immunostaining procedure demonstrating neurotransmitter and neuropeptide codistribution in the same brain section
Authors: Joseph Shirley A. , Piekut Diane T.
Published: February 2005

A method for sequential immunostaining of two antigens in the same section and the application to neuronal elements in the CNS is described. This method employs the peroxidase‐antiperoxidase (PAP) technique as a first sequence, followed by glucose oxidase‐antiglucose oxidase (GAG) complex as the second sequence, yielding rich brown and vibrant blue reaction products, respectively. Application of the PAP/GAG immunoreagents as dual markers for various neuropeptide and neurotransmitter systems is described in detail and the procedure is outlined. Comparisons with other dual staining methods are discussed and the application to other systems is presented.
Two‐color immunoperoxidase staining: Visualization of anatomic relationships between immunoreactive neural elements
Authors: Hancock M. B.
Published: February 2005

A method for differentially staining for two antigens in single sections is described. Paraffin‐embedded or Vibratome sections are incubated in two sequences of immunoperoxidase (PAP) reagents using a diaminobenzidine (DAB)‐nickel ammonium sulfate solution to localize the first antigen, and DAB alone to localize the second antigen. With these chromagens, black and amber‐colored reaction products are generated at the locations of the first and second antigens, respectively. The reaction products are stable and provide excellent color contrast. With this technique, the anatomical relationships between two sets of immunoreactive elements can be studied in individual sections. Intimate spatial associations that would probably not be detected by an examination of adjacent sections stained for each antigen can be visualized with this two‐color immunoperoxidase method.
A review of the study of protein secretion applying the protein A–gold immunocytochemical approach
Authors: Bendayan M. , Nanci A. , Herbener G. H. , Grégoire S. , Duhr M. A.
Published: February 2005

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A–gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph‐node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A–gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome‐like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans‐side and the trans‐most rigid one. Quantitative evaluations of the intensities of labeling have allowed for (1) demonstration of the high specificity of the different labelings; (2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and (3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double‐labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A–gold complexes formed by gold particles of different sizes. Protein A–gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high‐resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A–gold technique, which in combination with other procedures constitutes a powerful method in cell biology.
Quantification of the initial phases of rapid brain enlargement in the chick embryo
Authors: Pacheco Mary A. , Marks R. William , Schoenwolf Gary C. , Desmond Mary E.
Published: February 2005

Rapid brain enlargement requires a hydraulic mechanism in the chick embryo. Such a mechanism involves a closed, fluid‐filled system that generates positive pressure. For the chick embryo this study (1) determined when rapid brain enlargement begins, (2) assessed the relative contributions of cavity expansion and tissue growth to overall brain enlargement, and (3) evaluated mathematical models of overall brain enlargement and expansion and growth of the component parts. Three to five embryos were collected at each Hamburger and Hamilton stage (11, 12, 14, 16, and 18) and processed for paraffin serial sectioning. Brain growth was determined over a 24‐hr period (stages 11–18) by calculating volumes from area measurements of sections of brains from individual embryos by using a computerized image‐analysis system. Statistical analysis indicated that a linear model adequately described cavity expansion, and a linear model was rejected for the description of tissue growth and total brain enlargement. At the onset of brain enlargement, the cavity expands faster than the tissue grows; but after 12 hr the reverse is true. Initially (i.e., at stage 11), the cavity accounts for 60% of the total brain volume and tissue for 40%. At stages 12–16, cavity and tissue contribute 50% each. Finally at stage 18, cavity accounts for 55% and tissue for 45%. In order to better distinguish changes in cavity expansion and tissue growth over the 24‐hr period studied, this period was divided into four intervals (I‐IV). The rates of both cavity expansion and tissue growth increase between intervals I and II, decrease between intervals II and III, and increase between intervals III and IV. Overall, the brain enlarges 8.5 times, the cavity expands 9.8 times, and the tissue grows 7.6 times. Change in cavity expansion for fixed time intervals is constant; for tissue growth it increases continually. Our results describe the cavity and tissue kinetics that occur during rapid brain enlargement. Such information is important for understanding normal development of the brain and may provide insight into the mechanisms of genesis of certain congenital malformations such as microcephaly and hydrocephaly.
Upper genital tract abnormalities in the Syrian hamster as a result of in utero exposure to diethylstilbestrol. II. Scanning electron microscope analysis of endometrium cell surfaces
Authors: Gilloteaux Jacques , Steggles Alan W.
Published: February 2005

Using scanning electron microscopy (SEM), we show that an in utero exposure to diethylstilbestrol (DES) will induce endometrial abnormalities postnatally. These changes are magnified when a subsequent postnatal DES treatment is given. More specifically, changes in cell surface morphology are associated with alterations in cell size and shape (from columnar to cuboid/squamous), and in microvilli and mucus secretion. Uteri from hamsters treated postnatally with DES and derived from normal (CD) or DES‐treated mothers (DD) show that accumulated mucoid secretion products are not expelled in the uterine lumen but are “stored” in cystic dilated glandular spaces of the fibrocellular stroma of polyps and papillae filling the uterine lumen. Uteri from female hamsters that have been prenatally treated with DES (DC) show endometrial mucosal crypts containing mucus and other cell debris including migrating granulocytes. In all cases, DES‐treated uteri show mucosal cell surface pleomorphism in the sequence DD > CD > DC Merocrine and/or cystic secretions were observed. Microvilli cover cell surfaces and include some rare and peculiar long microvillous growth. In contrast, DC hypoplastic uteri present no secretory activity. Structures similar to those described for CD uteri can be observed only after 250 days of age in DC uteri. This report confirms and complements previous observations, favoring the choice of the hamster as an animal mode for the study of endometrial carcinogenesis.
Comparison of lectin‐staining pattern in testis and epididymis of gerbil, guinea pig, mouse, and nutria
Authors: Arya Manjula , Vanha‐Perttula Tapani
Published: February 2005

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine‐conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin‐stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis‐derived material were lectin‐positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea‐pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin‐stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.
Peri‐ and postnatal development of heterogeneity in the amounts of endoplasmic reticulum in mouse hepatocytes
Authors: Kanai Kazuo , Kanamura Shinsuke , Watanabe Jun
Published: February 2005

The surface density and area per cell of the endoplasmic reticulum (ER) in periportal and perihepatic hepatocytes from male ddY mice, “17‐, 18‐, and 19‐day‐old fetuses,” “newborn and 1‐, 5‐, 10‐, and 20‐day‐old animals,” and “adult animals” were analyzed by quantitative electron microscopy. The surface density of rough ER was not significantly different between periportal and perihepatic cells in all age groups examined, except for 19‐day‐old fetuses in which the value was greater in periportal cells than perihepatic cells. The surface density of smooth ER and total (rough and smooth) ER did not significantly differ between the periportal and perihepatic cells from 17‐day‐old fetuses to 5‐day‐old animals. In 10‐ and 20‐day‐old and adult animals, the values of smooth and total ER were greater in perihepatic cells than in periportal cells. When the data were expressed as area per cell, the patterns of subacinar distributions hardly differed, but age‐related changes differed considerably from the patterns seen in the surface density data. The differences were generally caused by the increase in hepatocyte volume between 20 days of age and adulthood, especially in perihepatic cells, and by the changes in volume during the perinatal period. The results show that differences in the surface density and area per cell of smooth and total ER between periportal and perihepatic hepatocytes evident in adult animals are not present in fetal and newborn animals but arise during postnatal development.
Morphometric analysis of circadian variations in the retinal photoreceptor synaptic terminals of the adult and fetal guinea pig
Authors: McCartney Mitchell D. , Dickson D. Howard
Published: February 2005

In order to test whether the alterations in photoreceptor synaptic terminal size and shape reported in lower vertebrates occur in a mammalian visual system, adult and fetal guinea pig retinas were exposed to an LD 12:12 lighting cycle, as well as to long‐term light (LL) and long‐term dark (DD) regimes. Representative random samples from all retinal quadrants, obtained at various times during these lighting regimes, were processed for electron microscopy. The synaptic terminals of all three photoreceptor cell types in this retina (alpha and paranuclear rods, and cones) were analyzed with computer‐assisted morphometrics for changes in their area, perimeter, synaptic vesicle density, and the degree of plasmalemmal infolding. The data showed all three types of adult receptor terminals to have increased area and vesicle density, as well as decreased membrane infolding, during the light period, while both types of rods showed increased perimeter measurements in the dark. Results from adults maintained under extended lighting conditions (LL and DD) showed no difference when compared with sample times during a typical LD 12:12 lighting regimen where clear statistical differences existed. Data from fetal retinas showed no significant sustainable pattern in any of the measured variables. These quantitative findings have led to the conclusion that while alterations in perimeter measurements may be explained by using the vesicle recycling hypothesis, observed changes in terminal size and shape may be controlled by a light‐initiated or light‐enhanced mechanism and effected through an annular configuration of cross‐striated fibrils found within these photoreceptor synaptic terminals.
The effects of orchidectomy and replacement therapy on the ultrastructure and gonadotropin‐releasing hormone content of the median eminence of the rat
Authors: Beckman W. C.
Published: February 2005

The effects of 2 weeks of orchidectomy and replacement therapy with testosterone upon the content and distribution of gonadotropin‐releasing hormone (GnRH) in the median eminence were determined by means of radioimmunoassay and electron microscopy. Photographic montages were prepared from electron micrographs of the lateral median eminence at the point of deepest invagination of the tuberoinfundibular sulcus. Morphometric analysis of photographs of tissues immunohistochemically stained for GnRH was performed to determine changes in the volume density of GnRH‐containing axon profiles following the experimental treatments. A decrease in GnRH content after orchidectomy was observed both by morphometric analysis of axon volume density and radioimmunoassay of total GnRH content. Testosterone treatment of orchidectomized animals prevented the postorchidectomy loss of GnRH. Morphometric analysis of conventional electron micrographs revealed an increase in the number of axons containing no dense‐core vesicles following orchidectomy, but no decrease in volume density of the neuropil. The results indicate that the change in volume density of immunostained axons was related to the loss of immunostainable dense‐core vesicles and not to a change in the size or number of axons. The area corresponding to the location of the highest concentration of GnRH‐containing axons was observed to be largely avascular and separated from the vessels of the tuberoinfundibular sulcus by a “border zone” composed of glial foot processes. The unique morphology of the GnRH area has suggested the name “compact zone” to distinguish it from the palisade zone with which it is continuous medially. GnRH axons in this region are probably part of a tract extending farther caudally rather than a terminal field.
In situ localization of lectin‐binding glycoconjugates in the matrix of growth‐plate cartilage
Authors: Farnum Cornelia E. , Wilsman Norman J.
Published: February 2005

In the distal hypertrophic zone of growth‐plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin‐binding glycoconjugates were analyzed in these two matrix compartments of growth‐plate cartilage from Yucatan swine. Nine lectin‐fluorescein conjugates were tested by a postembedment method on 1‐μm‐thick, nondecalcified, Epon‐embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA‐I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA‐I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N‐acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin‐binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin‐binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.
Factors that determine the form of neuromuscular junctions of intrafusal fibers in the cat
Authors: Kucera Jan , Walro Jon M.
Published: February 2005

The form of terminations of fusimotor (γ) and skeletofusimotor (β) axons on intrafusal fibers was analyzed in serial sections of 20 spindles of the cat tenuissimus muscle. Seven synaptic features were assessed either qualitatively or quantitatively from electron micrographs of transverse sections of 184 intrafusal and 30 extrafusal endings. Features were compared among endings that were terminations of γ or β axons on different types of intrafusal fiber at different distances from the spindle equator. These comparisons indicated that interactions of several factors, and not the motor axon alone, determine the form of motor endings. Intrafusal muscle fiber type is dominant to the motor axon in regulation of the number and depth of postsynaptic folds. Separation of the influence of the motor axon from the muscle fiber was less clear with respect to the size of ending. Complete expression of the muscle fiber–motor axon interaction reflected by the form of motor endings is dependent upon location of the ending relative to the sensory region. Both depth of the primary synaptic cleft and size of the soleplate of motor endings increased with increasing distance of the ending from the spindle equator. A system of classification of cat intrafusal motor endings that reflects the multiplicity of factors that determine the form of endings, and one that simplifies the current terminology, is proposed.