bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in epithelial glycocalyx of rabbit uteri during early pseudopregnancy and pregnancy, and following ovariectomy
Authors: Anderson Ted L. , Hoffman Loren H.
Published: February 2005

Pseudopregnant, pregnant, and ovariectomized rabbits were utilized to study hormonal mediation of uterine epithelial surface negativity and glycocalyx morphology, and to seek local effects of blastocysts at sites of implantation. A loss of surface negativity [polycationic ferritin (PCF) binding] by day 6 of pregnancy or pseudopregnancy was noted, accompanied by alterations in epithelial glycocalyx. Uteri from estrous animals, or ovariectomized animals receiving oil or estradiol injections, bound PCF and exhibited a “globular” glycocalyx. Uteri from day 6 pseudopregnant or pregnant animals, or ovariectomized animals receiving progesterone injections, did not bind PCF or exhibit a globular glycocalyx. Both PCF binding and the globular character of the epithelial glycocalyx were sensitive to neuraminidase and trypsin treatment, suggesting sialoglycoprotein contribution to surface negativity. Implanting blastocysts had no detectable local effect on surface negativity, but did induce local reduction of epithelial glycocalyx at sites of implantation. Results of this study suggest that uterine epithelial glycocalyx alterations during the preimplantation period reflect a general response to prosgesterone stimulation, primarily qualtitative in nature, related to the acquisition of receptivity to ovo‐implantation.
Carbohydrate determinants involved in mammalian fertilization
Authors: Ahuja Kamal K.
Published: February 2005

During fertilization in mammals, the male and female gametes undergo a form of highly complex cell‐cell recognition whereby a nonspecific initial binding is followed by a species‐specific penetration of the zona pellucida. Recent data from many species have demonstrated the involvement of surface carbohydrates in regulating fertilization at both these stages. The potential benefits as well as drawbacks of three major techniques used so far are discussed, and the need for a cautious interpretation of the data is emphasized. During capacitation, the carbohydrate components of the entire surface of spermatozoa undergo striking changes which may be linked to the concurrent metabolic events within motile spermatozoa, leading to the appearance of egg‐specific glycoconjugates in a time‐dependent manner. A multiple set of glycoproteins on the sperm surface, possessing oligosaccharides synthesized by the lipid‐linked pathway, are probably required during different stages of fertilization, including sperm‐oocyte fusion. The oviductal glycosaminolycans may also be involved in regulating the timing and species specificity of mammalian fertilization by masking the sperm receptor sites on the zona and triggering the physiological acrosome reaction. Future biochemical and highresolution localization studies involving specific probes for surface glycoconjugates, glycosyltransferases, and hydrolytic enzymes should greatly aid our understanding not only of the role of the individiual surface macromolecules but also of the surface domains to which they are localized.
Sperm surface components involved in the control of the acrosome reaction
Authors: Oliphant Gene , Reynolds Al B. , Thomas Ted S.
Published: February 2005

Several lines of evidence suggest that decapacitation of sperm occurs normally in the male reproductive tract, and as a result the acrosome is stabilized and the acrosome reaction is controlled. Since the defining experiments in 1951, where decapacitation was reversed in the female reproductive tract by capacitation, investigations have pursued the molecular events of this process. This review attempts to examine critically the older literature and compare that perspective with the current theories. The theories for decapacitation of sperm include the possible role of a peptide decapacitation factor, a glycoprotein‐mediated steroid transfer to the sperm, masking of a galactosyl transferase by some macromolecule‐containing carbohydrate, preclusion of calcium influx by a binding protein, and sperm interaction with the acrosome stabilizing factor. Although these theories are diverse, there are some unifying aspects. However, there remain some major unanswered questions. For example, although we point to some circumstantial evidence that infers a single decapacitation factor, this needs to be further substantiated. It is concluded that with the purification of a macromolecule involved in capacitation, specific proposals on the mechanism of capacitation, and new tools to evaluate the capacitation process, it is likely that another decade will not pass without emergence of a unifying molecular theory of sperm capacitation.
Mammalian gamete interactions: What can be gained from observations on living eggs?
Authors: Gaddum‐Rosse Penelope
Published: February 2005

Studies on fertilization have involved a variety of investigational techniques from the simple to the very complex. Perhaps the most direct approach has been the observation and photographic recording of the interactions of living gametes in vitro. The purpose of this paper is to review the major contributions that have been made, by means of this technique, to our knowledge of mammalian fertilization, and to examine its advantages and limitations.

Some of the events of mammalian fertilization that have been observed in living eggs and are reviewed herein include (1) sperm penetration through the zona pellucida, (2) contact of the fertilizing spermatozoon with the oocyte surface and the subsequent incorporation of the sperm head into the oocyte cytoplasm, (3) the formation and disappearance of the incorporation cone, (4) the gradual incorporation of the sperm flagellum, (5) surfac movements of the oocyte after activation, and (6) the formation of the second polar body. One advantage of studying living eggs is the opportunity it affords to witness events as they actually occur and, under favorable circumstances, to observe the whole series of events in individual eggs. Only by this mechanism can certain features of the process be fully appreciated and accurate data obtained on the timing of events. With the addition of time‐lapse photographic methods, some of the more subtle changes become more amenable to study. Among the limitations of the technique are its limited resolution and the necessity for examining the gametes outside their normal in vivo environment.
Mammalian fertilization as seen with the scanning electron microscope
Authors: Phillips David M. , Shalgi Ruth , Dekel Nava
Published: February 2005

For several years we have been looking at mammalian gametes and their interactions with the scanning electron microscope (SEM). Examining the images produced by the SEM has given us a three‐dimensional view of sperm, eggs, and egg investments. We are particularly impressed with the structural variation among gametes of different mammalian species. In this short report we examine the structure of mammalian spermatozoa, eggs, zonae pellucidae, and cumuli. Our observations and those of others have led us to believe that variation in gamete structure and function may have evolved as a mechanism for reproductive isolation of mammalian species.
The basement‐membrane‐like matrix of the mouse EHS tumor: II. Immunohistochemical quantitation of six of its components
Authors: Grant Derrick S. , Kleinman H. K. , Leblond C. P. , Inoue S. , Chung A. E. , Martin G. R.
Published: February 2005

The presence of six substances—laminin, type IV collagen, heparan sulfate proteoglycan, ontactin, fibronectin, and the amyloid P component—was investigated immunohistochemically in the matrix of the Engelbreth‐Holm‐Swarm (EHS) mouse tumor after it had been fixed in formaldehyde (with or without a brief preliminary glutaraldehyde fixation), embedded in Lowicryl K4M, and sectioned for processing through the protein A‐gold sequence. Enumeration of the number of gold particles per sequare micrometer of matrix sections demonstrated that the six substances were present in distinct amounts. The results for each substance were fairly consistent throughout the matrix in three experiments. Furthermore, the available evidence indicated that, with the exception of the amyloid P component, the substances were associated with the cord network of the tumor matrix. Finally, the use of a reconstituted basement membrane containing known amounts of laminin, type IV collagen, and heparan sulfate proteoglycan as a standard, led to the conclusion that, in the tumor matrix, the relative content of laminin to type IV collagen to the proteoglycan was in a ratio of 1:0.6:0.03, suggesting molar ratios of approximately 1:1:0.2, respectively.
The basement‐membrane‐like matrix of the mouse EHS tumor: III. Immunodetection of the amyloid P component in basotubules
Authors: Inoue S. , Leblond C. P.
Published: February 2005

Immunohistochemical methods were applied to the ultrastructural localization of the amyloid P component in the EHS tumor matrix. First, the preembedding approach was used by exposing frozen sections of tumor to antiserum against the mouse amyloid P component followed by the peroxidaseantiperoxidase sequence. Second, using the postembedding approach, Lowicryl K4M sections of the tumor were exposed to antiserum against the amyloid P component and subjected to the protein A‐gold procedure. In both cases, the immunostaining was restricted to structures which appeared in longitudinal section as fairly straight rods and in cross section as 7‐ to 10‐nm pentagonal or roughly circular profiles outlining a lumen with a central dot. Since these features are characteristic of basotubules, it is concluded that the basotubules of the tumor matrix possess the antigenicity of the amyloid P component and presumably contain this substance itself. Similar experiments carried out on the thick basement membrane known as Reichert's membrane demonstrated that its basotubules also possessed amyloid P‐component antigenicity. It is likely, therefore, that the amyloid P component is a constituent of basotubules.
How the fixation‐embedding protocol affects the specificity and efficiency of immunocytochemical stains for gonadotropin subunits
Authors: Childs Gwen V. , Unabia Geda , Tibolt Robert
Published: February 2005

This report describes a study designed to test factors that may affect the efficiency and specificity of stains for gonadotropins. These include chemical or freeze‐fixation and dehydration, heat polymerization of the plastic embedding compound, dehydration in organic solvents, and etching. Specifically, postembedding stains for LH or FSH subunits were applied to 1‐μm sections of (1) Araldite‐embedded pituitaries that were either chemically fixed and dehydrated or freeze‐fixed and freeze‐dried; (2) Aldehyde‐fixed pituitaries that were dehydrated in water‐soluble glycol methacrylate (GMA) and embedded in GMA at 4°C; and (3) p‐formaldehyde‐fixed pituitaries that were embedded in paraffin. A fourth group of pituitaries was dispersed and grown in monolayers for 1–3 days. These were stained following glutaraldehyde fixation. The optimal dilution of the primary antisera varied with the protocol; however, the percentage of cells staining for beta subunits did not change. In contrast, postembedding stains showed that alpha subunit reactivity is masked or destroyed in pituitaries that are fixed in glutaraldehyde and embedded in Araldite. Alpha chain reactivity was detected (in 14% of cells) either after freeze‐fixation and freeze‐drying followed by Araldite embedding, or after 4% paraformaldehyde fixation and GMA embedding (in 17% of cells). Staining in paraffin‐embedded pituitaries was seen in only 10% of the cells. Preembedding stains for alpha chains were strikingly sensitive, however, and immunoreactivity was seen in 18–26% of the population of monolayer cells. Thus, whereas the percentages of cells staining for beta subunits do not change following the use of most of the fixation and embedding protocols, alpha chain reactivity is destroyed by all but the mildest. These findings show that one can control or improve the specificity of the stains for LH and FSH by the fixation‐embedding protocol.
Observations on the solitary cilium of rabbit oviductal epithelium: Its motility and ultrastructure
Authors: Odor D. Louise , Blandau Richard J.
Published: February 2005

Solitary cilia have been observed on rabbit oviductal epithelial cells. In tissue cultures of fimbrial epithelium of 3‐ and 4‐day‐old animals observed by phase microscopy, most of these single cilia exhibited a vortical or funnel‐type movement while others had the usual to‐and‐fro motility. Primary cilia are usually considered immotile. Transmission electron microscopy of specifically identified single cilia revealed differences between the ciliary shafts and basal bodies of the single cilia as compared to those of mature oviductal ciliated cells. The basal body of the solitary cilium often had at least two triangular, striated, basal foot processes, lacked electron‐dense satellite material around its basal end, and occasionally had striated rootlets. In contrast, the cilia of mature ciliated cells had only one basal foot, exhibited much electron‐dense satellite material, and lacked rootlets. Cross sections of the single cilia showed patterns of microtubules different from the usual 9 + 2 axonemal complexes of normal cilia and included 9 + 0, 10 + 2 singlets, 7 + 2 doublets, and 8 + 1 doublet and 2 singlets; one did have the usual 9 + 2 arrangement. We postulate that the presence of more than one basal foot process may be responsible for the vortical motility observed. The primary cilia are shorter than normal cilia; the longest one measured was 1.86 μm in length, 0.28 μm in width at its base, and 0.14 μm at its tip. Based on the lightmicroscopic, scanning‐electron‐microscopic and transmission‐electron‐microscopic observations, such solitary cilia were observed more frequently in the oviductal tissues of the 3‐ to 4‐day postnatal rabbits grown in tissue culture and in ovariectomized and ovariectomized/progesterone‐treated adult animals than in estrous, ovulatory, or ovariectomized/estradiol‐treated rabbits.
Immunocytochemistry: Its evolution and criteria for its application in the study of epon‐embedded cells and tissue
Authors: Gosselin Edmund J. , Cate Charles C. , Pettengill Olive S. , Sorenson George D.
Published: February 2005

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen‐specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidinbiotin, and colloidal‐gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic‐embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen‐specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 μm does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two‐step technique that employs an antigen‐specific primary antibody followed by a gold‐labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include (1) substitution of specific antiserum with normal serum; (2) the use of antigen‐adsorbed antiserum; (3) the use of antisera with specificities for antigens not present in the tissue being studied; (4) the use of tissue previously shown to be stainable for the antigen; and (5) if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.
Use of pre‐embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells
Authors: Tougard Claude , Picart Renée
Published: February 2005

A pre‐embedding immunoperoxidase procedure performed directly on cultured cells in situ was used to localize several intracellular antigens at the electron‐microscope level. With this procedure, we compared the effect of various fixatives, with or without saponin permeabilization, on the immunoreactivity of a secretory product (prolactin) and membrane proteins in cultured prolactin cells. Prolactin was detected within all compartments of its intracellular secretory pathway. Membrane antigens of the rough endoplasmic reticulum, the Golgi apparatus, and lysosomes were localized in distinct intracellular compartments. These immunocytochemical results are discussed in relationship to others in the literature that describe the localization of similar types of antigens. The technique, here described, which preserves ultrastructural detail and antigenicity, should be applicable for the localization of other intracellular antigens in cultured cells.
Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system
Authors: Anthony Edythe L. P. , King Joan C.
Published: February 2005

A pre‐embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone‐releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine‐structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a “pretreatment” regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free‐floating sections and the peroxidase‐antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.
Ultrastructural characteristics of peptidergic neurons using pre‐embedding immunocytochemical methods
Authors: Piekut Diane T.
Published: February 2005

The immunocytochemical staining technique represents a valuable method for electron microscopic analysis with which to elucidate the ultrastructural characteristics, synaptology, and potential interactions of selectively labeled neuronal systems. The unlabeled antibody peroxidase‐antiperoxidase (PAP) technique is one of the most widely used immunocytochemical methods and has been applied to both pre‐embedding and postembedding EM procedures. This review provides a detailed protocol for the former method and discusses advantages and disadvantages associated with the use of both approaches. A descriptive fine structural analysis of peptidergic neurons localized in the hypothalamus of brain illustrates the applicability and specificity of utilizing this method for the localization of cellular antigens of other systems.
Some improvement in tissue preparation and colloidal‐gold immunolabeling for electron microscopy
Authors: Hisano S. , Adachi T. , Maegawa M. , Daikoku S.
Published: February 2005

The application of freeze‐substitution (FS) and freeze‐drying (FD) techniques and the protein A‐gold‐antibody complex immunocytochemical methods are described. The two tissue‐preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue‐preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A‐gold‐antibody complexes are easily prepared and label the antigens clearly. If the protein A–coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50‐nm gold particles, and immunoreactive methionine‐enkephalin, labeled with 15‐nm gold particles, in the axonal terminals of the FD‐prepared rat neurohypophysis. This study demonstrates the value of the use of gold‐antibody complexes for immunocytochemical labeling on FS‐ or FD‐treated tissues.
Postembedding immunogold labeling for electron microscopy using “LR White” resin
Authors: Timms B. G.
Published: February 2005

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White‐embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high‐resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.
Use of immunoperoxidase and immunogold methods in studying prolactin secretion and application of immunogold labelling for pituitary hormones and neuropeptides
Authors: Papka R. E. , Yu S. M. , Nikitovitch‐Winer M. B.
Published: February 2005

Two immunocytochemical methods, immunoperoxidase and immunogold (IG), were used in an attempt to study the dynamic process of prolactin release from stimulated rat pituitary mammotrophs. The immunogold method was also used to localize other pituitary hormones including growth hormone, follicle‐stimulating hormone, luteinizing hormone, and the neuropeptides substance P, neuropeptide tyrosine, leu‐enkephalin, and atrial natriuretic factor in peripheral nerves. Light‐microscopic immunoperoxidase staining of prolactin revealed a unique distribution of immunoreactive mammotrophs. Two groups of cells were seen, one centrally located and one forming a narrow peripheral rim on the gland. The two groups were separated by a zone of nonimmunoreactive cells. In addition, the distribution of immunoper‐oxidase‐stained material was not uniform in all mammotrophs. In some, prolactin immunoreactive material was clumped near the nucleus (in the Golgi cisternae); in others it was more diffused within the cytoplasm (but immediately surrounding the cisternae of rough endoplasmic reticulum). After stimulation of mammotrophs, via suckling, prolactin‐immunoreactive material was visualized in extracellular spaces. With immunogold methods, prolactin labelling was seen mainly in secretory granules; but some labelling of Golgi cisternae and rough endoplasmic reticulum also occurred. Immunogold labelling revealed that material immunoreactive for leu‐enkephalin and atrial natriuretic factor was present in nerve terminals in the rat paracervical ganglion. Material immunoreactive for substance P and neuropeptide tyrosine was present in nerve terminals in the guinea pig heart. Thus, in some situations the immunoperoxidase technique was useful and helped to visualize “grossly” the presence of specific antigens, but it was inadequate for fine ultrastructural localization of these antigens. The immunogold technique was excellent for precise localization of antigens and especially for the detection of colocalization of different antigens. This method can be used in very different structures, such as the adenohypophysis and peripheral nervous tissue, without any modification except for the nature of the antibodies.
Pitfalls in immunocytochemistry with special reference to the specificity problems in the localization of neuropeptides
Authors: van Leeuwen Fred
Published: February 2005

In this review, the different types of specificity in immunocytochemistry (ICC) are discussed. Some examples of misinterpretations on the basis of ICC results are given. A strategy is proposed to assess the specificity of antisera and to test them again after purification.
A comparative study of muscle spindles in slow and fast neonatal muscles of normal and dystrophic mice
Authors: Johnson Marjorie I. , Ovalle William K.
Published: February 2005

Muscle spindles from the slow‐twitch soleus and the fast‐twitch extensor digitorum longus (EDL) muscles of genetically dystrophic mice of the dy2J/dy2J strain were compared with age‐matched normal animals at neonatal ages of 1–3 weeks according to histochemical, quantitative, and ultrastructural parameters. Intrafusal fibers in both the soleus and EDL exhibited similar regional differences in myosin ATPase activity, and conformed to those noted previously in various adult species. In distal polar regions, all nuclear bag fibers resembled extrafusal fibers of the type 1 variety, whereas in capsular zones they could be divided into two subtypes. Nuclear chain fibers possessed a staining pattern similar to type 2 extrafusal fibers, and in contrast to the bag fibers they exhibited no regional variations. These features were consistently observed in both the normal and dystrophic muscles at all ages. Spindles varied only slightly in their number and distribution in the two types of muscle, and their location followed the neurovascular branching pattern in each. Irrespective of age or genotype, spindles in the soleus were more homogeneously dispersed, but those in the EDL were concentrated along the dorsal aspect of the muscle. No significant differences were noted in the total number of spindles between normal and dystrophic muscles. In addition, no dramatic differences were observed in the muscle spindle index for soleus and EDL. The first obvious disease‐related changes were noted in extrafusal fibers of the soleus of 3‐week‐old mice, and spindles were often located close to these areas of fiber degeneration. Despite alterations in the surrounding tissue, however, spindles appeared morphologically unaltered in dystrophy. These observations indicate that intrafusal fibers of spindles in neonatal mice appear enzymatically and histologically unaffected in incipient stages of progressive muscular dystrophy.
SEM observations of the elastic networks in canine femoral artery
Authors: Crissman Robert S.
Published: February 2005

Scanning electron microscopy was used to study the normal architectural arrangement of elastic tissue in a medium‐sized muscular artery. Selective NaOH sonication digestion or formic acid digestion was used to expose and isolate the elastic networks in the femoral arteries of four healthy dogs. The digested segments were neutralized and freeze‐dried before mounting for scanning electron microscopy (SEM) observation. The fenestrated internal elastic lamina (IEL) had a smooth surface with scattered regions of the fine elastic fibers that made up lacy networks protruding from the luminal surface. Prominent ellipsoid fenestrae, randomly scattered across the surface, were grouped into small and large sizes based on their mean diameter. The openings of most fenestrae were bridged by elastic fibers to give the fenestrae a sieve‐like appearance. Large, transversely oriented, fusiform gaps were randomly scattered along the length of the IEL. These gaps, filled in by an elastic fiber network, sometimes spanned as much as a quarter of the vessel circumference. It is suggested that these gaps represent splits in the IEL that have been repaired. The tunica media contained a complex network of anastomosing elastic fibers and lamellae that were primarily circumferential in orientation. A well‐defined external elastic lamina formed a solid sheet at the junction of the tunica media and the tunica adventitia. The tunica adventitia contained 8–10 incomplete lamellae of large, interconnecting, longitudinally oriented fibers. The architecture of the elastic network in canine femoral artery was compared with that previously described in medium‐sized canine veins and in the rat femoral artery.
Tooth induction and temporal patterning in palatal epithelium of fetal mice
Authors: Richman Joy Marion , Kollar Edward J.
Published: February 2005

The present study examined the effect of aging on epithelium and on its ability to respond to an inductive stimulus provided by murine dental papillae. In fetal CD‐1 mice, 15‐ to 17‐day molar mesenchyme was combined with 15‐ to 19‐day epithelium from the secondary palates. Enamel organs were separated from the dental papillae, and palatal epithelium was peeled away from its underlying mesenchyme after treatment with 1% trypsin. Recombinants of epithelium and papillae were initially cultured on a solidified complex medium for 24 hr followed by an additional 10–14 days of intraocular explanation. Control specimens consisted of isolated molar papillae. Nineteen of 88 isochronal, heterotypic recombinations formed teeth. None of the 46 heterochronal, heterotypic grafts of 18‐ and 19‐day palatal epithelium combined with 15‐ to 17‐day molar papillae‐produced teeth. Instead, keratin‐filled epithelial cysts and bone spicules were formed. Isolated control molar papillae often formed bone in the intraocular sites but did not form teeth or contain epithelium. These results show that palatal epithelium is first restricted to its developmental pathway at 18 days of gestation. Younger epithelium can convert to functional ameloblasts that secrete enamel protein. In addition to the change in gene expression, normal tooth morphology is attained. The loss of competence of the palatal epithelium at 18 days gestation coincided with the acquisition of stratum corneum and the attainment of the fully differentiated state. The oral surface of palatal epithelium appears to be determined histogenically and morphogenically at 18 days of gestation in mice.
Collagen types I, III, and V in human embryonic and fetal skin
Authors: Smith Lynne T. , Holbrook Karen A. , Madri Joseph A.
Published: February 2005

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult‐like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger‐diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermalepidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno‐SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70–75%, type III collagen was 18–21%, and type V was 6–8% of the total of these collagens at all gestational ages tested, compared to 85–90% type I, 8–11% type III, and 2–4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel‐ and nerve‐associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.
Mesenchyme formation from the trigeminal placodes of the mouse embryo
Authors: Nichols David H.
Published: February 2005

The trigeminal placode is a thickened region of ectodermal epithelium located along the side of the embryonic head. Mesenchyme escapes from the placode to form neurons of the trigeminal (V) ganglion. To further our knowledge of the morphogenesis of this escape, plastic thick sections were cut from mouse embryos and stained for light microscopy by using a technique which revealed escaping mesenchyme. The escape of trigeminal mesenchyme began at approximately 12 somites of age and was substantially complete by 30 somites. These results provided spatial/temporal orientation for a subsequent electron microscopic study. The first ultrastructural manifestation of escape was the penetration of an otherwise continuous basal lamina by small cell processes. The presence of longitudinally oriented microtubules within these processes suggests that mesenchymal cells escape through the basal lamina by using microtubules to direct/move their contents (e.g., the cell nucleus) into an enlarging process. Nuclei were distorted as they passed into these processes. This distortion suggests that basal lamina, together with a possible contribution from basal microfilaments, forms a rigid obstruction which is disrupted in the region from which a process is formed. In some cases a collar of basal lamina was observed around the necks of processes, but their distal membranes were invariably lamina‐free. This lamina‐free membrane is possibly that which is newly formed to accommodate the growing process. In later stages of escape, instances were observed in which the lamina was completely absent beneath an escaping cell and partially degraded beneath adjacent cells as well. These instances suggest that enzymatic digestion may play a role in degrading the lamina during mesenchymal escape. Apical desmosomes were often retained beyond the initial stages of escape. Mechanisms involved in their disruption are thus not among those which initiate escape.
Processing of concanavalin A‐receptor complexes by rat osteoclasts in vitro
Authors: Popoff Steven N. , Schneider Gary B.
Published: February 2005

The osteoclast is a large multinucleate cell that is widely accepted as the primary effector cell responsible for normal bone resorption. In a previous study, we demonstrated that concanavalin A (con A) has a dose‐dependent biphasic effect on the bone‐resorbing capacity of osteoclasts, using a 45Ca bone‐organ culture system; bone resorption was stimulated at low concentrations and inhibited at high concentrations. The mitogenic property of con A in lymphocyte cultures is well documented; therefore con A has been used extensively to study the manner in which lymphocytes and other mononuclear cells process the cell‐bound lectin. In this study, we have investigated the processing of con A‐receptor complexes by osteoclasts in culture, using con A—FITC to evaluate the redistribution of cell‐bound con A via epifluorescence microscopy and using con A—ferritin to determine whether the lectin receptor complexes are internalized. The osteoclasts were obtained from the long bones of newborn rats and allowed to attach to glass coverslips at 37°C. Following attachment, the nonadherent cells were removed by rinsing. The adherent osteoclasts were preincubated in 50 μg/ml con A–FITC or con A–ferritin at 4°C for 10 min, washed to remove unbound con A, and incubated at 37°C for 15 or 30 min in the absence of con A. Positive controls were fixed immediately after preincubation at 4°C; negative controls were preincubated in con A–FITC and α‐methyl mannoside, the haptenic inhibitor of con A binding. The results demonstrate that redistribution and endocytosis of con A–receptor complexes occurs within 30 min. These findings confirm the hypothesis that cell‐bound con A can alter the structure and activity of osteoclast membrane components in a manner similar to that observed in mononuclear cell cultures. The internalization of con A may be important in altering osteoclastic activity by mediating intracellular mechanisms involved in the bone‐resorbing process.
An ultrastructural comparison of neuromuscular junctions in normal and developmentally arrested Rana pipiens larvae: Limited maturation in the absence of metamorphosis
Authors: Lynch Kathryn , Homer Mary J. , Harris C. Darryl , Morrissey James
Published: February 2005

Neuromuscular junctions in the rectus abdominis muscles of normal and developmentally arrested Rana pipiens larvae were studied with freeze fracture and conventional electron microscopy to determine whether structural aspects of junctional maturation depend on metamorphosis. Comparison was made between junctions in premetamorphic larvae 1–3 months old and junctions in larvae that had remained in premetamorphosis for more than a year (more than four times as long as normal). In most respects, junctions from the two groups of larvae were similar. Unlike adult junctions, nerve‐muscle contacts in both larval groups were pleomorphic and often involved more than one neuronal process; Schwann cell processes very rarely extended between nerve and muscle. Active zone structure ranged from total disorganization to an adult pattern of highly ordered double rows of particles aligned over junctional folds. Only quantitative analysis revealed differences between junctions in old and young larvae. The older larvae had fewer nerve‐muscle contact sites involving multiple neuronal elements and a higher ratio of active zone length to presynaptic membrane area, although the mean active zone length was the same in the two groups. The results indicate that the maturation of junctional shape, the branching pattern of the axons, and the relationship of presynaptic axons to Schwann cells must be directly or indirectly dependent on the hormonal or behavioral changes associated with metamorphosis.
The microfibrils of connective tissue: I. Ultrastructure
Authors: Inoué S. , Leblond C. P.
Published: February 2005

The ultrastructure of connective tissue microfibrils was examined in two sites: the ciliary zonule of the eye and the foot pad, in 20‐day‐old mice perfused with glutaraldehyde. The microfibrils were classified into two categories, referred to as typical and atypical.

Typical microfibrils predominate in both sites; they are unbranched, straight or gently curving, tubular structures of indefinite length with an overall diameter of 12.8 ± 1.7 nm in the zonule and 13.8 ± 2.8 nm in the foot pad. They are composed of two parts: tubule proper and surface band. The tubule is 7‐ to 10‐nm wide and characterized in cross section by an approximately pentagonal wall and an electron‐lucent lumen containing a 1‐ to 2‐nm bead referred to as a spherule. When longitudinal sections of microfibrils are examined at high magnification, the wall of the tubule does not appear as a continuous line but as a series of successive dots. The interpretation of these findings is that the tubule is composed of successive annular segments with an approximately pentagonal outline. The surface band is a 3‐nm‐wide, ribbon‐like structure wrapped around the tubule. The band has dense borders called tracks. Along the tracks, densely stained, 4.6‐nm‐long “spikes” are attached at 4.0‐nm intervals. The wrapping of the bands is somewhat irregular. They may be in a transverse position across single or several microfibrils, in which case each band might constitute a distinct belt; more frequently, the bands are oblique and appear to form a continuous helix. It is proposed that surface bands play a role in holding together the juxtaposed segments making up a tubule. A model has been constructed to represent the association of tubule and band into a typical microfibril.

Atypical microfibrils, which are more common in foot pad than in ciliary zonule, appear wavy, lack a definite tubule, and are characterized by distorted, irregular surface bands. They are attributed to proteolysis of typical microfibrils.
Cell surface of mouse blastocysts at the trophectoderm‐uterine interface during the adhesive stage of implantation
Authors: Chávez Daniel J.
Published: February 2005

The molecular basis for the acquisition of adhesiveness between blastocysts and uterine luminal epithelium is an interesting problem in reproductive biology. It is rather difficult to study implantation‐stage blastocysts of mice because during the implantation period each blastocyst becomes lodged within a crypt formed by decidualizing stroma. After trophectoderm adheres to uterine luminal epithelium, it is not possible to flush intact blastocysts from the uterus by standard recovery procedures. By identifying implantation sites with the Evans blue technique and splitting or gently separating the apposed epithelium of finely trimmed sites, it was possible to expose nonadhesive and adhesive trophectoderm to polycationized ferritin (PCF) and a series of ferritin‐conjugated lectins. Examination by transmission electron microscopy revealed that both adhesive and nonadhesive trophectoderm bound PCF, concanavalin A, wheat‐germ agglutinin, Ricinus communis agglutinin I, and Limulus polyhemus agglutinin, but not Dolicos biflorus agglutinin or peanut agglutinin. Nonadhesive trophectoderm bound succinylated wheat germ agglutinin but adhesive trophectoderm did not. There was no apparent difference in the relative amounts of each lectin bound to adhering and nonadhering cells.
The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts
Authors: Womble Mark D. , Carlson B. M.
Published: February 2005

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine‐alpha‐bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP‐less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra‐junctinal AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP‐less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP‐less grafts as early as 20 days postgrafting. The results of this study support the existence of still‐unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well‐differentiated endplates.
Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo
Authors: Nichols David H.
Published: February 2005

Murine neural crest mesenchyme begins its escape from columnar epithelium near the tips of the midbrain‐rostral hindbrain neural folds at 4+ to 5 somites of age. At that time the tip of each fold is located dorsolateral to the pharynx. Once crest formation is complete at this earliest site, it leaves behind both crest mesenchyme and overlying squamous epithelium. Crest formation then progresses medially, into the lateral margin of the neural plate. At the same time, this lateral margin elevates as the tip of the neural fold. By the time crest formation ceases at approximately 10 somites, the result of these simultaneous activities is to passively distribute the earliest mesenchyme, formed from the lateralmost epithelium, dorsolateral to the pharynx and the later, more medially derived mesenchyme lateral to the neural tube. Once formed, the crest mesenchyme dorsolateral to the pharynx is displaced ventromedially in a narrow, transient subectodermal space functionally similar to that observed in the chick embryo. Displacement might result from cell motility or the formation of matrix‐filled spaces between cells of the mesenchyme. Displaced cells are closely associated with the overlying columnar epithelium. This association precedes their subsequent induction and may reflect preliminary patterning. The crest mesenchyme passively distributed lateral to the neural tube is subsequently displaced medially. Here the formation of enlarged (matrix‐filled?) spaces is clearly involved in the initial displacement. Displaced cells proliferate to form the anlage of the trigeminal ganglion. The other major contributor to this ganglion is the trigeminal placode. The placodal epithelium is located dorsolateral to the pharynx of the 12‐somite embryo. If the epithelia of the head maintain their relative positions, this placode is derived from the squamous epithelium formed together with the earliest crest mesenchyme. If not, an alternative source is the columnar epithelium located ventromedial to the tip of the 4 + ‐ to 5‐ somite neural fold.
Different pharmacological anatomy in the paraventricular hypothalamic nucleus, supraoptic nucleus, and suprachiasmatic nucleus of rats: Quantitative autoradiography on angiotensin II receptor binding sites
Authors: Hwang Bang H. , Wu Jang‐Yen , Wieczorek Caroline M. , Harding Joseph W. , Erickson Joel B. , Wamsley James K.
Published: February 2005

Angiotensin II (AII) and vasopressin (VP) play important roles in cardiovascular function. Using 125I‐[Sar1, Ile8]‐angiotensin II (125I‐SI‐AII), a potent AII antagonist, AII receptor binding sites were autoradiographically localized in three VP‐producing areas of the hypothalamus and compared in hypertensive and normotensive rats. Within three major VP‐producing areas, AII receptor binding was highest in the paraventricular hypothalamic nucleus and lowest in the supraoptic nucleus, suggesting that a differential AII regulation of separate VP systems exists in the brainstem. No statistical difference in 125I‐SI‐AII receptor binding was found between WKY and SHR rats in each of the three major VP‐producing nuclei studied. These results are consistent with a role of AII receptors in a subtle and complicated regulation of VP in cardiovascular function.