Contents

Review article:

1. The structure and function of folliculo-stellate cells in the anterior pituitary gland(INOUE, K.)

Original articles:

1. Three-dimensional ultrastructure of synoviocytes in the horse joint as revealed by the scanning electron microscope(SHlKlCHl, M.)
   
2. Changes in the orientation of collagen fibers on the superficial layer of the mouse tibial bone after denervation: scanning electron microscopic observations(SUDA, K.)
   
3. Three-dimensional structure of the synaptic contact of the neuromuscular junction in the rat lumbrical muscle(SOHN, A.)
   
4. Rapid loss of dorsal horn lectin binding after massive brachial plexus axotomy in young rats(SANT'ANNA-DA-COSTA, E.)
   
5. Scanning electron microscopic studies on the route of neutrophil extravasation in the mouse after exposure to the chemotactic peptide N-formyl-methionyll leucyl-phenylalanine (fMLP)(HOSHI, O. and T. USHIKl)
   
6. Changes in the distribution of peanut agglutinin (PNA) binding molecules during muscle reinnervation following nerve crush injury(ZHOU, C. J.)
   
7. The extracellular matrix in the mouse brain: its reactions to endo-alpha-N-acetylgalactosaminidase and certain other enzymes(MURAKAMI, T.)
   
8. Molecular chaperone calmegin localization to the endoplasmic reticulum of meiotic and post-meiotic germ cells in the mouse testis(YOSHINAGA, K.)
   

Summary

Title
The Structure and Function of Folliculo-Stellate Cells in the Anterior Pituitary Gland

Author
Kinji INOUE, Ernest F. COUCH, Kaiya TAKANO and Satoshi OGAWA

Address
Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan; and Department of Biology, Texas Christian University, Fort Worth, Texas, U.S.A.

Summary
The folliculo-stellate cells (FS cells) in the anterior pituitary gland are characterized by their star-like appearance and their ability to form follicles. Although FS cells do not produce any pituitary hormones, their special tendency to surrounding endocrine cells with their long cytoplasmic processes suggests that they regulate endocrine ceils by intercellular communication. In spite of many morphological and cytophysiological studies recently performed, a precise understanding of the major functions of FS cells in the pituitary gland remains obscure. We review here the morphological characteristics of FS cells and their suspected functions in the anterior pituitary gland. It is well established that the FS cell produces many kinds of growth factors, i. e., fibroblast growth factor, vascular endothelial cell growth factor and interleukin 6. The biological significances of these growth factors in the anterior pituitary gland are also discussed in this paper. The origin and differentiation of FS cells, especially the possibility that the FS cell is a kind of stem cell which has the potential to differentiate into endocrine cells, is also presented.

Prof. Kinji INOUE
Department of Regulation Biology
Faculty of Science
Saitama University
255 Shimo-ohkubo, Urawa
338-0825 Japan
Tel and Fax: +81-48-858-3422
E-mail: kininoue@seitai.saitama-u.ac.jp

Title
Three-dimensional Ultrastructure of Synoviocytes in the Horse Joint as Revealed by the Scanning Electron Microscope

Author
Mitsumori SHIKICHI, Hiroko P. KITAMURA, Haruko YANASE, Akihiro KONNO,

Address
Hiromi TAKAHASHI-IWANAGA and Toshihiko IWANAGA
Laboratory of Anatomy, Graduate School of Veterinary Medicine and Department of Anatomy, Graduate School of Medicine, Hokkaido University, Sapporo, Japan

Summary
The synovial membrane displays a superficial cellular lining composed of two types of synoviocytes: ''absorptive" macrophages (type A cells) and ''secretory" fibroblast-like cells (type B cells). The types are intermingled and extend a variety of processes, rendering the cellular architecture of the synovial membrane difficult to visualize. Previous electron microscopic and histochemical studies failed to demonstrate the entire shape of synoviocytes, except our immunohistochemical study for protein gene product 9.5 in the horse joint. The present SEM study is the first to demonstrate the three-dimensional ultrastructure of synoviocytes as well as their distribution in the synovial membrane, using macerated samples from the horse carpal joints. The equine synovial membrane was largely covered by conspicuously developed synovial villi. Type A synoviocytes were closely similar to macrophages in regard to surface structure, and showed uneven distribution with the densest occurrence around the tips of the synovial villi. In the basal half of villi, type B synoviocytes, which were situated in close proximity to the synovial cavity, projected thick processes horizontally and inter. twined to form a regular network of processes on the synovial surface. Those in the upper half of the villi were located in the abluminal layers and protruded an antenna-like process into the joint cavity with tips covered with long microvilli, in addition to forming the superficial plexus of processes. Type B cells were also provided with fine, membranous extensions that tended to cover the surface of synovial intima. The meshwork of horizontal processes, the antenna-like processes, and the membranous processes imply advantages in not only secretion but also sensation and regulation of the barrier function in the synovial membrane.

Prof. Toshihiko IWANAGA
Laboratory of Anatomy
Graduate School of Veterinary Medicine Hokkaido University
Kita 18-Nishi 9, Kita-ku
Sapporo, 060-0818 Japan
Tel: +81-11-706-5187
Fax: +81-11-717-7569
E-mail: tiwanaga@vetmed.hokudai.ac.jp

Title
Changes in the Orientation of Collagen Fibers on the Superficial Layer of the Mouse Tibial Bone after Denervation: Scanning Electron Microscopic Observations

Author
Kohta SUDA, Kazuhiro ABE and Kiyoshi KANEDA

Address
Department of Orthopaedic Surgery and Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan

Summary
This study was undertaken to evaluate the relationship between the mechanical stress loaded onto the bone and the orientation of collagen fibers formed by osteoblasts. The femoral, obturator, and sciatic nerves in the left posterior legs of 7-week-old mice were exposed and electroscissored to reduce the mechanical stress loaded onto the leg. Four weeks after operation, the tibial bones in the control and denervated legs were removed and observed by scanning electron microscopy (SEM) after NaOCl treatment. In the control right tibia, collagen fibers on the superficial bone matrix tended to be arranged parallel to the longitudinal axis of the bone. However, the arrangement of collagen fibers in the left tibia, which were immobilized for 4 weeks by denervation, was disorganized and ran in random directions. The findings suggest that the direction of collagen fibers in the bone changes in response to the mechanical stress loaded onto the bone, probably due to changes in the activity of osteoblasts in the denervated leg.

Dr. Kohta SUDA
Department of Orthopaedic Surgery
Hokkaido University School of Medicine Kita-15, Nishi-7, Kita-ku
Sapporo, 066-8638 Japan

Title
Three-Dimensional Structure of the Synaptic Contact of the Neuromuscular Junction in the Rat Lumbrical Muscle

Author
Akiko SOHN, Toshiyuki KAIDOH and Takao INOUÉ

Address
Department of Anatomy, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan

Summary
This study examined the three-dimensional structures of the synaptic contact in rat lumbrical muscles by scanning electron microscopy using three different methods : the aldehyde prefix-osmium-dimethyl sulfoxide-osmium method (A-O-D-O method), the cell-extraction method, and the NaOH-digestion method. These three methods visualized the motor nerve endings, subneural basal lamina and postsynaptic sarcolemma, respectively. The motor nerve endings were composed of a cluster of spherical and cylindrical terminals. Pores on the presynaptic membrane were considered openings of exocytotic vesicles. The post. synaptic side of the subneural basal lamina showed numerous ridges, corresponding to junctional folds. Most of the ridges rose vertically from their base. The ridges showed widening, narrowing, and branching. The subneural basal lamina appeared to be composed of small granular substances. The basal lamina of the primary synaptic clefts had pores 25-30 nm in diameter, which may facilitate the transport of acetylcholine (ACh) without being hydrolyzed by ACh esterase in the lamina. On the outer surface of the postsynaptic sarcolemma in a sole plate, the primary synaptic clefts were composed of a mixture of depressions and gutters; so far as we know, this represents the only example of such a phenomenon. These depressions and gutters seem to fit respectively into the spherical and cylindrical terminals of the motor nerve endings. The openings of the junctional folds consisted of a mixture of many slits and a few pits in the primary synaptic clefts.

Dr. Akiko SOHN
Department of Anatomy Faculty of Medicine
Tottori University
Yonago, Tottori
683-8503 Japan
Tel: +81-859-34-8004
Fax: +81-859-34-8167

Title
Rapid Loss of Dorsal Horn Lectin Binding after Massive Brachial Plexus Axotomy in Young Rats

Author
Elizabete SANT'ANNA-DA-COSTA, Sergio L. CARVALHO Rosalia MENDEZ-OTERO and Leny A, CAVALCANTE

Address
Instituto de Biofísica C. Chagas Filho; and Pós-Graduação em Ciências Morfológicas, ICB, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Summary
Lectins are proteins with binding affinities for specific sugars in complex glycoconjugates, some of which have been implicated in limiting synaptic plasticity or modulating nerve growth and guidance. We studied the expression of the glycoconjugate recognized by the isolectin B4 of Griffonia simplicifolia (Gs-IB4) in spinal dorsal horns after massive axotomy of the brachial plexus in weanling rats. Gs-IB4+ binding sites in Rexed's lamina II were rapidly reduced after massive peripheral axotomy. This rapid loss suggests that multiple nerve lesions minimize the number of intact fibers that converge with lesioned fibers into the same cord segments and thus may prevent the plastic changes accompanying the lesion of single nerves.

Profa. Leny A. CAVALCANTE, M. D., Ph. D. Instituto de Biofísica Carlos Chagas Filho Universidade Federal do Rio de Janeiro
21941-590 Rio de Janeiro, RJ, Brazil
Tel: +55-21-290-6897
Fax: +55-21-280-8193
E-mail : leny@biof.ufrj.br
              lacav@abc.org.br

Title
Scanning Electron Microscopic Studies on the Route of Neutrophil Extravasation in the Mouse after Exposure to the Chemotactic Peptide N-formyl-Methionyl-Leucyl-Phenylalanine(fMLP)

Author
Osamu HOSHl and Tatsuo USHIKI

Address
Department of Anatomy and Histology, Niigata University School of Medicine, Niigata, Japan

Summary
The present study was performed to demonstrate three-dimensionally the process of neutrophil extravasation induced by the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in mice. Thirty to 40 min after the injection of fMLP to the mouse lip, the tissues were fixed with glutaraldehyde and examined by scanning electron microscopy (SEM) as well as by transmission electron microscopy (TEM). Observation of fMLP-injected tissues showed many neutrophils adhering to the inner wall of postcapillary venules. Some of these adherent neutrophils attached to each other to form groups of two to six. There were also many neutrophils migrating through the endothelium. Most of these neutrophils took a transcellular route, in that they penetrated the cytoplasm of the endothelial cells. The junction of two neighboring endothelial cells did not open, and endothelial pores free from migrating neutrophils were scarcely observed. There were bulging portions on the vascular wall which were probably produced by the presence of underlayed neutrophils. Thus, the present study gave direct evidence of neutrophil migration via a transcellular route in response to fMLP. Our findings also indicate that the pores of the endothelium close quickly after neutrophil extravasation.

Dr. Osamu HOSHI
Department of Anatomy and Histology Niigata University School of Medicine Asahimachi-dori, Niigata 951-8510 Japan
Tel: +8l-25-227-2059
Fax: +8l-25-224-1767
E-mail: o-hoshi@med.niigata-u.ac.jp

Title
Changes in the Distribution of Peanut Agglutinin (PNA) Binding Molecules During Muscle Reinnervation Following Nerve Crush Injury

Author
Chong Jian ZHOU, Masaru KAWABUCHI, Jian Wen HE, Akio KURAOKA, Kazuho HIRATA, Songyan WANG and Osami NADA

Address
Department of Anatomy and Cell Biology Graduate School of Medical Sciences, Kyushu University; and Department of Medical Technology, Kyushu University school of Health Sciences, Fukuoka, Japan

Summary
Peanut agglutinin (PNA) staining during muscle reinnervation following a crushing injury of the sciatic nerve was performed in reference to the neural profiles immunolabeled with the PGP 9.5 antibody. PNA staining in the normal controls exhibited dots, granules, or lines along the length of the nerve fibers in the nerve trunk, but was faint or absent in the motor endplate. At seven days post-crush, PNA staining was detected around the vacuolated neural structures in the disorganized nerve trunk, but was still faint or absent in the motor endplate. At twenty-one days post-crush, when PGP 9.5..positive regenerating axons appeared in most of the motor endplates, PNA staining, either faint or strong, followed the pathway of the nerve fibers delineated by PGP 9.5-like immunoreactivity. During reinnervation to the motor endplates, PNA staining displayed signs of remodeling in the nerve trunk, such as marked variations in density and profile in the nerve fiber-associated dots or patches; it increased in intensity in the connective tissue covering the area of the motor endplate, as well as in the junctional myofiber surface. The structures recognizable by PNA coincided with components of the connective tissue such as collagen fibers and capillaries. Results suggest that: l) the expression of PNA.binding molecules is dependent on the state of innervation, and 2) the spatiotemporal relationship between neural profiles and PNA staining provides sequences of axonal extension and subsequent nerve terminal maturation during regeneration in the motor endplate.

Dr. Chong Jian ZHOU
Department of Anatomy and Cell Biology Graduate School of Medical Sciences
Kyushu University
Fukuoka, 812-8582 Japan

Title
The Extracellular Matrix in the Mouse Brain: Its Reactions to Endo-Alpha-N-Acetylgalactosaminidase and Certain Other Enzymes

Author
Takuro MURAKAMI, Aiji OHTSUKA, Wei-Dong SU, Takehito TAGUCHI, Toshitaka OOHASHI, Tetsuro MURAKAMI, Koji ABE and Yoshifumi NINOMIYA

Address
Departments of Anatomy Molecular Biology and Biochemistry and Neurology, Faculty of Medicine, Okayama University Medical School, Okayama, Japan

Summary
As our previous studies have indicated, the cingulate cortex of the adult mouse brain contains many neurons with rich cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The present study demonstrated that exclusive incubation with endo-alpha-N-acetylgalactosaminidase abolished the lectin Vicia villosa or Wisteria floribunda agglutinin (VVA or WFA) labeling of the nerve cell surface glycoproteins, while it neither interfered with the cationic iron colloid or aldehyde fuchsin stainings of the perineuronal proteoglycans nor abolished the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Double incubations with endo-alpha-N-acetylgalactosaminidase and collagenase did not eliminate the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, though they did eliminate the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans as well as the Go!mo!ri's ammoniacal silver impregnation of the collagenous ligands. Triple incubations with endo-alpha-N-acetylgalactosaminidase,collagenase, and endoalpha-N-acetylgalactosaminidase abolished the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, and also eliminated the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans and the Gömöri's ammoniacal silver impregnation of the collagenous ligands. These findings indicate that: the nerve ceil surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase; these galactosamines associated with the collagenous ligands or perineuronal proteoglycans are not digested by endo-alphaN-acetylgalactosaminidase; and the terminal N-acetylgalactosamines newly exposed by collagenase incubation are digested by this galactosaminidase. It was further demonstrated that hyaluronidase incubation neither digests the collagenous ligands nor revives the lectin VVA or WFA labeling of the nerve cell surface proteoglycans

Prof. Takuro MURAKAMI
Department of Anatomy
Faculty of Medicine
Okayama University Medical School 2-5-1 Shikata-cho, Okayama
700-8558 Japan
Tel: +81-86-235-7088
Fax: +81-86-235-7095
E-mail: em2kai@med.okayama-u.ac.jp

Title
Molecular Chaperone Calmegin Localization to the Endoplasmic Reticulum of Meiotic and Post-meiotic Germ Cells in the Mouse Testis

Author
Kazuya YOSHINAGA, Ichiro TANII and Kiyotaka TOSHIMORI

Address
Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical College, Miyazaki, Japan

Summary
Calmegin is a testis-specific Ca2+,binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultrastructural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.

Dr. Kazuya YOSHINAGA
Department of Anatomy and
Reproductive Cell Biology
Miyazaki Medical College
Kiyotake, Miyazaki
889-1692 Japan
Te}.: +81-985-85-1783
Fax.: +81-985-85-1363
E-mail: kyoshina@post.miyazaki-med.ac.ip