菊地 元史

Ossification or calcification of the ligamentum flavum (LF) is relatively common in the middle and lower cervical, thoracic, and lumbar spine but extremely rare in the upper cervical region. This clinical fact suggests that there exist local factors promoting or preventing ossification or calcification of LF. However, little is known about the differences in the ultrastructure and cellular alterations of the LF between the different spinal levels, even in the cervical spine. With electron microscopy, we examined samples of LF collected surgically from the upper and lower cervical spine regions we then studied the apoptotic appearance of ligament cells using a preferential labeling method. We found direct evidence of apoptosis of ligament cells in the LF. Apoptosis was more apparent in the upper region samples than in the lower region samples. The spaces around the normal fibroblasts were filled with thick collagen fibrils, but the collagen fibrils disappeared around the apoptotic bodies and thin fibrils were formed. The difference of the level of apoptosis may correlate to the ultrastructual difference of LF, and our data will benefit further investigations seeking to clarify the mechanism of various pathological conditions in the human LF. © The Japanese Society for Clinical Molecular Morphology 2005.

Different types of integrin alpha subunit were investigated immunohistochemically to determine the relationship between the cell adhesion molecule and the local morphology of the rat anterior pituitary using anterior pituitary tissues and primary cultured cells. Alpha 3 subunit was shown to be the main integrin subunit in the anterior pituitary. In vitro, immunoreactions of α3 subunit appeared mainly surrounding cell clusters. The distribution roughly overlapped with immunoreaction of laminin, while it was also observed where expression of laminin was negative. When dispersed cells were primary cultured for 2, 3 or 5 days, immunoreaction of α3 subunit was clearly detected in the vicinity of cell membrane throughout the period while that of α5 subunit was not observed on day 2, it was observed as dots in cytoplasm on day 3 and more distinctly as lines in the vicinity of the cell membrane on day 5. Alpha 5 subunit was not detectable in vivo. These results indicate the possibility that integrin α3 subunit not only binds the cells to the basement membrane but also to other extracellular matrix or directly to adjacent cells in the anterior pituitary. In addition, integrin α5 subunit may supplement the function of α3 subunit under certain conditions.

Seasonal temperate zone breeders respond to increasing day length to anticipate the approach of spring breeding conditions. Other (supplementary) environmental cues, such as temperature and precipitation, were historically thought to play unimportant roles in reproductive timing. We demonstrate variation in reproductive timing across small geographic distances by examining the vernal testicular recrudescence of adult song sparrows (Melospiza melodia morphna) breeding in coastal (0-10 in elevation) and montane (280-1220 in elevation) habitats. Each year, these birds experienced the same photoperiod, but were exposed to different supplementary cues that varied with altitude. Coastal birds experienced warmer and more stable temperatures during late winter and early spring than did montane birds. We measured bud opening, emergence of new green shoots, and arthropod biomass to monitor the pace of spring's approach. New spring shoots emerged 2 months earlier on the coast than in the mountains and buds on flowering trees and shrubs also tended to open earlier at the coast. Arthropod biomass was similar in both the mountains and the coast during early spring, and began to increase in early summer. Reproductive morphology (i.e. testis volume and cloacal protuberance length) developed up to 2 months earlier on the coast than in the mountains. Testicular recrudescence occurred earlier on the coast in most years and proceeded at a faster rate in I year. Circulating levels of luteinizing hormone, follicle stimulating hormone and prolactin increased through the season, but did not correlate with differences between sites. Both populations responded similarly when exposed to identical photoperiodic cues in the laboratory. Therefore, we suggest that an integrated response to cues characteristic of location and elevation account for differences in patterns measured in the field.

The structure of the ligamentum flavum has yet to be fully elucidated and no studies have investigated fine structural differences at different spinal levels in any animals. The aim of the present study was to clarify structural differences in the ligamentum flavum at different spinal levels (cervical: C3/4 and C5/6 upper thoracic: T2/3 lower thoracic: T9/10 lumbar: L3/4) using light and electron microscopy of rabbit specimens. Light microscopy using resorcin-fuchsin staining revealed that the distribution of elastic fibers was diffuse in the cervical and upper thoracic regions, but was generally dense in the lower thoracic and lumbar regions. Electron microscopy demonstrated that the cervical and upper thoracic regions were rich in collagen fibers. Conversely, the lower thoracic and lumbar regions were rich in elastic fibers. Quantitative image analyses displayed thick elastic fibers in the lower thoracic and lumbar regions, with high area ratios. Radiographic examinations revealed that ranges of motion were large at the cervical region, but small at the lower thoracic and lumbar regions. These findings suggest that structure of the ligamentum flavum varies at different spinal levels with respect to differences in motion.

To investigate the proliferative potencies of the two types of synovial lining cells (types A and B), we used immunohistochemical techniques under light and electron microscopy to survey the complete process of arthritis in a rat model system. Complete Freund's adjuvant (0.1 ml) containing 5 mg of heat-inactivated Mycobacterium butyricum was administered intradermally into the right hind paws of 25 male Sprague-Dawley rats. Five animals were killed at weeks 1, 2, 3, and 4 after the immunological challenge, respectively, and the infrapatellar adipose synovium was removed from each animal. Using proliferative cell nuclear antigen (PCNA) as a cell proliferation marker, we quantified the locations of proliferative cells under light microscopy and then identified the cell type with immunoelectron microscopy. The number of PCNA-positive cells was high in the deep layer at week 2, and most of them were determined to be B cells. At week 3, the number of PCNA-positive cells increased in the superficial layer, and most were identified as A cells. Our results support the idea that cells of the synovial lining proliferate in situ during the course of arthritis.

During aging, the male Japanese quail exhibits a loss of fertility, increased morphological abnormalities in the testes, and a higher incidence of Sertoli cell tumors. Although there is a co-incident loss of reproductive behavior, plasma androgen levels remain high until testicular regression occurs in association with senescence. The purpose of this study was to compare mean specific binding of chicken luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as a measure of testicular receptors during identified stages during aging. Males were categorized according to age (young = 9 months, middle aged = 24 months, or old = 36+ months) and sexual behavior (active or inactive). Testicular samples were collected immediately after perfusion with 4% paraformaldehyde from the following groups: young active (n = 8), young photoregressed (n = 5), young photoregressed plus testosterone implant (n = 4), middle-aged active (n = 8), middle-aged inactive (n = 4), old inactive (n = 5), and old inactive plus testosterone implant (n = 6). A crude plasma membrane fraction was prepared from the testes of each bird and an aliquot deriving from 10 mg of testicular tissue was used for binding assay. Specific binding of labeled LH or FSH was expressed as percentage of total radioactive hormone. Results showed significant (P < 0.05) age-related decreases in both FSH and LH receptor numbers. The highest FSH binding was found in young and middle-aged active males, with low binding in old inactive males. Testicular LH binding decreased during aging, with a sharp decrease in middle-aged males, which was similar to old males. Testosterone implants weakly stimulated FSH and LH binding in old males. Both LH and FSH binding decreased in photoregressed young males. However, testosterone implants stimulated increased LH binding, but did not affect FSH binding in young photoregressed males. These results provide evidence for separate regulation of testicular LH and FSH receptors, with testosterone stimulation of LH receptor, but not FSH receptor number in young males. However, during aging there appears to be a loss of this response, potentially because of the reduced efficacy of testosterone stimulation, thereby implying a diminished capacity for response with aging.

鳥類の性判別法として,性染色体上に存在するCHD(chromodomain helicase DNA binding protein)遺伝子を解析する方法が有用である。トキ(Nipponia nippon)のZおよびW染色体上のそれぞれのCHD遺伝子について特定領域の塩基配列を決定した。両者の間には数箇所のヌクレオチドの変異が見られた。これにより,PCR法によって試料中のCHD遺伝子の特定領域を増幅し,そのPCR産物を制限酵素解析することによって性を判別する方法をトキにも応用できることが明らかとなった。この方法を用いて,1999年に中国より贈られたつがいのトキから,佐渡トキ保護センターにおいて,1999年,2000年に産まれた計3羽の雛の性判別を行なった。DNAの抽出のための試料として,羽軸に付着した細胞の他,孵化後の卵殻内に残された胚由来の組織が有効であった。

We examined the topographic cell affinity of different types of hormone-producing cells in the rat anterior pituitary gland. Pituitaries were removed from 8-10 week-old male SD rats, and were fixed in sublimate-formalin. The 40 μm sections were obtained with a Microslicer, then double-immunolabeled (FITC and TexasRed methods) using antibodies against five types of pituitary hormones. Topographic affinity was examined three-dimensionally using a confocal microscope, and was expressed as attachment rate. Statistical analysis revealed that the different types of hormoneproducing cells did not exist at random locations in the gland, but rather showed specific topographic affinities. The following pairs of cell types were observed to have significantly high affinities: LH and PRL cells, ACTH and GH cells, and TSH and GH cells. In contrast, the following pairs of cell types were observed to have no significant affinities: GH and PRL cells, ACTH and LH cells, TSH and LH cells, ACTH and TSH cells, and cells of the same type. These specific topographic affinities between different types of anterior pituitary cells may be intimately related to cell-to-cell interactions in the gland.

To estimate gonadal and adrenocortical endocrine activities of birds without disturbing them, we have devised methods for extracting and measuring steroid hormones in excreted feces. Methods needed for proper extraction of steroid hormones differed among species and included a simple extraction with water, an extraction with ethanol-acetone followed by an LH-20 column chromatography or an extraction with ether followed by a reverse phase HPLC. Samples thus prepared were analyzed for steroid hormones by radioimmunoassays. Preliminary experiments using Japanese quail showed that fecal steroid levels reflect plasma levels and hence the endocrine activity as well. Based on this experience, we proceeded to examine reproductive capacities of Japanese crested ibis, an endangered species with only one male and female were surviving in Japan in 1995, and also to study seasonal changes in gonadal and adrenocortical activities of wild rock ptarmigan. Clear changes in sex steroids and corticosterone were observed in the rock ptarmigan.

A variety of orientation cues has been suggested for the migration to the breeding site in adult amphibians. We categorized the cues into the following 3 groups: 1) cues from the breeding pond such as male calling and pond odors, 2) celestial cues such as the sun light and the magnetic field of the earth and 3) cues from the area or route of the migration which compose a local map such as a visual and olfactory maps. To determine which of these is used by the toad, Bufo japonicus, we designed and conducted a displacement experiment in which migrating toads from one direction were transported to the ground in the opposite side of the pond. The displaced toads were completely disoriented and moved to random directions. We conclude that the toad uses a local map) to orient to the breeding pond and cues from celestial bodies and the pond are not used. We also found that adult toads tracked the same route on both trips from and to the pond. This suggests that the local map was memorized by newly metamorphosed toads at their first terrestrial trip from the pond. The next step of our study was to determine what sense is used to receive the cue . We found blind toads, whose upper and lower eye-lids were stitched together, could reach the pond at a similar rate with the sham-operated and intact toads. However, anosmic toads, whose olfactory mucosa were damaged by the treatment with a 5% silver nitrate solution, rarely reached the pond. Tracking experiments with blind toads and toads bearing a bar magnet on their heads revealed that neither blindness nor the magnet had any effect on either the direction or distance of the migration to the pond. In contrast, anosmic toads could not orient properly and moved in random directions. We speculate that newly metamorphosed toadlets remember the route or area of their first terrestrial migration by using the olfactory sense. In the spring, two to three years later, they start their breeding migration to the home pond by using a local olfactory map and reverse tracking of the memorized route.