X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

The molecular biology of Euglena gracilis. XV. Recovery from centrifugation-induced stratification

The contents of Euglena gracilis cells can be separated in vivo by ultracentrifugation. Within the unbroken cell, each set of components forms a distinct layer according to their respective densities. The degree of segregation increases with both the g-force and the time of centrifugation, up to a maximum at 100,000 x g for 1 h, when six distinct strata can be observed. When returned to normal growth conditions, essentially all the cells return to the normal state and growth pattern. Greater g-forces or longer exposures do not alter the observable strata, but the ability of the cells to recover is diminished. Smaller g-forces result in less separation of cellular contents and all cells recover, even after 18 h of exposure. Euglena cells stratified at 100,000 x g for 1 h were returned to normal growth conditions; recovery was followed microscopically and by the rate of utilization of oxygen as well as that of the single carbon source. The cells recovered their normal state within 1 to 2 h, which is only a tenth of the normal doubling time. The mechanism for this recovery involves a natural process of change in cell shape caused by contraction and relaxation of the pellicle, a cell surface structure.     

Behavior of in vitro grown normal human mucosal epithelial cells and tumorigenic rat cells inoculated into nude mice

The present study describes the behavior of in vitro grown normal human oral mucosal epithelial cells and that of a tumorigenic epithelial cell line following subcutaneous inoculation into nude mice. A successful recovery of viable human epithelial cell inocula was seen in 25-90% of mice and there was no improvement in recovery rates after addition of fibroblasts. These inocula resulted in cyst formation lined by a 2-6 cell layer unkeratinized squamous epithelium without rete ridges. There was no increase in recovery rate or size of cysts when coinoculated with fibroblasts. The tumorigenic cell inocula were successfully recovered in all cases. Tumors established from these inocula had a low grade of differentiation and were without signs of metastasis. Inocula of tumorigenic cells showed an increased size after addition of fibroblasts to the inocula. The model may be useful in studies of interactions between inoculations of heterologous normal and pathologic cells as well as in studies of differentiation of carcinogen-treated epithelial cells.     

SPECIFIC positive and negative selection of rat lymphocytes reactive to strong histocompatibility antigens: activation with alloantigens in vitro and in vivo.

This study compares the functional properties of rat thoracic duct lymphocytes (TDL) after stimulation with strong alloantigens of the major histocompatibility complex (MHC) either in vitro in preparative mixed lymphocyte interactions (MLI) or in vivo in systemic graft-vs-host (GVH) reactions. Comparisons were made of PHA responses and reactivity to the specific priming haplotypes or to third party haplotypes in analytical MLI and in GVH reactions either before or after the activated populations were "parked" in syngenetic T cell-deprived (B) rats. These comparisons can be summarized as follows: 1) TDL populations primed in bulk MLI cultures (MLI-TDL) slowed some evidence of specific positive selection when tested immediately; MLI responses to specific alloantigens were both relatively large and accelerated in tempo, whereas responses to third party alloantigens were diminished but also accelerated in tempo. Specific GVH responses were more marked than in third party recipients but they were also decreased relative to normal, and displayed an abberant dose/response slope. MLI-TDL populations tested after they had been stored in syngeneic B rats showed clear evidence of stable-specific positive selection; specific MLI and GVH responses were enriched relative to third party responses and also in comparison to normal, unselected TDL populations. This finding indicates that GVH and MLI reactivity are probably both functional capacities of the same lymphocyte subpopulation since positive selection by one function (MLI) also enriched for a second (GVH). 2) Parental strain TDL activated in vivo in the systemic GVH reaction in irradiated F1 animals and recovered from the thoracic duct 3 to 4 days later (late GVH-TDL) consisted mainly of blast cells, however, in contrast to MLI-TDL these populations showed no evidence of positive selection when tested before or after parking in B rats. MLI responses to specific alloantigens were minimal, and greatly reduced in magnitude compared to normal. GVH responses to specific haplotypes could be detected, but these were not enriched compared to normal, despite the content in the late GVH-TDL populations of a significant proportion of blast cells presumably activated by host alloantigens. 3) Early collections (less than 40 hr) of parental strain GVH-TDL collected from F1 recipients contained no blast cells and showed impressive degrees of negative selection; they were markedly depleted of both GVH and MLI activity to specific alloantigens but displayed normal reactivity to third party alloantigens. Moreover, specific negative selection was persistent in these populations parked for several weeks in B rats, and indication that a specific subpopulation of reactive cells had been physically eliminated. 4) PHA responses of both MLI- and GVH-activated TDL populations tested either before or after parking in B rats were approximately normal on a per T cell basis...     

Dexamethasone modulates the metabolism of type IV collagen and fibronectin in human basement-membrane-forming fibrosarcoma (HT-1080) cells.

The effect of dexamethasone on the synthesis and degradation of type IV collagen was studied in human fibrosarcoma cells, HT-1080. A dexamethasone concentration as low as 0.1 microM markedly increased collagen synthesis in HT-1080 cells labelled with [14C]proline. The increase in type IV collagen synthesis was not specific, since total protein synthesis was also increased. Further studies indicated that part of the increase was due to an increase in the specific radioactivity of the intracellular proline pool, after dexamethasone treatment. In fact, with dexamethasone concentrations of 0.1-10 microM the relative collagen synthesis was decreased, indicating that synthesis of other protein was increased more than that of type IV collagen. This was also confirmed by measuring the relative amount of type IV collagen RNA by using recombinant plasmid cDNA specific for the human procollagen pro alpha l (IV) RNA. The results indicated that relative collagen synthesis and the relative amount of type IV collagen messenger RNA was decreased similarly, indicating that dexamethasone affected type IV collagen synthesis at the pre-translational level. The dexamethasone-induced effect on total protein and collagen synthesis was maximal after 12-24 h. Dexamethasone induced a marked accumulation of collagen into the cell layer, leading to diminished deposition of soluble collagen into the medium. Since bacterial-collagenase treatment of the cell layer drastically decreased the collagen content of the dexamethasone-treated cells, this indicates that dexamethasone caused an accumulation of collagen into the extracellular matrix of the cell layer. In contrast, the amount of fibronectin was markedly increased in the medium. Dexamethasone decreased the type IV collagen-degrading activity in HT-1080 cells. The HT-1080 cells contained glucocorticoid receptors, as demonstrated by two different methods: by a whole-cell binding assay and by using a cytosol-gel-filtration method. The number of specific binding sites was similar to that in human skin fibroblasts. In conclusion, glucocorticoids affect the metabolism of type IV collagen and fibronectin in HT-1080 cells, and, since these cells contain specific glucocorticoid receptors, the effects are apparently receptor-mediated.     

Increased virus replication and virulence after serial passage of human immunodeficiency virus type 2 in baboons

Similar to human immunodeficiency virus type 1 (HIV-1) infection of humans, the natural history of HIV-2 infection in baboons (Papio cynocephalus) is a slow and chronic disease that generally takes several years before an AIDS-like condition develops. To shorten the amount of time to the development of disease, we performed five serial passages of HIV-2(UC2) in baboons by using blood and bone marrow samples during the acute phase of infection when viral loads were at high levels. After these serial passages, virus levels in plasma, peripheral blood mononuclear cells (PBMC) and lymphatic tissues in the acutely infected baboons were increased. Within 1 year of the HIV-2 infection, all of the inoculated baboons showed specific signs of AIDS-related disease progression within the lymphatic tissues, such as vascular proliferation and lymphoid depletion. The HIV-2(UC2) recovered after four serial passages showed increased kinetics of viral replication in baboon PBMC and cytopathicity. This study suggests that the HIV-2 isolate recovered after several serial passages in baboons will be useful in future studies of AIDS pathogenesis and vaccine development by using this animal model.     

Simulation of soil carbon cycling and carbon balance following clear-cutting in a mid-temperate forest and contribution to the sink of atmospheric CO2

A simulation model of soil carbon cycling was developed based on the data observed in a mid-temperate forest in Yoshiwa, Hiroshima Prefecture, Japan, and soil carbon cycling and carbon budget in a mature forest stand and following clear-cutting were calculated on a daily basis using daily air temperature and precipitation data. The seasonal change in the amount of the A₀ layer was characterized by a decrease from spring to autumn due to rapid decomposition of litter, and recovery in late autumn due to a large litterfall input. There was little change in the amount of humus in mineral soil. These estimates coincides closely with those observed in the field. Most flow rates and the accumulation of soil carbon decreased very markedly just after clear-cutting. The A₀ layer reached its minimum in 10 years, and recovered its loss within 50–60 years after cutting. A large loss of carbon was observed just after cutting, but the balance changed from negative to positive in 15 years after cutting. The total loss of soil carbon following cutting recovered within 30 years, and nearly the same amount of carbon as that stocked in the timber before harvesting accumulated 70–80 years after cutting. The calculation by the simulation model was made using the assumption that the increase in atmospheric CO₂ promoted the primary production rate by 10% over the last three decades. The result suggests that about 8 t C ha^-1 was sunk into soils of the mid-temperate forest over the same period. It indicates that forest soils may be one of the main sinks for atmospheric CO₂.     

Phenotypic changes and loss of N-CAM-mediated adhesion in transformed embryonic chicken retinal cells

Transformation of 6-d-old embryonic chicken retinal cells by Rous sarcoma virus (RSV) was found to cause significant changes in several cellular properties including adhesiveness, motility, and state of differentiation. The alterations in cell adhesivity were analyzed by means of specific antibodies to the calcium-independent neural cell adhesion molecule, N-CAM. In the RSV-transformed cells the amount of N-CAM present at the cell surface was significantly decreased relative to normal cells, as assessed by immunofluorescent staining, specific immunoprecipitation, and immunoblotting experiments. This decrease was reflected in a marked reduction in N-CAM-mediated adhesiveness measured in vitro. A different, calcium-dependent, adhesive system also present on neurons was not detectably altered by RSV transformation and, in contrast with previous studies on normal neurons, this adhesive system was detected without treatment by proteases. In culture, the transformed cells formed fewer and less compact colonies than the normal retinal cells. Observation of the RSV-transformed retinal cells by time-lapse cinematography confirmed the reduction in adhesiveness and also revealed that the transformed cells were more highly motile than their normal counterparts. In addition, RSV transformation appeared to alter the differentiation of the cultured retinal cells. Immunofluorescent staining studies indicated that in contrast to mature neurons, transformed neural retinal cells expressed the 34,000-mol-wt tyrosine kinase substrate and reduced amounts of a neuron-specific ganglioside recognized by monoclonal antibody A2B5. These characteristics are shared by untransformed glial cells. In double immunofluorescent staining experiments, many cells expressed both N-CAM and pp60src shortly after viral infection, which implies that the N-CAM-positive neuroepithelial cells were transformed by RSV. In addition, a highly purified population of N-CAM-positive neural retinal cells, selected using a fluorescence-activated cell sorter, was rapidly and extensively transformed by RSV at rates comparable to those of the unfractionated population. These results established that the transformed cells were largely derived from RSV-infected neuroepithelial cells rather than from a small population of retinal glial cells present in the primary culture. The findings suggest reconsideration of the possible origin of tumors classified by morphological criteria as derived from glia and raise the possibility that the normal homologue of pp60src may play a role in the commitment of neuroepithelial cells to neuronal or glial differentiation pathways.     

Morphofunctional characteristics of the endocrine cells of the stomach following administration of hydrocortisone and L-thyroxine

Electron microscopy with application of specific fluorescent histochemical reaction of Falck, as well as some methods of impregnation made it possible to indentify enterochromaffin cells in the stomach of hyperthyroid rats and the rats after cortisone injection under the conditions ox hyperfunction of the thyroid gland. After 20 days of L-thyroxin injection, and after 10 days of hydrocortisone injection, preceded by L-thyroxin, the amount of enterochromaffin cells in the epithelial layer of the gastric mucosa were noted to increase that was accompanied by simultaneous increase of the number of secretory argyrophil granules in their cytoplasm. Simultaneous injection of L-thyroxin and hydrocortisone, while not decreasing statistically significant amount of the cells, produced degradation of their cytoplasm.     

The female prostate and prostate-specific antigen. Immunohistochemical localization, implications of this prostate marker in women and reasons for using the term "prostate" in the human female

Prostate-specific antigen (PSA) is currently the most frequently used marker for the identification of normal and pathologically altered prostatic tissue in the male and female. Immunohistochemically PSA is expressed in the highly specialized apically-superficial layer of female and male secretory cells of the prostate gland, and as well as in uroepithelial cells at other sites of the urogenital tract of both sexes. Unique active moieties of cells of the female and the male prostate gland and in other parts of the urogenital tract are indicative of secretory and protective function of specialized prostatic and uroepithelial cells with strong immunological properties given by the presence of PSA. In clinical practice, PSA is a valuable marker for the diagnosis and monitoring of diseases of the male and the female prostate, especially carcinoma. In the female, similarly as in the male, the prostate (Skene's gland) is the principal source of PSA. The value of PSA in women increases in the pathological female prostate, e.g., carcinoma. Nevertheless, the total amount of PSA in the female is the sum of normal or pathological female prostate and non-prostatic female tissues production, e.g., of diseased female breast tissue. The expression of an antigen specific for the male prostate, i.e., PSA in female Skene's glands and ducts, and structural and functional parameters and diseases similar to that of the male prostate, have provided convincing evidence of the existence of a prostate in women and definitive preference of the term "prostate" over that of Skene's glands and ducts. The use of the term Skene's glands incorrectly implies that some other structure rather than prostate is involved, promoting the vestigial position of this female organ.     

Distinctive cellular immunity in genetically susceptible BALB/c mice recovered from Leishmania major infection or after subcutaneous immunization with killed parasites

Genetically susceptible BALB/c mice are refractory to further infection after recovery from Leishmania major infection after a sublethal dose of gamma-irradiation. In contrast, mice immunized with killed promastigotes s.c. develop exacerbated lesions after infection. Both groups of mice produce only a low level of specific antibody and no detectable cytotoxic T cells, but do have a strong antigen-specific DTH, which is adoptively transferable with Lyt-1+2-, L3T4+ T cells. Kinetic and histological studies revealed that mice immunized s.c. developed Jones-Mote hypersensitivity, peaking at 15 hr. with little mononuclear cell infiltration at the site of antigen administration; whereas mice that had recovered from infection developed tuberculin-type of reactivity, peaking at 24 to 48 hr, with intense mononuclear cell infiltration. Splenic T cells from recovered mice, when injected into the footpads of normal recipients together with live promastigotes, were able to retard lesion development; whereas T cells from s.c. immunized mice, when similarly transferred, accelerated disease progression. Antigen-specific culture supernatant of spleen cells from recovered mice also activated normal resident peritoneal macrophages to kill intracellular L. major amastigotes and tumor cells. Culture supernatants of spleen cells from s.c. immunized or normal mice were devoid of such activities. Part of the macrophage-activating potential can be inhibited by antibody specific for IFN-gamma. These results therefore demonstrate that whereas the Jones-Mote reaction is correlated with disease exacerbation, the tuberculin-type of DTH may be protective. Furthermore, in vivo immunity is directly related to the capacity of T cells to produce macrophage-activating factor.     

Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.     

Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology

The rat central nervous system (CNS) during experimental allergic encephalomyelitis (EAE) was analyzed immunohistochemically from the preclinical to recovery stage by using monoclonal antibodies specific for rat T lymphocyte subsets and Ia antigen. Through combination of the avidin-biotin technique and carefully selected fixative, cells with dendritic morphology (DC) and infiltrating mononuclear cells were clearly and intensely demonstrated in the CNS parenchyma during EAE. In normal and complete Freund's adjuvant (CFA)-injected controls, there were no inflammatory foci. Ia (OX3)-positive parenchymal cells were not detected, whereas W3/25 stained DC that were located mainly in the white matter and W3/13 stained axons. At the preclinical stage, 11 days after CNS/CFA sensitization, a few clusters of Ia+ DC were detected in some sections of the spinal cord. The number of Ia+ DC increased as clinical signs developed (P less than 0.001). In rats with a clinical score of 1 or 2, Ia+ DC were mainly located in the perivascular region and closely associated with infiltrating T lymphocytes. However, at moribund state (score 3), Ia+ DC were evenly distributed in gray and white matter on almost all sections of the spinal cord. In recovered rats, the numbers of inflammatory foci and Ia+ DC were less than those in clinical EAE rats (P less than 0.001). Rats without clinical signs throughout the course also contained a few clusters of Ia+ DC. Double immunofluorescent staining with OX3 and anti-glial fibrillary acidic protein (GFAP) antiserum demonstrated that Ia+ DC were negative for GFAP. Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells, suggesting that Ia+ DC are microglia. In contrast to DC, no astrocytes or endothelial cells express detectable levels of Ia antigen in control and clinical EAE rats. These findings suggest that brain cells other than Ia+ DC may not be involved in the local immune interaction. Ia+ DC may play a significant role in antigen presentation in the CNS with EAE.     

Recovery of Cell-Bound Interferon

Interferon could be recovered from homologous cells to which it was applied but could not be recovered from heterologous cells. The amount of interferon that could be recovered from cells corresponded to the sensitivity of the cells to the antiviral activity of the interferon: mouse embryo fibroblasts, which were 5 to 10 times as sensitive as L-929 cells to interferon, bound 5 to 10 times more interferon than the latter, whereas Lpa cells, which were only one-third as sensitive as L-929 cells to interferon, bound only one-third as much as the latter. The concentration of cell-bound interferon was as much as 150 times the extracellular concentration of interferon applied to the cells. Interferon bound to cells at 4 C with the same efficiency as it did to cells at 37 C, and actinomycin D-treated cells bound interferon as well as normal cells. Even though the total amount of interferon bound to cells was as much as 30% of the amount of interferon applied to them, no loss of antiviral activity was detectable from the medium.     

Migration of Merkel cells in the labial mucous epithelium of adult rabbits following mental nerve resection

Summary Merkel cells in the lower labial mucosa of adult rabbits were studied electron microscopically, 9, 21, 28, and 50 days after resection of the mental nerves. By day 9, nerve fibers were completely retracted from the epithelial layer of the mucosa. On and after day 21, Merkel cells were located not only in the basal layer but also in the prickle or more superficial cell layers. The ultrastructure of the migrating Merkel cells was unchanged, both as to the amount and location of the specific cored granules in the cytoplasm, until the cells reached the granular cell layer. The position of the migrating Merkel cells differed from cell to cell, and migration continued for at least 50 days. A remarkably large number of immature Merkel cells was observed in the basal and suprabasal cell layers of the denervated epithelium even by day 50. Therefore, the possibility of the reproduction of Merkel cells exists. The migrating Merkel cells, as well as the keratinocytes in the same cell layer, had degenerated drastically in the parakeratinized cell layer. This seems to indicate that the Merkel cells belong to the line of keratinocytes.     

Cytoskeletal dynamics of the teleostean fin ray during fin epimorphic regeneration

Teleost fishes can regenerate their fins by epimorphic regeneration, a process that involves the transition of the formerly quiescent tissues of the stump to an active, growing state. This involves dynamic modifications of cell phenotype and behavior that must rely on alterations of the cytoskeleton. We have studied the spatial and temporal distribution of three main components of the cytoskeleton (actin, keratin and vimentin) in the regenerating fin, in order to establish putative relationships between cell cytoskeleton and cell behavior. According to our results, the massive rearrangement undergone by the epidermis right after injury, which takes place by cell migration, correlates with a transient down-regulation of keratin and a strong up-regulation of actin in the epidermal cells. During the subsequent epidermal growth, based on cell proliferation, keratin normal pattern is recovered while actin is down-regulated, although not to normal (quiescent) levels. The epidermal basal layer in contact with the blastema displays a particular cytoskeletal profile, different to that of the rest of the epidermal cells, which reflects its special features. In the connective tissue compartment, somatic cells do not contain vimentin, but keratin, as intermediate filament. Proliferative and migrative activation of these cells after injury correlates with actin up-regulation. Although this initial activation does not involve keratin down-regulation, blastemal cells were later observed to lack keratin, suggesting that such cytoskeletal modification might be needed for connective tissue cells to dedifferentiate and form the blastema. Cell differentiation in the newly formed, regenerated ray is accompanied by actin down-regulation and keratin up-regulation.     

Biophysical properties of corneal cells reflect high myopia progression

Myopia is a common ocular disorder with significant alterations in the anterior ocular structure, including the cornea. The cell biophysical phenotype has been proposed to reflect the state of various diseases. However, the biophysical properties of corneal cells have not been characterized during myopia progression and their relationship with myopia remains unknown. This study characterizes the biophysical properties of corneal cells in normal, myopic, and recovered conditions, using two classical myopia models. Surprisingly, myopic corneal cells considerably reduce F-actin and microtubule content and cellular stiffness and generate elevated traction force compared with control cells. When myopia is restored to the healthy state, these biophysical properties are partially or fully restored to the levels of control cells. Furthermore, the level of chromatin condensation is significantly increased in the nucleus of myopic corneal cells and reduced to a level similar to healthy cells after recovery. These findings demonstrate that the reversible biophysical alterations of corneal cells reflect myopia progression, facilitating the study of the role of corneal cell biophysics in myopia.     

Studies on the Structure,Afterripening and Cytochemistry of Seeds in Eleutherococcus brachypus Harms

Seeds of Eleutherococcus brachypus Harms were flat-kidney-shaped and their seed coats were only composed of one layer of cells. Embryos with abundant protein in their cells were just at the heart-shaped stage and were capped by sacs formed from degenerating endosperm cells when seeds shed from their maternal plants. A large amount of stored protein grains and lipids existed in endosperm cells but no polysaccharide grains were present either in endosperm cells or in embryo cells. Viable seeds were only 9.27% of the total. The plump seeds germinated in the cultivated field after 18～19 months and their germinating rate was 1.67%. Besides, the content of protein decreased gradually and a few polysaccharide grains were stored in embryo cells during the process. The afterripening process of seeds stratified at different temperatures ended after 6 months and the cytochemistry features of the seeds were that the content of protein decreased gradually and numerous polysaccharide grains had been stored in embryo cells at the late heart-shaped embryo stage and retained till the mature embryo stage. The structure, afterripening and cytochemistry of seeds were compared between Eleutherococcus brachypus and Eleutherococcus senticosus. The poor quality of the seeds, longer time of afterripening in a natural state and much lower germination rate of E. brachypus are considered to be important reasons for the endangerment of this species. Somemeasures are suggested for its conservation based on the above facts.