Innate immunity in the edible ascidian Halocynthia roretzi developing soft tunic syndrome: Hemolymph can eliminate the causative flagellates and discriminate allogeneic hemocytes.

The infection of the kinetoplastid flagellate Azumiobodo hoyamushi causes soft tunic syndrome that often results in mass mortality in the aquaculture of the edible ascidian Halocynthia roretzi. In the diseased ascidian individuals, the flagellates are exclusively found in the tunic matrix that entirely cover the epidermis, and never invade into internal tissues, such as a mantle. The present study for the first time demonstrated that the ascidian blood plasma and hemolymph have an activity to agglutinate and disintegrate the flagellates, suggesting the innate immunity protects the internal tissue from the invasion of A. hoyamushi. This activity is indifferent between the healthy and the diseased individuals. Allo-specific recognition and cytotoxic reaction among ascidian hemocytes, so-called contact reaction, occur among the individuals of healthy-healthy, healthy-diseased, and diseased-diseased combination, and therefore, the hemocytes from diseased individuals still retain the allo-reactivity. Moreover, the allo-reactive combinations are not changed under the presence of the flagellates, indicating the flagellates neither suppress nor induce the effector system of the contact reaction. These results suggest that the infection of A. hoyamushi does not impair the innate immunity in the ascidian hemolymph.

Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794.

Fatty Acid Binding Protein 3 Is Involved in n-3 and n-6 PUFA transport in mouse trophoblasts.

BACKGROUND: Low placental fatty acid (FA) transport during the embryonic period has been suggested to result in fetal developmental disorders and various adult metabolic diseases, but the molecular mechanism by which FAs are transported through the placental unit remains largely unknown. OBJECTIVE: The aim of this study was to examine the distribution and functional relevance of FA binding protein (FABP), a cellular chaperone of FAs, in the mouse placenta. METHODS: We clarified the localization of FABPs and sought to examine their function in placental FA transport through the phenotypic analysis of Fabp3-knockout mice. RESULTS: Four FABPs (FABP3, FABP4, FABP5, and FABP7) were expressed with spatial heterogeneity in the placenta, and FABP3 was dominantly localized to the trophoblast cells. In placentas from the Fabp3-knockout mice (both sexes), the transport coefficients for linoleic acid (LA) were significantly reduced compared with those from wild-type mice by 25% and 44% at embryonic day (E) 15.5 and E18.5, respectively, whereas those for α-linolenic acid (ALA) were reduced by 19% and 17%, respectively. The accumulation of LA (18% and 27% at E15.5 and E18.5) and ALA (16% at E15.5) was also significantly less in the Fabp3-knockout fetuses than in wild-type fetuses. In contrast, transport and accumulation of palmitic acid (PA) were unaffected and glucose uptake significantly increased by 23% in the gene-ablated mice compared with wild-type mice at E18.5. Incorporation of LA (51% and 52% at 1 and 60 min, respectively) and ALA (23% at 60 min), but not PA, was significantly less in FABP3-knockdown BeWo cells than in controls, whereas glucose uptake was significantly upregulated by 51%, 50%, 31%, and 33% at 1, 20, 40, and 60 min, respectively. CONCLUSIONS: Collectively FABP3 regulates n-3 (ω-3) and n-6 (ω-6) polyunsaturated FA transport in trophoblasts and plays a pivotal role in fetal development.

Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-ß) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

Fatty acid-binding protein 4 (FABP4) and FABP5 modulate cytokine production in the mouse thymic epithelial cells.

Thymic stromal cells, including cortical thymic epithelial cells (cTEC) produce many humoral factors, such as cytokines and eicosanoids to modulate thymocyte homeostasis, thereby regulating the peripheral immune responses. In this study, we identified fatty acid-binding protein (FABP4), an intracellular fatty acid chaperone, in the mouse thymus, and examined its role in the control of cytokine production in comparison with FABP5. By immunofluorescent staining, FABP4(+) cells enclosing the thymocytes were scattered throughout the thymic cortex with a spatial difference from the FABP5(+) cell that were distributed widely throughout the cTEC. The FABP4(+) cells were immunopositive for MHC class II, NLDC145 and cytokeratin 8, and were identified as part of cTEC. The FABP4(+) cells were identified as thymic nurse cells (TNC), a subpopulation of cTEC, by their active phagocytosis of apoptotic thymocytes. Furthermore, FABP4 expression was confirmed in the isolated TNC at the gene and protein levels. To explore the function of FABP in TNC, TSt-4/DLL1 cells stably expressing either FABP4 or FABP5 were established and the gene expressions of various cytokines were examined. The gene expression of interleukin (IL)-7 and IL-18 was increased both in FABP4 and FABP5 over-expressing cells compared with controls, and moreover, the increase in their expressions by adding of stearic acids was significantly enhanced in the FABP4 over-expressing cells. These data suggest that both FABPs are involved in the maintenance of T lymphocyte homeostasis through the modulation of cytokine production, which is possibly regulated by cellular fatty acid-mediated signaling in TEC, including TNC.

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation.

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes.

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)(+) fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7(+) cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5(+) cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4(+) cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.

Fatty acid-binding protein regulates LPS-induced TNF-alpha production in mast cells.

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.

Ultrastructural alterations of the cortical epithelial cells of the irradiated and recovering rat thymus.

To understand the roles of cortical thymic epithelial cells (CTECs) in T-lymphocyte development, we analyzed rat thymi recovering from irradiation (6Gy), at the ultrastructural level. The morphological alterations in the CTECs were most prominent during the third to fifth day of recovery, when proliferating thymocytes were observed in the vicinity of the CTECs. The most striking finding among the alterations in the CTECs after irradiation was a cytoplasmic vacuolization with an increased amount of granular and membranous content. The granular content was observed as loosely aggregated structures or finely dispersed granules and dense bodies. The membranous content appeared in various forms including vesicular, tubular, and irregular membranous structures and myelin figures. The above features are characteristic of the hyperfunctional state of CTECs with increased secretion activities, which suggests their important roles in the repopulation and maturation of the cortical thymocytes during recovery after irradiation.

Semiquantitative detection of cytokine messages in X-irradiated and regenerating rat thymus.

We investigated the expression of cytokine mRNA derived from thymocytes or thymic epithelial cells in X-irradiated (8 Gy) and recovering rat thymuses, according to our previous observation (Mizutani et al., Radiat. Res. 157, 281-289, 2002). The changes in mRNA expression level of interleukin 2 (Il2), Il4, tumor necrosis factor alpha (Tnf), interferon gamma (Ifng), and transforming growth factor beta (Tgfb) were examined. The mRNA expression of Il2 and Il4 decreased from day 5 to day 14 after irradiation. Thereafter, the expression level of Il2 mRNA recovered to normal control levels; however, the expression of Il4 mRNA tended toward significantly low levels. Tnf mRNA expression decreased on day 5 after irradiation and then showed a gradual increase back to normal control levels. Tgfb mRNA expression did not change significantly. Ifng mRNA expression was transiently enhanced from day 11 to day 14. The mRNA expression levels of Il10 increased significantly from day 3 to day 7 after irradiation. In addition, the mRNA expression of thymic epithelial cell-derived Il7 showed a transient decrease on day 3; however, then it showed a continuous increase from day 5 to day 21, finally reaching twice the normal control levels after X irradiation. These observations suggest that the expression of cytokine messages in the irradiated thymus changed significantly and did not return to normal for a long time after 8 Gy irradiation.

Expression of PAC1 receptor in rat thymus after irradiation.

In the present work, PAC1-R (G-protein-coupled receptor specific for PACAP) was detected on cells in the normal thymus. Immunohistochemically PAC1-R was expressed strongly in stromal cells of the thymic medulla. Positive cells were also observed in the thymus of fetal and old adult rats. After 8 Gy irradiation to 9-week-old rats, PAC1-R expressions in the thymus decreased and almost recovered by day 21. The expression of PAC1-R mRNA was weak in the thymus and decreased further after irradiation. The expression almost recovered by day 28. Hip and hip/hop variants, which were not expressed in the normal thymus, were expressed in the thymus on days 3, 5 and 21 after irradiation. The expressions of IL-6 and IL-10 tended to increase initially after irradiation then decreased. Histologically, the thymic structures were destroyed on day 3 after irradiation and the thymus almost recovered by day 21. Thus PACAP is thought to be one of the important factors for cross-talk between cells involved in thymic regeneration.

Morphological and immunohistochemical changes to thymic epithelial cells in the irradiated and recovering rat thymus.

The present study analyzed morphological and immunohistochemical changes of thymic epithelial cells in the irradiated and recovering rat thymus. Observations showed the number of thymocytes was initially severely reduced after irradiation but abruptly increased on days 3 to 5 after 6 Gy and on days 7 to 11 after 8 Gy irradiation. To analyse the mechanisms for this abrupt recovery of the thymocytes after irradiation, the expression of p63 in the normal and irradiated thymus was immunohistochemically studied as the expression of this antigen may be related to the proliferation of epithelial cells. In the fetal thymus tissue, thymic epithelial cells were the principal cell type that stained strongly positive for p63. The sporadic expression of p63 was also observed in the normal adult thymus tissue, especially in the subcapsular region. An increased number of p63- positive cells in the thymus after irradiation indicates that repair or renewal of the thymic epithelial cells may be taking place because p63 is more specific to transient amplifying thymic epithelial cells. A RT-PCR analysis of p63 expression in irradiated and regenerating thymus tissue also showed an increased expression of p63 after irradiation compared with that of the normal thymus. These results suggest that changes in the thymic microenvironment-especially in relation to the repair and renewal of thymic epithelial cells- may have an important influence on thymocyte proliferation in the normal thymus as well as in the irradiated and recovering thymus.

Study of color variation in the solitary ascidian Halocynthia roretzi, collected in the Inland Sea of Japan.

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.

Identification and characterization of major histocompatibility complex class II compartments in cortical thymic epithelial cells.

Previous studies have concentrated on elucidating the subcellular localization of major histocompatibility (MHC) class II molecules mainly in B cells, macrophages, and dendritic cells. Despite very rich cell-surface expression of MHC class II molecules by cortical thymic epithelial cells (cTECs), little is known regarding the expression of these molecules by cTECs at the subcellular level. In the present study we focused on the identification and characterization of MHC class II compartments (MIICs) in cTECs in situ by immunogold electron microscopy (IEM). We found that MHC class II molecules were located exclusively in the cytoplasmic vacuoles, and we identified these MHC class II molecule-containing cytoplasmic vacuoles as MIICs in cTECs. These MIICs were immunopositive for early endosomal, late endosomal, and lysosomal markers. Moreover, in these MIICs, MHC class II molecules were colocalized with cathepsin L, H2-DM, class II-associated invariant chain (Ii), and class II-associated invariant chain peptide (CLIP). Similarly, Ii molecules were colocalized with endosomal and lysosomal markers, cathepsin L, and H2-DM in the vacuoles. Taken together, these results suggest that MIICs in cTECs represent conventional endocytic compartments. The colocalization of MHC class II molecule or Ii with cathepsin L and H2-DM in the MIICs suggests that MIICs in cTECs may be sites of Ii degradation and peptide loading.

Differential expression of MHC class II antigens and cathepsin L by subtypes of cortical epithelial cells in the rat thymus: an immunoelectron microscopic study.

To better comprehend the thymic microenvironment, it is necessary to identify the antigenic profile of cortical thymic epithelial cells (cTECs) that are involved in the development of major histocompatibility complex (MHC)-restricted T cells. Ultrastructurally, cTECs can be classified into four morphologically distinct subtypes: subcapsular/perivascular (EC1), pale (EC2), intermediate (EC3) and dark (EC4) cells. Several immunohistochemical studies were done on cTECs at the light and electron microscopic levels, but not with reference to the above subtypes. In the present paper, we analysed the expression of MHC class II antigen and cathepsin L by individual cTEC subtypes at the electron microscopic level. We show that (1) MHC class II antigens are expressed on the cell surfaces except on the basal surface of EC1, both on the cell surface and in intracytoplasmic vacuoles of EC2, and only in the intracytoplasmic vacuoles of EC3 and EC4, and (2) that cathepsin L is expressed strongly and uniformly throughout the cytoplasm of EC2, but weakly and non-uniformly in the cytoplasm of EC1, EC3 and EC4. These results show that MHC class II antigen expression and cathepsin L expression is heterogeneous in cTEC subtypes and suggest that EC2 might play a significant role in the development of CD4+ T cells.

Spatial relation between major histocompatibility complex-restricted antigen receptor-bearing thymocytes and subtypes of thymic epithelial cells.

R73, a monoclonal antibody that recognizes a rat T cell surface antigen of TCR(alphabeta), was used to identify thymocytes that express major histocompatibility complex-restricted antigen receptors, and to define the spatial relation between these receptor-bearing thymocytes and individual thymic cortical and medullary epithelial subtypes by ultrastructural immunohistochemistry. We show that in both the cortex and medulla 1) the thymocytes that reacted with R73 antibodies exhibited three staining patterns: cytoplasmic-only staining, simultaneous cytoplasmic and surface membrane staining, and surface membrane-only staining; 2) the subcapsular/perivascular epithelial cells (ECs) were usually associated with thymocytes expressing perinuclear staining only; and 3) the surface membrane areas of thymocytes that expressed antigen receptors made contact with pale and intermediate ECs, but not with dark cells. These results suggest that thymic selection of major histocompatibility complex restriction and/or tolerance may occur by interaction of the receptors on maturing thymocytes with major histocompatibility complex antigens on thymic ECs in general, and on pale and intermediate subtypes in particular.

Inflammatory and anti-inflammatory cytokines regulate the recovery from sublethal X irradiation in rat thymus.

We investigated the regeneration of rat thymus after sublethal X irradiation (6 Gy). The number of thymocytes was much lower on day 3 after irradiation, and many apoptotic cells were observed. However, by day 5, there had been a rapid proliferation of thymocytes. Since cytokines are considered to be important regulatory factors in postirradiation recovery, we performed in vivo cytokine assays using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and found serial changes in the cytokine message. The messenger RNA (mRNA) expression of the pro-inflammatory cytokines interleukin 1 beta (Il1b), Il6 and tumor necrosis factor alpha (Tnf) was higher than normal on day 3, lower on day 5, and higher again on day 7. In particular, Tnf was completely absent on day 5 and was expressed again on day 7. Of the anti-inflammatory cytokines Il4, transforming growth factor beta (Tgfb) and Il10, only the Il10 message changed substantially. Il10 expression was very high on day 5 but was completely absent on day 7. Thus the Tnf and Il10 messages were expressed alternately. The changes in the distribution of macrophages detected by the immunohistochemical analysis may be related to the changes in the cytokines. Analysis of cytokine messages in the regenerating thymus in vivo may provide new insights into potential therapies for radiation-induced damage.

Dynamics of Mitotic Apparatus Formation and Tubulin Content during Oocyte Maturation in Starfish: (tubulin/starfish/oocyte maturation/cytoskeleton/meiosis).

To follow the topo-temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1-methyladenine (1-MeAde) stimulation, "pre-meiotic asters" were observed on the germinal vesicle at the animal pole. 1-MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS-PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.

Cortical contraction and ooplasmic movement in centrifuged or artificially constricted eggs ofCiona intestinalis.

To discover the force causing bipolar ooplasmic segregation just after fertilization in ascidian eggs (Ciona intestinalis), cortical contraction and cytoplasmic movement were examined by centrifugation and by artificial constricting techniques. In the centrifuged eggs, the surface constriction appeared independently of cytoplasmic stratification. The yolk layer and the sub-centripetal layer moved toward the vegetal pole in the peripheral region. In the eggs which were artificially constricted by partially broken chorion and then fertilized, the inner cytoplasm always flowed from the vegetal sphere into the animal sphere during bipolar segregation. The direction of this cytoplasmic movement was independent of sphere size. This shows that the force causing cytoplasmic movement is supplied by the contraction of the vegetal-side cortex. It is suggested that the contracting cortex pushes the inner cytoplasm toward the animal pole and drags the peripheral cytoplasm toward the vegetal pole.

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs.

The egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.