Mice deficient in N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase are unable to synthesize chondroitin/dermatan sulfate containing N-acetylgalactosamine 4,6-bissulfate residues and exhibit decreased protease activity in bone marrow-derived mast cells.

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.

Sperm penetration and transformation of sperm entry site in eggs of the freshwater teleost Rhodeus ocellatus ocellatus.

Eggs of bony fishes are enveloped by an egg envelope (chorion) in which a micropyle is present near the animal pole. Therefore, sperm penetration into the eggs is limited to the sperm entry site (SES), a region of plasma membrane just beneath the micropyle. In rose bitterling eggs, the SES transforms from a tuft of microvilli into a swollen mass (SM) that continues to plug the micropyle after sperm penetration. The present observations using the rose bitterling Rhodeus ocellatus ocellatus were conducted to examine: 1) whether or not sperm penetration is necessary for formation of the SM and 2) whether or not actin microfilaments are involved in the formation of the SM. Water activation without sperm transformed the SES from a tuft of microvilli into the SM, although it took a longer time for the transformation and the SMs were smaller than in the case of inseminated eggs. The SES presumably has the ability to transform into the SM upon activation of eggs in the present species. Cytochalasin B, which acts on actin microfilaments, did not prevent formation of the SM, irrespective of insemination or activation. The present observations suggest that sperm penetration is not necessary for SM formation and actin microfilaments do not participate in SM formation. © 1996 Wiley-Liss, Inc.

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. I. WGA Affects the Ion Fluxes Associated with the Acrosome Reaction: (sea urchin/acrosome reaction/wheat germ agglutinin/ion flux/species specificity).

The lectin wheat germ agglutinin (WGA) inhibited the egg jelly-induced acrosome reaction (AR) of sperm of the sea urchin, Strongylocentrotus intermedius. Fluorescein-conjugated WGA applied to sperm bound to the acrosomal region, to the midpiece, and to the tip of the flagellum. These effects were not observed in the presence of N-acetly-D-glucosamine. When the egg jelly was replaced by artificial AR inducers such as A23187 or nigericin, the AR was not inhibited by WGA. Results obtained using a Ca2+ indicator fura-2, a pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) and a membrane potential sensitive dye 3,3'-dipentyl 2,2'-dioxacarbocyanine [diO-C5 (3)] showed that WGA suppresses the egg jelly-induced influx of Ca2+ and slightly suppresses the efflux of H+ caused by the egg jelly, whereas the depolarization of the plasma membrane by the egg jelly is remarkably amplified by the treatment with WGA. These results suggest that WGA affects the regulatory system of the ion fluxes associated with the AR. The target protein of WGA (WGA-binding protein) was a membrane glycoprotein of 260 kD under non-reducing condition.

Effects of Forskolin on Fine Structures of Medaka Follicles: (forskolin/medaka follicle/cell organelle/cell junction/oocyte maturation).

Effects of forskolin (FK), which stimulates production of 17α, 20ß-dihydroxy-4-pregnen-3-one and estradiol-17ß, on the fine structure of preovulatory follicles of Oryzias latipes were examined. Granulosa cells incubated in culture medium containing FK exhibited dislocation of the nucleus from the chorion side to the basement membrane side, vesiculation of conspicuous dilated endoplasmic reticulum (ER) with electron-dense material and Golgi lamellae, and development of large oval mitochondria with an electron-dense matrix. Moreover, a thin vesicular layer adherent to the outermost layer of the chorion was found in all immature oocytes at the end of incubation in the presence of FK. Intercellular junctions between granulosa cells and the oocyte gradually decreased during incubation in the presence of FK, and were finally lost with closure of the radial canals in the chorion at the end of the incubation. On the other hand, intrafollicular oocytes that were first incubated with FK for 10 hr, matured normally when they were incubated an additional 8 hr in plain medium. In granulosa cells of these follicles, the dilated ER and vacuolated Golgi lamellae were no longer detectable. These observations suggest that the development of dilated ER and vacuolated Golgi lamellae is characteristic of granulosa cells induced by FK.

FINE STRUCTURE OF LOACH OOCYTES DURING MATURATION IN VITRO.

The morphological changes during in vitro maturation of Misgurnus anguillicaudatus oocyte are described. The process of oocyte maturation can be divided into three provisional stages based on morphological events. Fully-grown, immature oocytes are opaque yellowish-white. The morphological characteristics of their ooplasm are the existence of annulate lamellae, a mass of long mitochondria and an electron dense layer beneath the vitelline surface. Three hr after a 1 hr exposure to corticosterone, these structures disappear and the cortical ooplasm becomes semi-transparent. In this stage of the maturation process (Stage I), the germinal vesicle, without a nucleolus, moves toward the animal pole, and scattered cytoplasmic inclusions approach the vitelline surface. Six hr after exposure to the hormone (Stage II), the whole ooplasm becomes semi-transparent and large yolk platelets are seen in the animal pole region. Tubular endoplasmic reticula develop throughout the ooplasm and some cortical alveoli (CA) become aligned beneath the vitelline surface. Nine hr after exposure to the hormone (Stage III), the oocyte chorion separates from the follicle cells. Most CA align beneath the vitelline surface and cytoplasm accumulates in the cortical region of the animal hemisphere.

INITIATION OF CLEAVAGE IN FISH EGGS BY INJECTION OF FLAGELLA OR MICROTUBULES OF SEA URCHIN SPERMATOZOA.

Flagella and their microtubules obtained from sea urchin (Hemicentrotus pulcherrimus) spermatozoa were injected into unfertilized eggs of the medaka (Oryzias latipes) with a micropipette. Upon activation, some of the eggs began the first cleavage with three or more irregular blastomeres, and developed to the morula stage. It is suggested that sperm flagellar microstubule material is one of the cleavage initiation substances.