Identification and cDNA cloning of alveolin, an extracellular metalloproteinase, which induces chorion hardening of medaka (Oryzias latipes) eggs upon fertilization.

Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes.

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase.

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca(2+) and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at

Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs.

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal-vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes.

Specific antibodies against the major chorionic glycoproteins (ZI1-2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozoa, play an important role in sperm guidance into the micropyle.

Development of a motor nerve in the caudal fin of the medaka (Oryzias latipes).

The entire process of the development of the motor nerve to the middle interradial muscle (MIR muscle) in the tail region of the medaka (Oryzias latipes) was examined by light and electron microscopy. The motor nerve to the MIR muscle was observed to develop in a series of four steps. (1) Trunk-formation step: from stage 30 to stage 31, when the MIR muscle had not yet differentiated to the myotube stage, the motor axons starting from the caudal spinal cord, including spinal segment 27, formed a nerve trunk, the ventral caudalmost nerve (VCN). The VCN bypassed the place where the MIR muscle would differentiate and extended further caudad. (2) Wandering step: at stage 32, nerve processes of the motor axons (short branches) wandered from the course of the VCN. (3) Connection step: from stage 33/34 to stage 35, the mesenchyme cells differentiated into the MIR myotubes in the vicinity of the short branches, and the nerve and the muscle became connected. (4) Modification step: after stage 35, the morphological pattern of the motor nerve was modified by the growth and the shift of the short branches, and by the disappearance of the caudalmost part of the VCN. These results suggest that the complex pattern of the motor nerve in the adult fish is gradually constructed by multiple steps, each of which may have its own mechanism.

[A case of quadricuspid aortic valve associated with mitral regurgitation].

A case of a 60-year-old man associated with quadricuspid aortic valve and mitral regurgitation is reported. The aortic valve consisted of three larger cusps and a small accessory cusp situated between the right and the non-coronary cusps. The aortic regurgitation resulted from malcoaptation of the four cusps and the mitral regurgitation resulted from annulus dilatation and thickening of the anterior leaflet. The surgical treatment was performed successfully by the aortic and mitral valve replacements with St. Jude Medical valves. The patient is doing well fifteen months postoperatively. Sixteen Japanese cases of quadricuspid aortic valve which were corrected surgically are reviewed.

[A case who developed mechanical pulsus alternans after Bentall's operation for annuloaortic ectasia].

Left ventricular pulsus alternans developed immediately after Bentall's operation in a 37-year-old patient with Marfan's syndrome accompanied by severe left heart failure due to annuloaortic ectasia. Echocardiographic examination suggested that this symptom was caused by alternating contractility based on left ventricular myocardial failure. Left heart failure disappeared early after operation, but pulsus alternans persisted until 7 months after operation, when EDVI decreased from 225 ml/m2 to 113 ml/m2.

Structural studies of a novel type of tetraantennary sialoglycan unit in a carbohydrate-rich glycopeptide isolated from the fertilized eggs of Indian Medaka fish, Oryzias melastigma.

A novel carbohydrate-rich sialoglycopolyprotein of apparent molecular mass approximately 7000 Da was isolated from the fertilized eggs of the Medaka fish species, Oryzias melastigma. The glycoprotein was identified as a member of the L-hyosophorin family because it exhibited the following several distinctive features of L-hyosophorin molecules: (a) it contains a high proportion of carbohydrate (90% by weight), and (b) the amino acid sequence of the apopeptide was identical with that of the Oryzias latipes L-hyosophorin which has previously been demonstrated to be derived from a high molecular weight form of hyosophorin, i.e. H-hyosophorin, present in the cortical vesicles of unfertilized eggs. The apoprotein of H-hyosophorin is composed of tandem repeats of the L-hyosophorin apopeptide, i.e. it is a polyprotein. The structure of the carbohydrate portion of purified L-hyosophorin of O. melastigma was studied by composition and methylation analysis, selective chemical (periodate-Smith degradation; hydrazinolysis-nitrous acid deamination), and enzymatic (endo-beta-galactosidase; peptide:N-glycanase) degradation, together with instrumental methods (fast atom bombardment-mass spectrometry and 1H NMR). O. melastigma L-hyosophorin was found to contain two types of large, branched tetraantennary glycan units capped with sialic acids. The two glycans differ with respect to the branching pattern of the trimannosyl core (x = 4 or 6 in Eq. A). [formula: see text] The possible physiological significance of the hyosophorin family is discussed in the light of their unique structural features.

[A case of Marfan's syndrome that required emergency Bentall's operation due to acute dissecting aortic aneurysm (DeBakey type II) following surgery of abdominal aortic aneurysm].

Graft replacement was performed in a 29-year-old man for an abdominal aortic aneurysm associated with Marfan's syndrome. Since the dissecting aortic aneurysm (DeBakey type II) accompanied by disruption of the right coronary artery developed 74 days after operation, the emergency Bentall's operation was successfully carried out with bypassing of the right coronary artery using a saphenous vein graft. Fifteen Japanese cases operated on for abdominal aortic aneurysm associated with Marfan's syndrome are reviewed, and the problems concerning surgery of abdominal aortic aneurysm and Bentall's operation are discussed.

[Subdural hematoma following surgery of an aneurysm of the diverticulum of the ductus arteriosus with partial cardiopulmonary bypass].

The patient was a 72-year-old man, who had an aneurysm of the descending thoracic aorta with left recurrent laryngeal nerve palsy identified by preoperative examination. Thoracotomy revealed an aneurysm of a diverticulum of the ductus arteriosus measuring 2.7 X 3.4 cm. The aneurysm was excised under partial cardiopulmonary bypass and the defect of the aortic wall was repaired with a Dacron patch. Two days after surgery, he developed mild deterioration of consciousness and right hemiparesis. An acute subdural hematoma which covered the left frontal, parietal, and temporal lobes was found by CT examination. Craniotomy was performed 16 days after the previous surgery, and 100 g of hematoma was removed. Following craniotomy, complete recovery of cerebral function was observed. This appears to be a valuable report of a patient who developed acute subdural hematoma following surgery of aneurysms of the descending thoracic aorta with partial cardiopulmonary bypass.

Cortical alveolus breakdown in the eggs of the freshwater teleost Rhodeus ocellatus ocellatus.

Cortical alveoli and their breakdown in the eggs of the rose bitterling were morphologically investigated. Cortical alveoli of various sizes existed in multiple layers within the cortical cytoplasm of each egg. Two types of cortical alveoli were recognized--one possessed homogeneous contents; the other possessed heterogeneous contents with electron-less dense spots. The eggs were activated by a penetrating spermatozoon or by immersion in fresh water. The immersion of the eggs into fresh water induced a wave of cortical alveolus breakdown (CABD), which was propagated from the vegetal pole to the animal pole. When eggs were inseminated in fresh water, CABD progressed from both the vegetal and the animal poles of the egg, and was complete within about 20 min. The contents of the cortical alveoli were released into the perivitelline space through a large opening formed by the fusion of the plasma and cortical alveolar membranes. For a short time after CABD, the smooth inner surfaces of the cortical alveoli were readily discernible from the rougher original plasma membrane. About 30 min after immersion in fresh water, the inner surfaces of the cortical alveoli were indistinguishable from the original egg surfaces. Aequorin-injected eggs became luminescent immediately after their immersion in fresh water. The luminescence reached a maximum level about 15 min later and subsided after roughly 30 min. The result shows that increase in intracellular free Ca ions occurs during CABD.

[Successful mitral valve replacement for infective endocarditis in pregnancy].

A 27 weeks' pregnant women exhibited infective endocarditis due to alpha-streptococcus. As echocardiography showed vegetations on both mitral leaflets, cesarean section was performed at 35 weeks' gestation and a healthy male infant weighing 2,430 g was delivered. Antibiotic therapy was continued for fever after the cesarean section but macrohematuria and Osler's nodes developed. Emergency mitral valve replacement was performed successfully despite the presence of active infective endocarditis. She was discharged after completion of a 10-week course of antibiotic therapy.

Stage-dependent expression of the chicken delta-crystallin gene in transgenic fish embryos.

To study the regulation of gene expression of vertebrate crystallin genes, the chicken delta-crystallin gene was introduced into a small freshwater fish, medaka (Oryzias latipes), which lacks this gene, and its expression was examined immunohistologically at several developmental stages before hatching. The gene expression was detected in the central fiber cells of the lens at an early stage, showing a stage-dependent expression. In non-lens tissues, the expression was barely detectable before tissue differentiation. It first became substantial mainly in mesodermal tissues and then later in a greater variety of tissues, including ectodermal and endodermal ones. Thus, the non-lens expression of delta-crystallin was also stage-dependent, with the stage being dependent on the tissue type. These results from lens and non-lens tissues are discussed in relation to tissue differentiation and two categories of delta-crystallin expression.

Ultrastructural changes in micropylar cells and formation of the micropyle during oogenesis in the medaka Oryzias latipes.

Ultrastructural changes in differentiating micropylar cells and in the micropyle occur during oogenesis in the medaka, Oryzias latipes. A micropylar cell is not detectable in previtellogenic ovarian follicles. In the early vitellogenic phase, the micropylar cell becomes differentiated from neighboring granulosa cells by its electrondense cytoplasm. The micropylar cell in this phase characteristically displays an increase in rough endoplasmic reticulum, Golgi complexes, and tonofilaments around the nucleus. By the late vitellogenic phase, the enlarged micropylar cell extends a broad cytoplasmic process to the oocyte surface. A conspicuous feature of the process is a large bundle of microtubules oriented perpendicular to the oocyte surface. The inner surface of the micropylar canal has a spiral structure and is covered with the outermost layer of the chorion. In the postvitellogenic phase, the main cell body possesses many tonofilaments, mitochondria, Golgi complexes, and rough endoplasmic reticulum, and the winding cytoplasmic process contains a twisted large bundle of microtubules with a bundle of tonofilaments as its core. The spiral structure of the micropylar vestibule and the micropylar canal reflects the twisting associated with elongation of fibrous bundles of the micropylar cell anchored on the chorion at the animal pole of the oocyte.

Mechanism of Ca2+ release in medaka eggs microinjected with inositol 1,4,5-trisphosphate and Ca2+.

The reaction time of Ca2+ release from cytoplasmic stores induced by microinjection of inositol 1,4,5-trisphosphate (IP3), calcium ionophore A23187, Ca2+, Sr2+, Ba2+, and cyclic guanosine 5'-monophosphate (cGMP) in Oryzias latipes eggs in Ca2+-free medium was measured by the luminescence of aequorin injected into the egg. Microinjection of IP3 or calcium ionophore induced rapid Ca2+ release without a time lag, while microinjection of either Ca2+ or cGMP required a time lag of 5-30 sec for Ca2+ release. Following microinjection of both IP3 and Ca2+, Ca2+ release commenced in a cytoplasmic region close to the egg surface. These results suggest that in the medaka egg, cytoplasmic Ca2+ induces Ca2+ release from cytoplasmic stores indirectly, probably via a membrane factor such as IP3.

Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicle in response to exogenous substrates.

Changes in the capacity of medaka, Oryzias latipes, ovarian follicles to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) or testosterone to testosterone, estradiol-17 beta, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were examined using 18-hr incubations. Under a constant long photoperiod (14 hr light-10 hr dark) at 26 degrees medaka spawn daily within 1 hr of the onset of light. Under these conditions, vitellogenesis and oocyte maturation of individual follicles occur within 72 hr, allowing accurate determination of the time of oocyte maturation and ovulation. In the absence of substrates, vitellogenic follicles isolated between 28 and 16 hr before spawning produced increased amounts of estradiol-17 beta, while postvitellogenic follicles between 14 and 8 hr produced a large amount of 17 alpha,20 beta-diOHprog. However, under the same conditions, testosterone levels were very low in follicles from all stages of development. The capacity of follicles to produce estradiol-17 beta in response to 17 alpha-OHprog or testosterone increased as follicles developed from the early to late vitellogenic stage, but declined during oocyte maturation. Maximum estradiol-17 beta production was observed in follicles at 20 hr before spawning. In contrast, the conversion of 17 alpha-OHprog to 17 alpha,20 beta-diOHprog was low in vitellogenic follicles and increased in follicles isolated immediately prior to or during oocyte maturation, with maximum 17 alpha,20 beta-diOHprog production occurring in follicles isolated at 14 hr before spawning. These results demonstrate a distinct shift in the activities of steroidogenic enzymes from C17-20 lyase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and aromatase to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) occurring in the medaka ovarian follicles immediately prior to oocyte maturation.