ก F ก F กF ก Hatching Envelope Formation in the Black Tiger Shrimp, Penaeus monodon F Wanita Putthawat F ˈ F ก ก ก ก F F A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Anatomy Prince of Songkla University 2553 F (1) F ก F ก F F ก F กF ก F F ก F ก ก ก ...................................................................... ......................................... ( . F F ) ( . F F F ) ก ก .....................................................ก ( . F F ) ก ...................................................................... .....................................................ก ( F . . ก) ( F . . ก F ก FF ก) ...................................................................... .....................................................ก ก ( . F F) ( F F F . ก F ก ก F F ก ˈ F ก ( F . ) F F F ... ) (2) F ก F ก F F ʾก ก ก 2553 กF ก F F F F ก ก ก F ก F (hatching envelope) กF ก F lectin ˈ (probe) F F F lectin hatching envelope ก ก ก F F hatching envelope F Concanavalin A (Con A F glucose mannose), Lens culinaris agglutinin (LCA F mannose) wheat germ agglutinin (WGA F N-acetyl glucosamine) F ก Con A WGA ˈ probe ก ก F hatching envelope F F F fluorescein-lectin labeling F F (thick sections) ก Fก F F F F (fluorescent microscope) gold-lectin labeling F F (thin sections) ก Fก F F F F (transmission electron microscope) ก thick sections F fluorescein-Con A labeling ก cytoplasm F F F F F ก ก F ก (cortex) F ก ก ˈ F hatching envelope 8 F fleorescein-WGA labeling cytoplasm F F F F ก F 1-20 F ก perivitelline space ก hatching envelope 30 F กก ก F TEM F membranous structures, dense vesicles flocculent vesicles cytoplasm F membranous structures ก F ก ก granular materials ก ˈ ก hatching envelope F ก exocytosis gold-Con Alabeled dense vesicles ก ˈ hatching envelope F gold-WGAlabeled flocculent vesicles F exocytosis ก F perivitelline space F ก ก ก hatching envelope ก กF FF hatching envelope mannose/glucose ˈ F ก perivitelline space N-acetyl glucosamine ˈ F ก (3) Thesis Title Author Major Program Academic Year Hatching envelope formation in the black tiger shrimp, Penaeus monodon Miss Wanita Putthawat Anatomy 2010 ABSTRACT The aim of this study is to reveal the process of hatching envelope formation in the eggs of the black tiger shrimp Penaeus monodon. Lectins were used as molecular probe in this study. Screening test for lectin affinity to isolated hatching envelope revealed the binding of Concanavalin A (Con A, specific binding to glucose and mannose), Lens culinaris agglutinin (LCA, specific binding to mannose) and wheat germ agglutinin (WGA, specific binding to N-acetyl glucosamine). Con A and WGA were chosen for labeling eggs at different period after spawning, as fluorescein-lectin labeling in thick section observed under fluorescent microscope and as gold-lectin labeling in thin section observed under transmission electron microscope. In the thick sections with fluorescein-Con A labeling, diffused fluorescent dots were distributed throughout the ooplasm at spawning, migrated to the cortical area and became part of the hatching envelope at 8-min post-spawning. With fleorescein-WGA labeling, diffused fluorescent dots were also distributed throughout the ooplasm at spawning but at 1-20 min postspawning, they migrated into the perivitelline space, at the same time of elevation of the hatching envelope; and they associated with hatching envelope at 30-min postspawning. Under TEM, at time of spawning, membranous structures, dense vesicles and flocculent vesicles were observed in the cytoplasm of the oocyte. Membranous structures were released and coalesced with granular materials, and became the outer layer of the hatching envelope. This was followed by exocytosis of gold-Con A-labeled dense vesicles, which became the inner layer of the hatching envelope. Finally, goldWGA containing flocculent vesicles were released into the perivitelline space, accompanying the elevation of the hatching envelope. The results suggest that the inner layer of the hatching envelope contains mannose/glucose and the perivitelline space contains N-acetyl glucosamine. (4) F ก ก F 1. 2. ก 3. ก 4. F 5. ก F ก F ก ก F (7) (8) (10) 1 19 23 38 41 42 47 53 (6) ก F 1. Lectin 11 2. F ก 3. ก F กF ก F ก ก hatching envelope ก F กF Sicyonia ingentis F 17 lectin F 23 hatching envelope 37 (7) ก ก F 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 1 กF ก F F ก F 3 5 6 6 กF ก ก ก F F กF ก F thelycum กF ก ก F F กF ก ก F กก F F light microscope ก FกF ก F ก light microscope FกF ก ก ก egg activation S. ingentis F F lectin lectin F 3 Fluorescent micrograph lectin Con A, LCA, WGA Fluorescent micrograph FกF ก F fluorescein-labeled Con A Fluorescent micrograph FกF ก F fluorescein-labeled WGA Transmission electron micrograph Transmission electron micrograph F Transmission electron micrograph ก 8 9 10 13 16 16 F ก hatching envelope 24 F F incubate 26 F กF F ก FกF ก FกF ก F incubate 0 15-45 1 F F 27 28 29 30 (8) ก ก ( F ) F 17. 18. 19. 20. 21. 3 Transmission electron micrograph Transmission electron micrograph Transmission electron micrograph TEM gold-Con A FกF ก TEM gold-WGA FกF ก FกF ก กF F ก กF F ก 30 30 1-2 3 30 F F F F F 31 32 33 35 36 (9) 1 1 1.1 ก ก F กF ก (Penaeus monodon) ก ก F 20 ʾ ˈ F F ก ʾ F F ก กF ก F (intensive farming) F F ก ( 4 F 35-45 /ก ก ) FกF F ก ˆ F ก F กF (White spot syndrome virus, WSSV) (Yellow head virus, YHV) ˈ F ก ก ˆ กF ก F (Withyachumnarnkul et al., 2004) ก ก ก F F F ก ก กF F ˈ F F F F ก F Fก ก F F กF Litopenaeus vannamei ก F กF Fก กF F ก F กF ก ก กF ก ก F F ˈ กF F ˈ ก F 20 ʾ ก ก F กF ก ก F F ˈ ˈ 10 ʾ F ˈ 3 ʾ กF ก F ˈ F ก ก กF F ก (super intensive farming) F F 3-5 / F F 3 F F F ก FF ˈ กก F กF ก (60-100 /ก ก ) F F ก กกF ก ก ก F กF ก F ก Fก กF ก ˈ กF F F กF ˈ กF F F ก กก ก F กF F ก ˆ F ก ก กF ก F ก ก ก กก ก ก กF ก ก ก ก F F F กF ก F Fก F FกF ( ก.) F ก F (BIOTEC) ก F F ( .) ก ก ก F กF ก F F ก. กก F F ก ก F ก ก F ก ก ก F กF ก F F F F F F F กกF ˈ F ก F ก F กF ก ˈ ˆ ก ˆ ก ˆก F ˈ (hatching rate, HR) F ˈ F F FกF 2 ก HR 80-90% กF F ก ก F ˈ กF (domesticated shrimp) HR 50-80% F F F ˈ ˆ F F ก ก F ก กกF F กF F F ก ก F ก กกF F F กF F F F ก ก กกF ก ˈ กF (harvesting size) ก HR ก 50% ˈ 80% ˈ ก F ก HR domesticated shrimp ˈ F Fก ก ก F F ก HR ก ก 1) F oocyte F F 2) sperm F F / 3) F F ก ก F F ก ก F F pH alkalinity F magnesium ก (Pongtippatee et al., 2010) ˆก ก F ˆ ก ก F ก ก ก ก ˆ F F F ก F F / กF F F ก ˆก F HR ก ก (fertilization rate, FR) F กก ก F Fก F F ก ก F 4-5 F HR ก ก F FR 2-5% ก F HR ก F FR F ก F F ก F ก F F F ก F F HR ก F F F กก F 2-5% F ˈ F F ก F FR ˈ ก F FR 1-2 ก F ˈ F F ก ก F F F 4-8 F ˈ F FF F ก F 1-2 ก ˈ F ก FR F F ก F ก ˆก ก FR F 05.00 . ก ˈ ก ก ˈ F ก FR F 1-2 ก F กF ก F ก FR F F F F ก ก F F F ก F ก F ก ˆก ˈ ˈ F F FR ก F F 1-2 F ก ก F HR ก ˆ F ก Fก F F HR ก F กก F 2-5% ก ก F ก F (hatching envelope formation) ˈ ก ก egg activation egg activation ˈ ก ก ก F F ก ก ก F hatching envelope ˈ ˂ ก F F F F ก F ก กก F 3 ก F ก ก polyspermy F ˈ ˈ F F ก กก F hatching envelope กF F F (Pongtippatee et al., 2004) ก ก hatching envelope กF ก ก (fluorescent microscope) ก F F microscope, TEM) ก F F F F Fก 1.2. ก ก F F F F ก ก ก F F F F ก F F (transmission electron Fก F FR HR F ก ก กF ก 1. กF ก (http://www.fao.org/fishery/culturedspecies/Penaeus_monodon/en) : กF ก ก กF ก กF ก กF กF กF : Giant tiger prawn, Black tiger shrimp F : Penaeus monodon fabricius กF ก (antennule) (periopod) 5 F ก ˆ F F F กF F 6-8 ˆ F 2-4 (pleopod) 5 F F (abdomen) carapace F ( 1) F F ก 1.2.1 F ก (rostrum) ก (carapace) 4 กF ก F F F F ˆˉ F F ก ˈ FF กก F ˈ ก 20-50 ก กก ก F ก ʽก F ก ʽ ก กก ก F ก ก ก F ก (larva) F ˁ กF ก F กF F F ก ก F ˁ F F ก ก ˆˉ กF ก F กF F ก F (Motoh, 1981) กF ก (nauplius) 12 F ก ก F Fก F 2 ก ก 6 F F (protozoea) ก ก กF ˈ 3-4 F F (mysis) ˈ F F ก กF (eyestalk) F ˈ F ก ก 3 (postlarva, PL) ก F ก กF ก ก ก F F F (juvenile) ก F F F F F F F 10 ก F F ก ʽ F 1.2.2 ก กF ก ˆก ˈ กF F กF ก F ก F FกF ก กF F F F ˆก ˈ yolk F 0.30-0.33 กF 0.60 F F 1.00-3.30 ก 3 F 3.30-5.00 กF ก กF 3-4 F F F ก ก ก F ก F 15 F F F ˈ (Motoh, 1981) กF ก ก F Fก ก ก F F กF F (petasma) ก กก (endopodite) F F ก endopodite 2 F F ˈ F กF (thelycum) ก F F (sternal plate) FF ก F 7 8 4-5 ˈ ก Fก ก ก F ก F F F (spermatophore) ก F ก F ก Fก ก ˈ F F F (Motoh, 1981) 5 1.2.3 F กF 1.2.3.1 F กF ก F F (ovary) 1 F F F (oviduct) 1 F thelycum 1 F กF F 2 F F F F ก F anterior lobe, lateral lobe posterior lobe ( 2a) lateral lobe F cephalothorax F F F ก ก F Fก ก ก F กF F กF F F Fก F ก F F F F ก F ( 2b) F 2. a. lobe b. F ก ก posterior lobe ก F F F กF ก (Hall et al., 1999) ก F anterior lobe, lateral Fก 6 F ก F lateral lobe ovary gonopore F F 3 3. F ก F F กF ก ( F ) (Taweepreda, 2003) Thelycum ก ˈ F F ก F median plate 1 lateral plate 1 F thelycum กF ก (closed thelycum) F ก spermatophore Fก ก F F (Motoh, 1981; Taweepreda, 2003) 4. F ก thelycum กF ก ( 2 F F ˈ ʽ F ) (Taweepreda, 2003) 7 กก F กˆ F F ( 6) 1.2.3.2 F กF ก F F ก FกF ก ˈ ( 5) 1 (I) Undeveloped and/or spent ก ก ˈ ก (connective tissue capsule) F connective tissue capsule F กF F F ก F oogonia accessory cells ( F ) ( 6a) ก F F F ก 5 F ก F ก ก F ˈ ก stage F F F ก F F F soft vascular area F germinal epithelium F ก F follicular cell nurse cell 2 (II) Developing stage F F F F F F ก F oogonia F F mitosis germinal epithelium ก F F F ก ˈ ก F zone of proliferation oogonia F F F first meiotic division ก F haploid F ก yolk F ก oocytes F previtellogenic oocytes ( 6b) 3 (III) Nearly ripe stage F ˈ F F F ก ก F ก ʾก F F oocytes F ก ovarian lobe F ก ก F (ovulation) F ก follicular cell ก F oocytes F F yolk Fก oocytes ก ก vitellogenesis ก carotenoid pigments F F F F ( 6c) F 4 (IV) Ripe stage Fก F ก ก ˈ F ก ก ˈ ʾก Fก F 3 ˈ F F F ˈ vitellogenesis oocytes ก F cortical granules jelly-like substance ก F ก F กF ก ก ˈ cortical rod ( 6d) 8 5 (V) Post-spawn ก F ก F ก 5. ก F F ˈ ก (Hall et al., 1999) F F (Hall et al., 1999) F กF ก กก F ก F F ก 9 6. light microscope ก (Hall et al., 1999) a. b. c. d. ก F F F F FกF ก 1 2 3 4 F 10 1.2.3.3 FกF ก FกF ก ก ก F F Fก 270-280 µm F ก ˈ ก F cortical crypts F F Fก 10-15 µm F corticoid rods ก ˈ F ˆ F F ก ก F ก ก F investment membrane F F yolk granule, mitochondria cortical granules F F ก F F ก F (hatching envelope) FกF ก ก ก ก ก กF F ก F ก ก egg activation ก ก ก FกF ก (Pongtippatee et al., 2004) 7. light microscope ก FกF ก 0 ก F 1.2.3.4 ก ก ก egg activation ก ก egg activation ˈ ก ก ก F ก ก F ˂ ก ก ก polyspermy ก F ก F F Fก F F (Epel, 1975; Clark et al., 1980; Pongtippatee et al., 2004) ก ก ˂ ก ก ก polyspermy F ก F ก ก F F F F ก F ก F กF zona pellucida ก ก acrosome reaction ก zona pellucida glycoprotein ก F ZP3 F ˈ sperm receptor F Nacetylglucosamine ZP3 ก ก galactosyltransferase (GalT) ก F F ก acrosome reaction ก ก ก ก acrosome reaction ก F protease ก F acrosin ก acrosome ˈ F F ก F 11 F F F F zona pellucida F F ก ก plasma membrane Fก F F F ก ก zona reaction cortical reaction ก cortical granule cytoplasm F ก ˈ ก F protease ก ก exocytosis (ก membrane cortical granules ก cell membrane F granule ก ก F) F ก zona pellucida receptor F zona pellucida F F F F ก F Fก (Bleil and Wassarman, 1980; Shur and Hall, 1982a, b; Florman and Wassarman, 1985) F (sea urchin) ก กก egg jelly F ก F resact ก ˈ chemotaxis F F F ก jelly coat F F ก acrosome reaction hydrolytic enzyme F acrosome ก F jelly coat F ก polymerization globular actin F cytoplasm F ˈ actin filament ˈ F ก กF ก F acrosomal process F ˈ F F ก F jelly coat F ก vitelline envelope F acrosomal process bindin F bindin receptor vitelline envelope bindin ก ก species specific Fก ก ก cell membrane F ก กก ˂ ก polyspermy ก F cortical granule reaction F ก ก exocytosis cortical granules ก F cytoplasm F F proteases ก F F vitelline envelope cell membrane F F F vitelline envelope cell membrane F F ก กก ก F ก F bindin receptor F ก ก F cortical granules F exocytosis mucopolysaccharides F osmotic gradient F F F F F vitelline envelope cell membrane ˈ F vitelline envelope ก ก ˈ fertilization envelope cortical granules ก exocytosis F peroxidase F ก (hardening) fertilization envelope ก crosslink F tyrosine residues F ก F cortical granules hyalin ก F F F F (blastomeres) F F ก F (Vacquier et al., 1973; Foerder and Shapiro, 1977; Hylander and Summers, 1982; Crossley et al., 1988; Terasaki, 1995) 12 Goudeau Becker (1982) F ก ก F Carcinus maenas ก ก ก cortical reaction F ก ก F กก cell membrane F F F C. maenas cortical vesicles 2 F ก granule ก (fine granular material) granule F (ring-shaped granules) ก vitelline envelope 2 F ก F F ก ก ก F envelope F ก F ˈ fertilization envelope กก ก ring-shaped granule F vitelline envelope ก ก 7-8 ก ˆก F ก F ก Fก F F ก F Fก ˈ ˆก F ก (Little and Kitching, 1996) F ก ก ก F fertilization envelope lobster F ก F ก F envelope 2 ก ก F F F ovary ก กก ก cortical reaction กF F cortex oocytes vesicles (granules) 4 F กF high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV) ring vesicles (RV) HDV LDV exocytosis ก perivitelline space ก MDV RV ก exocytosis ก ก F ˈ envelope (Talbot and Goudeau, 1988) กF egg activation ก ก F ก Penaeus japonicus, Penaeus setiferus, Penaeus aztecus Sicyonia ingentis F ก jelly precursor ก ก F F jelly precursor ก ก ก ˈ jelly layer F F ก F ก hatching envelope ก กก exocytosis vesicle F F 2 (Duronslet et al., 1975; Chandler and Heuser, 1979; Clark et al., 1980, 1984, 1990; Yano, 1988; Pillai and Clark, 1990; Lynn et al., 1992) กF S. ingentis F Fก F F F F F seminal receptacle ก F thelycum ˈ ʽ (closed thelycum) F F F F F ก กก F ก ก Pillai (1990) F ก ก S. ingentis ( 8) F กF F F ก ก vitelline envelope (VE) F FF anterior spike F jelly precursor (JP) F extracellular crypt F Fก ก ก egg activation ก F jelly precursor ก ก cortical crypt F vitelline envelope ก ˈ jelly F F F ก ก vitelline envelope ก F 13 F 20 ก F F jelly layer F F acrosomal filament formation acrosome reaction ก ก F hatching envelope F กก exocytosis cortical vesicle 2 dense vesicle ring vesicle F outer inner layer hatching envelope 40-45 F F ก ˆก 24 ก กF F (Hertzler and Clark, 1992) ก ก F hatching envelope S. ingentis F ก glycoprotein F ก ก ก threonine glutamine, lysine asparagine กก SDS-PAGE carbohydrate staining F hatching envelope S. ingentis ก F mannose, sialic acid, Nacetyl glucosamine glucose (Pillai et al., 1990) 8. ก ก egg activation S. ingentis (Clark et al., 1994) A. ก vitelline envelope (VE) F F anterior spike F jelly precursor (JP) F extracellular crypts B, C. ก acrosome reaction ก F VE F F F jelly precursor ก F F VE F VE ก 14 D. 15 F jelly precursor ˈ jelly layer VE E. 20 F F acrosomal filament formation acrosome reaction F. F F cytoplasm F 30 F polar body ก F (PB I) ก ก 35-45 G. cortical granule (CG) 2 cytoplasm F H. CG ก ก exocytosis F hatching envelope (HE) ก I. CG exocytosis Fก HE ก HE F ˈ ก ก hatching envelope elevation J. 45 ก F HE F ก polar body 2 (PB II) F F pronucleus F ก ก Clark (1980) F ก ก ก ก cortical reaction F P. aztecus ก F ก F hatching envelope F F ก cortical rod F F ก ก F F 45 F F investment coat ก cortical rod ก ก ก ˈ ก F 5-7 F ก cortical rod ก F hatching envelope ก 15 F P. monodon ก ก egg activation ก F ก cortical rods ก ก cortical crypts F 45 F ก ˈ jelly layer F กก ก Kruevaisayawan (2007) F cortical rod F ก ก ˈ F ก ก acrosome reaction acrosomal mass formation ˆ F jelly layer ก ก F hatching envelope F F 1 F F 13-15 F polar bodies ก ก ก 3-5 10-15 F (Pongtippatee et al., 2004) 15 1.2.4 Lectin ก F ˈ probe ก F hatching envelope Lectin ˈ ก F (carbohydrate-binding protein) F F F ก F ก ก ˆ cell membrane F ก F lectin ก F F F 2 sugar binding site ก ก F monosaccharide oligosaccharide F ( 9) ก lectin ก membrane glycoprotein F F ก F lectin ก F ก F (Sheeler and Bianchi, 1987) ( 10) lectin F F lectin F F 1 (Clark et al., 1990) cell membrane F F F ก ˈ carbohydrate glycoprotein F cell membrane oocyte mouse กF F S. ingentis glycoprotein chitin ˈ F ก hatching envelope ก chitin hydrolytic enzyme chitinase N-acetylglucosaminidase incubate ก FกF 10 F ก ก F ก ก hatching envelope (Glas et al., 1996) กก ก ก ก cortical reaction S. ingentis Clark (1990) ก F F lectin ˈ probe F ก F lectin ก cortical vesicle F 2 lectin WGA (Wheat germ agglutinin) ก D-GlcNac sialic acid ก dense vesicle F ก hatching envelope lectin LCA (Lens culinaris) mannose ก ring vesicle F hatching envelope ก ก ก ก cortical granules oocyte mouse ก ก F LCA ˈ probe F mature oocytes cortical granule F F 2 F ก F LCAbinding cortical granule (Liu et al., 2003) 16 9. F F lectin (http://en.wikipedia.org/wiki/File:Gs4_sugar_all.png) Cell membrane 10. (Sheeler and Bianchi, 1987) lectin F F 17 1. Lectin 11 F Lectin F ก Sugar specificity Lens culinaris (LCA) Mannose Concanavalin A (CON A) Mannose/glucose Wheat germ agglutinin (WGA) D-GlcNac/sialic acid Griffonia simplicifolia (GS-11) D-GlcNac Ulex europaeus (UEA-1) L-fucose Maclura pomifera (MPL) D-galactose Soybean agglutinin (SA) D-galactose Griffonia simplicifolia (GS-1) D-galactose Arachis hypogaea (PNA) D-GalNac Dolichos biflorus (DBA) D-GalNac Bauhinia purpurea (BPA) D-GalNac 18 1.3 F ก ก กก F hatching envelope FกF ก ก ก exocytosis cortical vesicles F lectin ˈ probe F ก F F (epifluorescent microscope) กF F (transmission electron microscope, TEM) Fก F F F 19 2 ก 1. ก ก F F F FกF F FกF ก (Penaeus monodon) ก F ก F FกF . . F F ก ˆก (maturation tank) 30 ppt. F F F F F F ก F 3 ก FกF ก F ก (artificial insemination, AI) (spermatophore) F F F thelycum F F ก F ก F F F FกF Fก F 4 ก ก FกF F F 500 F F FกF F ก ก FกF F F ˈ ก ก F F F FกF F F ก ก F F FกF F ก 50 FกF F F F (dropper) FกF F 8% paraformaldehyde ( F Fก 8% paraformaldehyde F F F F F ˈ 4% paraformaldehyde) F F ˈ F 0 ก F ก Fก ก 3 F FกF ก F ก ก 15 ก ก F F F 8% paraformaldehyde ก FกF 15, 30 45 1, 2, 3, 5, 8, 15, 20, 30, 45, 60, 90 120 F ก ก ก hatching envelope F ก F F F o F ก F F F 80-90% ก F 4 C FกF 30 ˈ ก F hatching envelope F กF ก o F 1 mM phenylmethanesulfonyl fluoride (PMSF) 4 C ก F F ก hatching envelope 20 2. cortical vesicles hatching envelope F กF ก F ก ก cortical vesicles ก hatching envelope TEM F F lectin probe F กF ก F ก ก embed F F ก LR white resin F ก ก F lectin Fก morphology ˈ F ก ก vesicles hatching envelope F ก embed F F epoxy resin F F morphology F F F กF FกF F ก F fix F F 4% paraformaldehyde 2 F (tissue processing) F F FกF F 0.1 M PBS 3 5 post-fix F 1% OsO4 1 F F ก 2-3 F F 2% uranyl acetate 20 ก dehydration F 50% ethanol 10 F 70% ethanol 10 F 95% ethanol 2 10 absolute ethanol 2 10 F ก infiltration F propylene oxide 2 15 F propylene oxide : epoxy resin F 1:1 1 F propylene oxide : epoxy resin F 1:2 2 F epoxy resin 2 ก embedding ก FกF ˆ epoxy resin F capsule beam polymerization ก F F 80 oC ˈ 24 ก sectioning F F ultramicrotome (MT-XL RMC) thick section 400 nm ( F กF ) thin section 90 nm ( F copper grid F F acetone mesh cement F ก F F F) F F F staining thick section F F 0.5% toludine blue F ก ก thin section F F 5% uranyl acetate lead citrate F 10 F F ก F TEM (JEOL, JEM-100 CXII, Tokyo, Japan) 21 3. ก ก hatching envelope FกF FกF ก 30 F F F phosphate buffered saline (PBS; 0.14 M NaCl, 0.008 M KCl, 0.006 M Na2HPO4, 0.001 M KH2PO4, 0.001 M CaCl2 0.0004 M MgCl2, pH 7.4) 4 oC 3 ก F autopipette F 300 µl ( F 300 ) F 1 ml microcentrifuge tube (Appendolf tube) lysis buffer 300 µl (10 mM Tris-HCl, 2 mM EDTA, 0.4 M NaCl 0.01% Nonidet P-40, pH 8 1 mM PMSF) F grinder o shake 72 rpm 4 C ก ก hatching envelope ก 10 ก F 10 µl F Fก F F (light microscope) F ก ก hatching envelope กก F 80% F ก F ˈ ก ก F กF F F F (artificial sea water, ASW; 460 mM NaCl, 55 mM MgCl2, 10 mM KCl 10 mM CaCl2) o 4 C 10 F F ก F ˆˉ ก 200x g 5 F ก hatching envelope ก F F ก 2 ก F ก resuspend F ASW 200 µl 1 mM PMSF ก F -20 oC ก F 4. ก lectin F hatching envelope lectin F F hatching envelope ก Pillai and Clark (1990) F F lectin 11 ก hatching envelope lectin F ˈ probe F ก hatching envelope F 100 µl F F PBS PBS 100 µl F 20 mg/ml Bovine serum albumin (BSA) ˂ ก ก ก non-specific reaction F 1 F ˆˉ ก 200x g ˈ 5 ก F ก F PBS 1 ก incubate ก fluoresceinlabeled lectin (F-lectin) 11 (Vector Laboratories, Burlingame, CA) ( 1) F F 5 µg/ml 1 ก F F F PBS F ก F epifluorescent microscope (DP 50, Olympus, Tokyo, Japan) กF Fก incubate ก F F lectin ( 1) กF incubate ก lectin F F 200 mM ก F F WGA MPL F 500 mM lectin F positive F hatching envelope F ˈ probe ก F 22 5. ก ก formation FกF cortical vesicles exocytosis กF F ก F F F ก hatching envelope F lectin probe F fix F F 4% paraformaldehyde 2 F F F FกF F 0.1 M PBS o F ก F 0.05 M PBS 2 2 20 4 C o 20 4 C F ก dehydration F ก F FกF 50%, 70%, 80% 90% ethanol F F 20 4 oC 95% ethanol o 2 20 4 C F ก infiltration F F FกF 95% ethanol : LR white resin (London Resin Company, Ltd., England, UK) F 2:1 o 1 4 C 95% ethanol : LR white resin F 1:2 1 o o 4 C F F LR white resin 24 4 C F F LR white resin F 3 ก embedding ก F กF ˆ LR white resin F capsule beam polymerization F ก F o F 60 C ˈ 24 ก sectioning F F ultramicrotome thick section 400 nm thin section 90 nm ( F nickel grid) staining Thick section F ˂ ก ก ก non-specific reaction F 20 mg/ml BSA ˈ 20 ก F F 0.1 M PBS F F fluorescein-labeled lectin F positive ( กก lectin F F hatching envelope) F F 5 µg/ml ˈ 1 F F F PBS 2 10 F F F mount F 75% glycerol F Fก F epifluorescent microscope negative control Fก pre-incubation lectin F F lectin F กF incubate ก thick section Thin section F ˂ ก ก ก non-specific reaction F 20 mg/ml BSA ˈ 20 ก F F 0.1 M PBS F F fluorescein labeled lectin F positive ˈ 30 F F 0.1 M PBS ก incubate ก mouse monoclonal anti FITC conjugated gold ˈ 1 F F 0.1 M PBS F F F 5% uranyl acetate lead citrate F 10 F F ก F TEM 23 ¸É 3 ¨µ¦¨° µ¦°· ° lectin ¸ÉεÁ¡µ³ n° hatching envelope Hatching envelope ¸É¥¨oª¤¸ªµ¤µ¤µ¦Äµ¦´´ Con-A ¦¼¸É 11a) ¨³ LCA ¦¼¸É 11c) ¤µ¸É» 宦´ WGA ¤¸ªµ¤µ¤µ¦Äµ¦´´ hatching envelope Á¡¸¥Á¨Èo°¥ ¦¼¸É 11e) ¨³ GSL II, MPL, GSL I, DBA, SA, UEA, PA ¨³ BPL Ťn¤¸ ªµ¤µ¤µ¦´´ hatching envelope ¹Éªµ¤ÎµÁ¡µ³n° Con-A ¨³ LCA ¼¥´¥´Êoª¥ Ê嵨 mannose ¸ÉÁ·¤¨ÅÄ lectin n°ÎµÅ incubate ´ hatching envelope ¦¼¸É 11b ¨³ d) ¨³ªµ¤ÎµÁ¡µ³n° WGA ¼¥´¥´Êoª¥ N-acetyl glucosamine ¦¼¸É 11f) µ¦µ¸É 2) µ¨µ¦¨°¸ÊÅoÄo Con A ¨³ WGA Á} probe Ä ´Ê°n°Å µ¦µ¸É 2. ªµ¤µ¤µ¦Äµ¦´´ hatching envelope ° lectin Ân¨³· · ° lectin Con-A LCA WGA GSL-II MPL GSL-I DBA SA UEA PA BPL ªµ¤µ¤µ¦Äµ¦´´ lectin Strong Strong Weak None None None None None None None None 24 ¦¼¸É 11. Fluorescent micrograph ªµ¤µ¤µ¦Äµ¦´´ hatching envelope ¸ÉÂ¥ ¨oª ° fluorescein-labeled ConA a), LCA c) ¨³ WGA e) 宦´ µ¦ pre-incubation oª¥Êεµ¨ÎµÁ¡µ³ ° lectin ´Êµ¤·ÂĦ¼ b, d ¨³ f µ¤¨Îµ´ 25 µ¦´ ° lectin thick sections °Å n ¡ Con A ¤¸ªµ¤ÎµÁ¡µ³n° mannose/glucose) ´´ cortical vesicles ¦³µ¥´ªÁ}»Á¦º°Â¦³¦µ¥´Éª cytoplasm °Å n´ÊÂnÁª¨µ 0 ª·µ¸®¨´ªµÅ n ¦¼Â¸Ê¦µ°¥¼n¹Áª¨µ 1 µ¸®¨´ªµÅ n ¦¼¸É 12a) Con A ¥´µ¤µ¦´´ cortical rod Ã¥Á®È¨´¬³Á¦º°Â¹ÉÅo¤¸¦µ¥µÅªoÃ¥ Kruevaisayawan ¨³³ 2007) µ´Ê ¸ÉÁª¨µ 2-5 µ¸®¨´ªµÅ n »Á¦º°ÂŦª¤´ª°¥¼n¦·ÁªÄ¨o °Å n periphery) ¦¼¸É 12b) ĵ¸¸É 8 ®¨´ªµÅ n ¦µÂÁ¦º°Âµ Ç °¥¼n¦°·ªÅ n ¨³µ¸¸É 15 ®¨´ªµÅ n Á¦º°Â ° hatching envelope ¦°Å n¤¸ªµ¤®µÂ¨³´Á¤µ ¹Ê ¦µ¦°¥®¥´¸É ¦¼¸É 12c) ¸É 20 µ¸®¨´ªµÅ nÂÁ¦º°Â ° hatching envelope Â¥´ª°°µ·ªÅ n ¥¼ ¹Ê¦µ perivitelline space °¥¼n oµÄo ¨³¦µ»Á¦º°ÂÁ}¨»n¤Ç ¦³µ¥´ª°¥¼n Ä perivitelline space ¨³µ¨»n¤¨°¥ ¹ÊÅ·´ hatching envelope ¦¼¸É 12d) ¸É 30 µ¸®¨´ªµÅ n µ¦¦oµ hatching envelope Á¦È¤¼¦r ¦µ¨»n¤Á¦º°Â·°¥¼noµÄ ° hatching envelope Ťn¦µÄ perivitelline space ¨³Ä cytoplasm °Å n ¦¼¸É 12e) Ĩ»n¤ª»¤Åo¸ÉÁ·¤ mannose ¨ÅÄ lectin n°µ¦ incubate ´ thick section °Å n Ťn¡¨´¬³Á¦º°Â ¦¼¸É 12f) 宦´µ¦´ ° WGA ¤¸ªµ¤ÎµÁ¡µ³n° N-acetyl glucosamine) ¡ ¨´¬³Á¦º°Â¦³µ¥´ªÄ cytoplasm °Å n´ÊÂn´¸¸ÉªµÅ n¨³n°¤µ¦µÁ} Á¦º°ÂÁ}Áo°¥¼n¸É °Å nÃ¥¦°¹Áª¨µ 5 µ¸®¨´ªµÅ n ¦¼¸É 13a) ¨³¸ÉÁª¨µ 8 µ¸®¨´ªµÅ n´ÁÁ®È»Á¦º°Â¦³µ¥´ª ¹ÊµÂÁ¦º°Â ¦¼Á¨ÈĦ¼¸É 13b) ¨³ »Á®¨nµ´Ê¦³µ¥´ªÁ oµ¼n perivitelline space ¸ÉÁª¨µ 15-20 µ¸®¨´ªµÅ n ¦¼¸É 13b) n°¤µ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n¹ÉÁ}Áª¨µ¸Éµ¦¦oµ hatching envelope Á¦È¤¼¦r ¡ ÂÁ¦º°ÂÁµ³·°¥¼n´ hatching envelope Á}¦³¥³Ç åŤn¡»Á¦º°ÂÄ perivitelline space ¦¼¸É 13c) Ĩ»n¤ª»¤¸ÉÁ·¤ N-acetyl glucosamine ¨ÅÄ lectin n°µ¦ incubate ´ thick section °Å n Ťn¡¨´¬³Á¦º°Â ¦¼¸É 12d) 26 ¦¼¸É 12. Fluorescent micrograph ÂÅ n»o»¨µÎµ¸ÉÁª¨µnµÇ a-e) ®¨´ªµÅ n ¸É incubate oª¥ fluorescein-labeled Con A ¨»n¤ª»¤¤¸µ¦Á·¤Ê嵨 mannose n°Îµ¤µ incubate ´Å n f) ¦¼Á¨ÈoµÄ£µ¡ ¥µ¥ °¨´¬³Á¦º°Â 27 ¦¼¸É 13. Fluorescent micrograph ÂÅ n»o»¨µÎµ¸ÉÁª¨µnµÇ a-c) ®¨´ªµÅ n ¸É incubate oª¥ fluorescein-labeled WGA ¨»n¤ª»¤¤¸µ¦Á·¤ N-acetyl glucosamine n°Îµ¤µ incubate ´Å n d) 28 ¨´¬³ cortical vesicles ¨³µ¦Á· hatching envelope µµ¦«¹¬µoª¥ TEM ´¸¸ÉªµÅ n £µ¥Ä cytoplasm °Å n¡ yolk granules 2 µm), dense vesicles 500 nm), flocculent vesicles 200-1,000 nm) ¨³ membranous materials ¦¼¸É 14) Á¤ºÉ°Äoε¨´ ¥µ¥¼ ¹Ê Ä flocculent vesicles ¡Ã¦¦oµ¦¼ªÂ®ª®¦º°Ã´°¥¼n £µ¥Ä¨oµ¥´ ring vesicles ¸É¦µ¥µÄ Pillai and Clark 1988) µ vesicles ¤¸µ¦¦ª¤´ª ´¨µ¥Á} vesicles ¸ÉÄ®n ¹Ê¨³¤¸¦¼¦nµÅ¤nÂn° ¦¼Á¨ÈĦ¼¸É 14) ¡ Cortical rods °¥¼n£µ¥Ä cortical crypts ¨³£µ¥Ä ° cortical rods ¤¸ æ¦oµ bottle brushes ¨oµ¥Â¦¨oµ ª) ¦¦»°¥¼n ¹ÉÄn°ªnµÂÇ ¦³®ªnµ cortical rod ¨³Á¥ºÉ°®»o¤Á¨¨rÅ n¡ vesicles Á¨ÈÇ ¦³µ¥´ª°¥¼n vesicles Á®¨nµ¸ÊÁ¦¸¥ªnµ granular materials ¹É¡Ä»o S. ingentis Ã¥ Pillai and Clark 1988) °¥nµÅ¦È¸Ä»o S. ingentis ¤¸ µ¦¡ granular materials Á¡µ³¸É¦·Áªn°ªnµ¦³®ªnµ vitelline envelope ¨³Á¥ºÉ°®»o¤ Á¨¨rÅ nÁnµ´Ê¹ÉÁ}¦·Áª°ºÉ¸É°¥¼n·ª ° cortical rod ŤnÄnÁ}¦·Áª¸É°¥¼nÄ cortical crypt ¦¼¸É 14. Transmission electron micrograph ÂÅ n»o»¨µÎµ¸ÉÁª¨µ 0 ª·µ¸®¨´ªµÅ n £µ¡Á¨ÈÂε¨´ ¥µ¥¼ ° flocculent vesicles: CR, corical rod; DV, dense vesicle; GM, granular materials; Mb, membranous structures; FV, flocculent vesicles; Y, yolk 29 ¸ÉÁª¨µ 15-45 ª·µ¸®¨´ªµÅ n Á¤ºÉ° cortical rods èn¡o cortical crypts Ã¥ ¤¼¦r³¤¸µ¦¨n°¥µ¦µ cortical rods ¨µ¥Á}´Ê jelly layer °¥¼n¦°Å n ´ÁÁ®È membranous structure ¸É°¥¼nÄ cytoplasm °Å nèn°°¤µµ·ªÅ n ¦¼¸É 15a) ¨³¸Éµ ° jelly layer ¡ membranous structure °¥¼nĨo´ granular materials ¹Éε¨´Á¦¸¥´ª Á}ª¨³ ¦¼¸É 15b) ¸ÉÁª¨µ 1 µ¸®¨´ªµÅ n granular materials Á¦¸¥´ªÁ}ªÁ¸É¥ª ÁºÉ°¤·´ ¦¼¸É 16a) Á¥ºÉ°®»o¤ granular materials ¸É·´¨µ¥Á}æ¦oµÁo¹¥µª° ´Ê¸É¨´oª¥Âµ°¥¼n¦¨µ æ¦oµ¸ÊÅo¨µ¥Á}´Ê° ° hatching envelope ¦¼¸É 16b, c) ¦¼¸É 15. Transmission electron micrograph °Å n»o»¨µÎµ¸ÉÁª¨µ 15-45 ª·µ¸®¨´ªµÅ n µ¦°°¤µ ° membranous structure ¸É·ªÅ n a, ¨¼«¦) ¨³µ¦¦ª¤´ª´ granular materials °Å n: GM, granular materials; JL, jelly layer 30 ¦¼¸É 16. Transmission electron micrograph °Å n»o»¨µÎµ¸ÉÁª¨µ 1 µ¸®¨´ªµÅ n  µ¦¦oµ´Ê° ° hatching envelope Ã¥ granular materials ÁºÉ°¤·´Á}ªÁ¸¥ª a) εĮoÁ·Ã¦¦oµ¸É¤¸ªµ¤®µÁ}¹°´Ê¨´´Âµ¸É°¥¼n¦¨µ ¨³ ¦µ dense materials Áµ³°¥¼noµÄ ° hatching envelope b, c): DM, dense materials; GM, granular materials 31 ¸ÉÁª¨µ 1-2 µ¸®¨´ªµÅ n ¡µ¦ e ocytosis ° dense vesicles ¦¼¸É 17a) ¨³¨n°¥ dense materials ¸É°¥¼n£µ¥Ä°°¼n perivitelline space ¹Én°¥Ç ¹ÊÅ Áµ³·´oµÄ ° hatching envelope ´Ê°¸É¦µn°®oµ¸Ê ¦¼¸É 17b) ¨³ ¨µ¥Á}´ÊÄ ° hatching envelope ¦¼¸É 17. Transmission electron micrograph °Å n»o»¨µÎµ¸ÉÁª¨µ 1-2 µ¸®¨´ªµÅ n µ¦ e ocytosis ° dense vesicles a) dense materials ¸É¼¨n°¥°°¤µÁ¨ºÉ°¸É Á oµÅ°¥¼nÄ perivitelline space ¨³¨°¥ ¹ÊÅ·´oµÄ ° hatching envelope ´Ê° b): DV, dense vesicles 32 ¸ÉÁª¨µ 3 µ¸®¨´ªµÅ n flocculent vesicles Á¨ºÉ°¸ÉÅ¥´ °Å nÁ oµÄ¨oÁ¥ºÉ° ®»o¤Á¨¨r ¦¼¸É 18a) µ¦¸É°¥¼n£µ¥ÄÁ¨¸É¥ÅÁ}¨´¬³Ã¦n¨³¼ e ocytose ¨n°¥ °°ÅÄ perivitelline space oµ ¦¼¸É 18b) ¦¼¸É 18. Transmission electron micrograph °Å n»o»¨µÎµ¸ÉÁª¨µ 3 µ¸®¨´ªµÅ n  flocculent vesicles ¸ÉÁ¨ºÉ°¸ÉÁ oµÄ¨oÁ¥ºÉ°®»o¤Á¨¨rÅ n a) ¨³¤¸µ¦ e ocytosis Ä®o¨n°¥µ¦ ¨´¬³Ã¦nÂÁ oµ¼n perivitelline space b): FV, flocculent vesicles; HE, hatching envelope; PVS, perivitelline space 33 ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n µ¦¦oµ hatching envelope Á¦È¤¼¦r¹É ¦³°oª¥°´ÊÅoÂn ´Ê°Â¨³´ÊÄ ¦¼¸É 19a) ´Ê°¤¸ªµ¤®µ¦³¤µ 25-30 nm ¦³°oª¥Â¸Á o¤ ° electron dense °Â Â¥µ´oª¥Ân°ªnµ ° electron lucent ´ÊĦ³°oª¥´Ê¸µ ° electron dense ®µ¦³¤µ 50 nm ¦¼¸É 19b) ¦¼¸É 19. Transmission electron micrograph °Å n»o»¨µÎµ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n  hatching envelope ¸É¦oµÁ¦È¤¼¦r a) ¦³°oª¥´Ê° O) ¨³´ÊÄ I) b): HE, hatching envelope; PVS, perivitelline space 34 µ¦Á· hatching envelope µµ¦«¹¬µoª¥ gold-lectin labeling TEM Á¤ºÉ° incubate ´ª°¥nµ¸ÉÁ} thin section oª¥ gold-Con A ¡µ¦Áµ³· ° gold particles ¸É dense vesicles ¦¼¸É 20a) ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n¡ gold particles Áµ³·´¨»n¤ ° dense materials ¹É e ocytose °°¤µµ dense vesicles ¦³µ¥°¥¼n Ä perivitelline space ¨³Ä ³Á¸¥ª´¡ gold particles Áµ³·´´ÊÄ ° hatching envelope ¦¼¸É 20b, c) ´Ê° ° hatching envelope ¨³¸Éªnµ¦³®ªnµ dense materials Ťn¡ gold particles ¨³¸ÉÁª¨µ 1 ´ÉªÃ¤®¨´ªµÅ n¡ gold particles ¹ÊÅÁµ³·°¥¼n¸É ´ÊÄ ° hatching envelope ¦¼¸É 20d) 宦´¨»n¤ª»¤¸ÉÁ·¤ mannose ¨ÅÄ goldCon A n°µ¦ incubation ¡µ¦¥´¥´Êµ¦Áµ³· ° gold particles ¦¼¸É 20e) ÂÄ®o Á®Èªnµ dense vesicles ¤¸µ¦ e ocytosis Ä®oÁ} dense materials Á oµ¼n perivitelline space ¹Én°¤µÅo ¹ÊŦª¤´ hatching envelope ¨³¨µ¥Á}´ÊÄ ° hatching envelope 宦´ gold-WGA ¡µ¦Áµ³· ° gold particles ¸É flocculent vesicles ¦¼¸É 21a) ¹É e ocytose ¨n°¥µ¦¸É°¥¼n£µ¥ÄÁ oµ¼n perivitelline space ¦¼¸É 21c) ¨³¸É Áª¨µ 1 ´ÉªÃ¤®¨´ªµÅ n¡ gold particles ¹ÊÅÁµ³·°¥¼n¸É´ÊĨ³´Ê° ° hatching envelope ¦¼Á¨ÈĦ¼¸É 21c) 宦´¨»n¤ª»¤¸ÉÁ·¤ N-acetyl glucosamine ¨ÅÄ goldWGA n°µ¦ incubation Ťn¡µ¦Áµ³· ° gold particles ¦¼¸É 21b) µ¦ incubate ´ª°¥nµ thin section oª¥ gold particles ¸ÉŤnÅoÄn lectin Ťn¡µ¦Áµ³· ° gold particles ¸Éæ¦oµÄÇ 35 ¦¼¸É 20. £µ¡ TEM gold-Con A °Å n»o»¨µÎµ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n ¡ gold particles Áµ³·´ dense vesicles a) ¨³¨»n¤ ° dense materials ¸É°¥¼nÄ perivitelline space ¨³´ÊÄ ° hatching envelope b, c) n°¤µÁ¤ºÉ°Áª¨µ 1 ´ÉªÃ¤®¨´ªµÅ n¡ gold particles ¹ÊÅÁµ³·Á¡µ³´ÊÄ ° hatching envelope Ánµ´Ê d) 宦´¨»n¤ª»¤¸É Á·¤ mannose ¨ÅÄ gold-Con A n°µ¦ incubation ¡µ¦¥´¥´Êµ¦Áµ³· ° gold particles e); DM, dense material; DV, dense vesicle; HE, hatching envelope; PVS, perivitelline space 36 ¦¼¸É 21. £µ¡ TEM gold-WGA °Å n»o»¨µÎµ¸ÉÁª¨µ 30 µ¸®¨´ªµÅ n ¡ gold particles Áµ³·´ flocculent vesicles a) n°¤µ¡ gold particles ¸É flocculent vesicles ¸Éε¨´ e ocytosis ¨n°¥µ¦Á oµ¼n perivitelline space c) ¨³Á¤ºÉ°Áª¨µ 1 ´ÉªÃ¤®¨´ªµÅ n¡ gold particles ¹ÊÅÁµ³·¸É´ÊĨ³´Ê° ° hatching envelope ¦¼Á¨ÈÄ c) 宦´¨»n¤ ª»¤¸ÉÁ·¤ N-acetyl glucosamine ¨ÅÄ gold-WGA n°µ¦ incubation Ťn¡µ¦ Áµ³· ° gold particles b): FV, flocculent vesicle; HE, hatching envelope; PVS, perivitelline space 37 µ¦µ¸É 3. µ¦Á¦¸¥Á¸¥°r¦³° °nªnµÇ Ä hatching envelope °»o»¨µÎµ ¨³»o Sicyonia ingentis ´Ê ° hatching envelope ´Ê° ´ÊÄ Perivitelline space Sicyonia ingentis (Pillai and Clark, 1988 & 1990) N-acetyl glucosamine plus granular materials) mannose ? »o»¨µÎµ (µ¦¨°¸Ê) ? from granular materials) glucose/mannose N-acetyl glucosamine 38 4 Cortical vesicles FกF ก ก F ก กF S. ingentis กF ก cortical vesicles cytoplasm F F กF S. ingentis cortical vesicles 30 F F (Pillai and Clark, 1988) cortical vesicles กF ก F ก F F mature oocyte F F Kruevaisayawan (2010) cortical vesicles mature oocyte F กF vesicles (lightly stained vesicles) F (densely stained vesicles) ก F กF ก cortical vesicles F F flocculent vesicles dense vesicles F ก flocculent vesicles ก F large lightly stained vesicles mature oocyte dense vesicles ก F small densely stained vesicles mature oocyte (Kruevaisayawan et al., 2010) ก F ก S. ingentis ก cortical vesicles ก F FกF ก F ก ก cortical vesicles F F กF F F F F F กF lobster (Talbot and Goudeau, 1988) ก ก egg activation กF ก ก ก F F ก F ก (Pongtippatee et al., 2004) S. ingentis F ก egg activation 30-45 F (Pillai and Clark, 1988) ˈ FFก F F hatching envelope กF ก 1 F (Pongtippatee et al., 2004) F ˈ F F cortical vesicles F F F mature oocyte F ก ก ก ˆก F กF ก F F ก F กF S. ingentis 2 F กF ก F ก ˆก F ˈ 12-13 ก F กF F (Hall et al., 1999) กF S. ingentis F 24 ก ˆก F (Hertzler and Clark, 1992) ก F ก ˆก F ก ก egg activation กF ก F กF ก ก F กF S. ingentis ˈ F ก F ก F cortical vesicles ก F hatching envelope ก ก membranous structures granular materials F F F F ก F กก ก F TEM ก F ก ก F ก ก F ก F granular materials ก F membranous structures F membrane granular materials F ก ก 39 ก ก F ˈ F ˈ F ก hatching envelope Fก F F F ˈ F ก hatching envelope ก ก ก F ก hatching envelope F กF ก ก F granular materials F F กF S. ingentis granular materials ˈ F surface coat ˈ template hatching envelope (Pillai and Clark, 1988) F granular materials กF S. ingentis F F ก กF ก F F granular materials F Strongylocentrotus purpuratus ก vitelline envelope (Cheng et al., 1991) S. ingentis F F surface coat (template hatching envelope) ก exocytosis ก ก dense vesicles F กF N-acetyl glucosamine sialic acid ก ˈ hatching envelope ก F ก กก ก F TEM F F F gold-Con A labeling ก ก F fluorescent hatching envelope ˈ FF F ˈ hatching envelope F F Pillai and Clark (1988) ก F F ก hatching envelope S. ingentis F ก ก surface coat ก ก dense vesicles ˈ N-acetyl glucosamine Fก ก Pillai and Clark (1990) ก F ก F gold-WGA (N-acetyl glucosamine binding) labeling ก hatching envelope F F F F ก hatching envelope S. ingentis F ก N-acetyl glucosamine F F ก กก F hatching envelope กF ก F exocytosis ก ก dense vesicles ˈ glucose/mannose ( F F Con A) ( LCA F mannose ก F ก F F F ก ) ก ก F S. ingentis ก Pillai and Clark (1990) F F LCA (mannose) F ก ring vesicles ( flocculent vesicles ก ) F F ก ring vesicles F F hatching envelope S. ingentis ก hatching envelope ˈ F ก F ˈ glucose/mannose กF ก S. ingentis F F vesicles ก ก exocytosis F กF ก ก F ก กF F กก F flocculent vesicles ก F F F perivitelline space Fก ก ก hatching envelope (hatching envelope elevation) F ก fluorescent TEM ˈ gold-WGA labeling กF F F F ก N-acetyl glucosamine F ก ก hatching envelope ก 40 exocytosis flocculent vesicle F perivitelline space กก ก F fluorescent ก perivitelline space F 30 F ก ˈ F hatching envelope ˈ FF ก N-acetyl glucosamine ก hatching envelope ก F FกF F F ก ก F ก perivitelline space F F ก ก F กก ก S. ingentis Pillai and Clark (1988 & 1990) ก ก ก กก F ก ก F F ก ก ก F hatching envelope กF ก ก กF S. ingentis (Pillai and Clark, 1988 & 1990) F 3 cortical vesicles F F S. purpuratus F ก ˈ β-1, 3- glucanase ovoperoxidase F ก cross-link F F ก F hatching envelope (Cheng et al., 1991) ก F ovoperoxidase ˈ glycoprotein mannose N-acetyl glucosamine ˈ F ก (Deits et al., 1984) ก กF F flocculent vesicles dense vesicles FกF ก ก F F F vesicles F กF F 41 5 F ก F hatching envelope ˈ egg activation กF ก ก ก F กก ก F FกF ก ก F hatching envelope ก membranous structures granular materials F 2 F ก F ˈ hatching envelope ก ก กF F F 1 ก cortical vesicle ก 2 flocculent vesicle dense vesicle exocytosis dense vesicles F ก ˈ mannose/glucose exocytosis กF F dense materials F ก hatching envelope F hatching envelope ก ก F hatching envelope flocculent vesicles F ก ˈ N-acetyl glucosamine F ก ก hatching envelope ก ก ก 30 F 42 ก F Bleil, J.D. and Wassarman, P.M. 1980a. 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Global aquaculture advocate. 7(2): 88-89. Yano, I. 1988. Oocyte development in the Kuruma prawn Penaeus japonicus. Mar. Biol. 99: 547-553. 47 ก 48 ก 1. 5% uranyl acetate F Uranyl acetate 5.0 g 70% methanol 100 mL ก 1. uranyl acetate 5.0 g F ก ก F 2. 70% methanol F ก F F F 100mL 3. F ก F ก ก F 4 C F F 2. 2% uranyl acetate F Uranyl acetate 2.0 g 70% methanol 100 mL ก 1. uranyl acetate 2.0 g F ก ก F 2. 70% methanol F ก F F F 100mL 3. F ก F ก ก F 4 C F F F F F1 F F F ก ก F F1 F F F ก ก F 49 3. 50% ethyl alcohol F 95% ethyl alcohol 500 ml Distilled water 450 ml ก 1. 95% ethyl alcohol 2. ก 450 ml F F 500 ml F ก F F 950 ml ก F F F 4. 70% ethyl alcohol F 700 ml 250 ml 95% ethyl alcohol Distilled water ก 1. 95% ethyl alcohol 2. ก 250 ml F F 700 ml F ก F F 950 ml ก 5. embedding mixture F Mixture 1 - EMBed-812(EMS, PA) 5 ml - DDSA (EMS, PA) 8 ml Mixture 2 - EMBed-812 8 ml - NMA (EMS, PA) 7 ml Final embedding mixtures: - mixture 1 13 ml - mixture 2 15 ml - DMB 30 (EMS, PA) 0.56 ml F F F 50 ก 1. 2. 3. 4. F EMBed-812 F EMBed-812 5 ml ก DDSA 8 ml ก NMA F 1 DMB 30 F 2 0.56 ml 8 ml F Fก F Fก 7 ml F Fก F F Fก F F F ก F F 6. lead citrate F Lead nitrate (AnalaR, England) 1.33 Sodium citrate (Electron Microscopy Science, PA) Distilled water 30 mL ก 1. ก 30 mL F F (flask) 2. F lead nitrate 1.33 g 3. sodium citrate 1.76 g F Fก 5-10 ) F F F ˈ 4. F 1 N NaOH 5. F F ก F1 ก F ก ก F F 7. mesh cement F 50% ethanol (J.T.Baker, Malaysia) 95 mL Acetic acid (J.T.Baker, USA) 5 mL ก 1. 50% ethanol 95 mL F ก 2. acetic acid 5 mL F F ก g 1.76 g F Fก F F ( F F F F 30 8 mL F F 50 mL ก 0-4 °C F ʽ F 4 F F Fก ก F F F F 51 8. 4% osmium tetroxide F 1.0 g Osmium teroxide (OsO4) Distilled water 25 mL ก 1. F กF (ampule) OsO4 F F F F ก F ก F F F F ก F 2. F กF F ก F F 3. กF F F 2 F ก F ก (reagent bottle for OsO4) 4. ก ( F ก F ก ก ก F) 25 mL F F OsO4 F 4% OsO4 5. F ก F 1% OsO4 F ก 4% OsO4 F 0.1 M PBS F 1:3 F 4°C F 4% OsO4 F ก F F F ก 1% OsO4 F 9. 8% paraformaldehyde F Paraformaldehyde (EMS, PA) 10.0 g Sodium dihydrogen phosphate (NaH2PO4) 5.3 g 50% sodium hydroxide (NaOH) 1.8 mL Distilled water 125 mL ก 1. ก 100 mL F ก ก F 2. F paraformaldehyde 10.0 g NaH2PO4 5.3 g 3. F Fก F F F F C 4. F 50% NaOH F 5. F pH F F 7.2 F ก F F 6. F ก F ก ก F1 ก F F 0-4 C F F 60 125 mL 52 10. 0.1 M Phosphate buffer saline pH 7.2 F Solution A : sodium phosphate monobasic 27.6 g ก F F 1000.0 ml Solution B : sodium phosphate dibasic 28.4 g ก F F 1000.0 ml ก 1. F F ก F solution A : solution B = 23 : 77 2. pH F F 7.2 F ก F 1:1
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