DEVELOPMENTAL
BIOLOGY
46, 404-412 (1975)
Heterogeneous Distribution of “Lysosomal” Hydrolases in Yolk Platelets Isolated from Unfertilized Sea Urchin Eggs by Zonal Centrifugation l HERBERT
SCHUEL.
WALTER
L. WILSON.
JEAN
R. WILSON.
AND ROBERT
S. BRESSLER
Department of Biochemists, Downstate Medical Center, SLTNY, Brooklyn. N. Y. 11203; Biology Department. Oakland University, Rochester, Michigan, Pathology Laboratory, Pontiac General Hospital, Pontiac, Michigan; Department of Anatomy, Mt. Sinai School of Medicine, CUNY, New York, N. Y.; and the Marine Biological Laboratory, Woods Hole, Massachusetts Accepted
May 20. 1975
Three typical “lysosomal” glycosidases, a-L-fucosidase. N-acetyl glucosaminidase and Nacetyl galactosaminidase. were localized within the yolk platelets of unfertilized Strongylocentrotus purpuratus eggs. Homogenates of eggs were fractionated by rate-zonal centrifugation, and the isolated particles were subjected to integrated biochemical and morphological (electron microscopic) analysis. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase). and cortical granules (B-l.3 glucanase) in the sucrose density gradient. Yolk platelets were isolated in a high state ot purity, with contamination by mitochondria and cortical granules at trace levels. Enzymatic heterogeneity exists within the yolk platelet population. Acid nitrophenyl phosphatase and a-L-fucosidase activities appear to be uniformly distributed within all the yolk platelets. while N-acetyl glucosaminidase and galactosaminidase activities appear to be preferentially distributed within the slower sedimenting sub-population of yolk platelets. Another band of hexosaminidase containing particles sedimented slightly slower than the bulk of the yolk platelets. coincident with the mitochondria. The acid hydrolases packaged in the yolk platelets may participate in the mobilization of yolk material after fertilization. The yolk platelet thus appears to be a highly complex and structured “lysosome-like” storage organelle. INTRODUCTION
from eggs by the discharge (exocytosis) of the cortical granules at fertilization promotes the establishment of the block to polyspermy (Epel. 1975; Gwatkin et al., 1973; Longo and Schuel, 1973; Longo et a/., 1974; Schuel et al.. 19’73; Vacquier et al.. 1973). The cortical granules and yolk platelets in eggs exhibit many structural and functional characteristics usually associated with lysosomes in adult tissues, and the! have been characterized as “lysosomelike” organelles for this reason (Allison. 1969; Bluemink, 1970; Dore and Cousineau, 196T: Pasteels, 19’i3: Schuel et al., 1969 and 19’iZb). Mucopolysaccharides and other glycoproteins are known to be biochemical constituents of the yolk platelets. cortical granules. and the extra-cellular investments of the eggs of sea urchins and other organisms (Dauwalder et al.. 1972:
Mature unfertilized eggs contain two distinct species of sub-cellular organelles which are assembled from derivatives of the Golgi apparatus during oogenesis, the yolk platelets and the cortical granules (Anderson, 1968; Beams and Kessel. 1968; Dauwalder et al., 1972; Norrevang, 1968). The yolk platelets are storage granules for metabolites required during embryogenesis (Kavanau, 1954). The cortical granules are true secretory organelles (Schuel et al., 19’72b). The secretory products released ‘This investigation was supported in part by grants from the American Cancer Society (No. P-616) and the SUNY Research Foundation (No. 7125A). and the Population Council (No. MT4.034Cl by a research contract from the National Institute of Child Health and Human Development (No. 69-2202). and by a Research Career Development Award (No. 5K4CA-8606) from the National Cancer Institute to H. Schuel. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
SCHUEL et al.
L.wosomal
Epel, 1975; Immers. 1960; Runnstrom, 1966; Schuel et al., 1974; Tyler and Tyler, 1966). Lysosomes in adult tissues contain a variety of glycosidic enzymes which hydrolyze mucopolysaccharides (Baggiolini, 1972; Barrett, 1972; Clarke, 19651. Previous studies have shown that the typical lysosoma1 enzyme acid phosphatase is localized within the yolk platelets of unfertilized sea urchin eggs, while p-1.3 glucanase is found in the cortical granules (Schuel et al., 1972bl. The experiments described in the present communication were conducted to elucidate the enzymatic properties of the cortical granules and yolk platelets in order to understand the functions of these organelles at fertilization, by determining the subcellular distribution of several typical glycosidases capable of de“lysosomal” (Barrett, grading mucopolysaccharides 1972) in unfertilized sea urchin eggs. A preliminary report of part of this study has been presented previously (Schuel et al.. 1972aJ. METHODS
Eggs obtained from the sea urchin Strong>,locentrotus purpuratus were used in this study. The animals were supplied by the Pacific Bio-Marine Supply Co., Venice, CA. Homogenates of unfertilized eggs were prepared and then fractionated in the type A-XII zonal centrifuge (Anderson et al., 1966: Cline and Ryel, 1971) as described previously (Schuel et al., 1972b3. The total force applied to the subcellular particles during centrifugation was monitored in terms of the time integral of the rotor’s angular velocity (w* .t) using the I. E. C. model 637lA electronic digital speed squared integrator (Cline and Ryel. 1971). The fractions recovered from the zonal centrifuge were analyzed biochemically. Automatic procedures described previously (Schuel et al., 1964; 1968; 1969; 1972b) were used to determine the distribution of subcellular particles (U. V. absorbance at 254 nm), protein, mitochondria (enzymatic marker:cytochrome oxidase), yolk platelets
Hvdrolases
in Yolk Platelets
405
(enzymatic marker:acid nitrophenyl phosphatase), and cortical granules (enzymatic marker: p-1.3 glucanasel. The sucrose concentration in the isolated fractions was measured with a refractometer (Schuel et al., 1969). Automated procedures. originally employed to study rat liver lysosomes (Schuel et al., 1968; Tappel. 19721. were adapted to measure the distribution of several typical “lysosomal” hydrolases (Barrett, 1972) in the fractions isolated from sea urchin eggs (Fig. 1 and Table 1). The following enzymes were assayed using nitrophenpl ester substrates: acid phosphatase, CI-L-fucosidase, N-acetyl glucosaminidase, and Nacetyl galactosaminidase. The non-ionic detergent Turgitol NPX (Schuel and Anderson, 1964) was included in the enzyme incubation media as indicated in order to measure the total hydrolase activities present in the egg homogenates and the isolated subcellular fractions. The morphology of the particles in the fractions isolated by zonal centrifugation was examined under the electron microscope as described previously (Schuel et al., 1969 and 1972b). RESULTS
The results of a typical experiment involving fractionation of unfertilized sea urchin eggs by zonal centrifugation is shown in Fig. 2. The sample zone in fraction No. 3 was sharply indicated in terms of the high concentration of protein which did not sediment into the gradient (Fig. 2al. Mitochondria (enzymatic marker:cytochrome oxidasel were distributed in the gradient just beyond the area occupied by the slowly sedimenting microsomal particles, with the center of the peak in cytochrome oxidase activity located in fraction No. 10. Yolk platelets (enzymatic marker:acid phosphatase) were distributed primarily in the middle region of the sucrose gradient, with the center of this symmetrical peak in fraction No. 14. Pure preparations of cortical granules (en-
406
DEVELOPMENTAL Bro~oc\
zymatic marker: p-1.3 glucanasei were present in the most rapidly sedimenting band of particles, which accumulated at the interface between the heavy end of the gradient and the cushion of 55(r, sucrose P_OPORTIONING PUMP
FIG. 1. Schematic diagram of automated assay system for lysosomal hydrolases utilizing modules of the Technicon Autoanalyzer and Beckman DB spectrophotometer. Flow rates are based on specifications provided by Technicon Instruments Corp. for the manifold tubing on the proportioning pump module. Details of the conditions for the enzyme assays using nitrophenyl ester substrates are presented in Table 1.
VOLCIME 16. 1975
(Schuel et al., 19Eb). Another peak of p-1,3 glucanase activity was also associated with the slowly sedimenting microsomal particles. The lysosomal glgcosidases were distributed in the middle region of the gradient occupied by the yolk platelets (Fig. 2bl. The distribution pattern for a-L-fucosidase was very similar to that observed for acid nitrophenyl phosphatase. N-acetyl glucosaminidase and galactosaminidase activities were distributed primarily within the slower sediment ing population of yolk platelets, peaks in fraction No. 12. Moreover, the hexosaminidase activities did not show a normal Gaussian distribution, since a shoulder on the curve indicated the presence of another slightly slower sedimenting band of particles containing these glycosidases in the gradient between the microsomal and main yolk regions, coincident with the mitochondria. This bimodal distribution pattern for hexoaminidase activities is even more striking when the data are
TABLE ASSAY CONDITIONS Enzyme
Phosphatase
a-r.-fucosidase
N-acetyl-glucosaminidase
N-acetyl-galactosaminidase
1
FOR AUTOMATED DETERMINATION OF ACID HVDROL~SE ACTKITIEL FRACTIONS LISINC. NITROPHENI L ESTER SLWSTRATE~
Substrate
Reagent linesb buffer-detergent
Color de\reloper
Buffer
PH
Trigitol NPX (5 V/V)
p-Nitrophenyl phosphate ( 10 mM)
Na-Acetate
5.0
0.1
p-Nitrophenyl a+fucoside (5 mh4
Na-Citrate
p-Nitrophenyl N-acetyl-Doglucosaminide (5 mM)
Na-Citrate
p-nitrophenyl N-acetyl-fir)galactosaminide (5 mM)
Na-citrate
(0.1 M)
(0.2Ml
3.0
0.5
’ 4.0
0.5
(0.2 M 1.0
IN SEA URCHIN
0.5
(0.2 Ml
a Assay conditions used with automated manifold shown in Fig. 1. DConcentration of reagents shown for lines entering automated assay system.
NaOH
Etc.
Bath (coil)
1J-Length
(0.2 Ml
Single
NH,OH
Full-length
(2.0 M)
Double
NH,OH
Full-length
(2.0 M)
Double
NH,OH
Full-length
(2.0 Ml
Double
SCHL~ELet al.
L.w~somal
407
H.vdrolases in Yolk Platelets
a IF 1
iSd -
FR>CTlShlS
i’.
b
,wm,,
Fro. 2. Fractionation of homogenate of unfertilized sea urchin (S. purpuratus) eggs by zonal centrif’ugation in type A-XII rotor at 3500 rpm for 60 min at 5°C’. (w”.t = 5.2069 x lOa * 0.0985 8 10*1. Sucrose density gradient constructed in 0.5 A4 KCI. 10m3 M EDTA at pH 7.0. A. Distribution of subcellular components (absorbance at 254 nm). protein, cytochrome oxidase. and 8-1.3 glucanase activities in gradient. B. Distribution of acid nitrophenyl phosphatase, o-L-fucosidase, N-acetyl glucosaminidase, and N-acetyl galactosaminidase activities in gradient. C. Distribution of specific activities for acid nitrophenyl phosphatase, a-t.-fucosidase, N-acetyl glucosaminidase. and N-acetyl palactosaminidase activities in gradient.
presented in terms of specific activity (Fig. 2~). The peaks for specific hexosaminidase activities were found in fractions No. 9-10, with a prominent shoulder also present in the main yolk region of the gradient. The specific activities for acid nitrophengl phosphatase and a-L-fucosidase, on the other hand, both showed simple normal distribution patterns in the middle region of the gradient occupied by the yolk platelets. An estimate was also made of the distribution of the biochemical constituents in the egg homogenate in various regions of the gradient (Table 2). For the purpose oi this calculation, the gradient was divided on the basis of the inflection points in protein concentration between the peaks in distribution of subcellular components (Schuel et al.. 1973). Approximately 65% of
the total egg protein was associated with particles that sedimented beyond the starting sample zone in the gradient, while 85-95Y of all the hydrolase activities studied were sedimentable under the low-speed centrifugation conditions employed. The yolk platelets contained about 30’+ of the total egg protein, while about 70-80% of the acid phosphatase and lysosomal glycosidases were in the yolk region of the gradient. The cortical granule fractions contained about 7%’ of the egg protein, and about 300; of the total p-1,3 glucanase activity. Approximately 5O’“r of the total p-l,3 glucanase activity was distributed between the yolk and cortical granule fract ions. In these experiments essentiall> 100%’ of all these biochemical constituents in the egg homogenate originally introduced into the zonal rotor was recovered in
408
DEVELOPMENTAL BIOLOGY TABLE
VOLUME 46, 1975 2
DISTRIBL~TIONAND RECOLEHY OF BIOCHEMICAL CoNsrrr~%vrs IN HOMOGENITES OF UNFERTILIZED SEA URCHIN EGGS (S. purpuratus) FRACTIONATED B\ ZONAL CENTRIFI.GI\TION” Percent ot total in gradient
Biochemical constituent
Recovery (percent)
Non-Sed. (soluble,
Sedimentable
Yolk
Cortical granules
Protein I14 exps)
35.3 * 4.5
64.; i 4.5
31.5 * 3.5
7.1 * 2.9
98.7 zt 19.8
Acid phosphatase (14 exps)
7.8 I 1.9
92.” * 1.9
71.1 * 2.7
3.4 * 1.1
99.2 * 18.5
a-L-fucosidase exps)
16
5.2 + 2.2
94.8 * 2.2
83.0 3: 2.3
3.0 3: 1.7
105.7 =t 5.9
N-acetgl-glucosaminidase (6 exps I
9.2 + 1.6
90.8 rt 1.5
68.6 * 3.6
3.7
3.3
104.5 Lt 11.1
N-acetylgalactosaminidase (6 exps)
8.6 * 2.1
90.9 * 2.7
69.9 * 3.9
3.1 * 2.2
104.6 + 7.7
j3-1,3 glucanase (13 exps)
15.1 * 1.7
84.9 k 1.7
15.8 :t 3.7
33.1 & 2.7
98.2 * 18.2
l
DConsult text for details concerning the division of the gradient into specified regions. Data calculated show mean (kfJ values with standard deviation I +-SD) obtained in series of experiments.
the gradient after centrifugal fractionation. Subcellular organelles separated by zonal centrifugation were examined in the electron microscope. A sample taken from fraction 14 (see Fig. 2, above) contained an almost completely pure suspension of yolk platelets, with a trace level of contamination by mitochondria and cortical granules (Fig. 3). The distribution and ultrastructure of organelles which sedimented slower vesicles, mito( ribosomes, microsomal chondria, etc.) or more rapidly (cortical granules) than the yolk platelets were similar to that reported in previous studies, and the mitochondrial region of the gradient was contaminated by yolk platelets and microsomal vesicles (Schuel et al., 1969; 1972b). These observations also showed that yolk platelets were ubiquitously distributed through the entire sucrose density gradient, and even contaminated the cortical granule fractions at trace levels. DISCUSSION
The observations described above provide direct biochemical evidence for the
to
distribution of a-L-fucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase activities within the yolk platelets of unfertilized sea urchin eggs. Furthermore, since these glycosidases are not constituents of the cortical granules. it is unlikely that they act upon the acid mucopolysaccharides present in these secretory granules (Schuel et al., 1974) or in the extra-cellular investments of the egg (Immers, 1960; Runnstrom, 1966: Tyler and Tyler, 1966) during fertilization. Ultra-structural observations indicated that yolk platelets were isolated from homogenates of unfertilized sea urchin eggs in a high state of purity by rate zonal centrifugation. The center of the yolk peak was contaminated by mitochondria and cortical granules at trace levels. These morphological observations thus probably account for the low levels of cytochrome oxidase (enzymatic marker for mitochondria) as well as B-1,3 glucanase and tryptic protease (enzymatic markers for cortical granules) activities detected within this region of the density gradient (Schuel et
al., 19’i”b and 2973). However. significant numbers of yolk platelets were also distributed through the entire gradient, and were present at trace levels only in the center of the cortical granule peak rSchuel et al.. 1969; LS’i’rb 1. The quantitative biochemical data oh-
FIG. :$. Electrm~ experiment depicted level contamination
that tained in the present study indicate the lrolk platelets contain about 30$ of’ the total egg protein. while the cortical granules contain ahout iC; The former is consistent with morphometric estimates of the percent of the egg volume occupied b\y yolk (Costello. J.%EJ; Harye!.. I932), while the
micrvgraph ot’ pellet obtained from the center ot the yolk peak ~haction 141 [tom the in Fig. 2. This trarticm contains a very puresu~pension ot intact yolk platelets. with a trace by mitochondria (arrow) and cortical granules tarrorr’ head). Bar, 1 bm x 14,4~0.
410
DEVELOPMENTAL BIOLOGY
later (Schuel et al., 1972b) is consistent with estimates of the percent of total egg protein released into the ambient sea water following discharge (exocytosis) of the cortical granules (Bryan, 1970). Furthermore, all the hydrolases studied appeared to be associated with sedimentable particles. The low levels of hydrolase activities remaining in the startmg sample zone (soluble phase) following centrifugation suggest that these cytoplasmic organelles were not damaged during the homogenization and fractionation procedures. The data also support suggestions made by previous investigators (Gross et al., 1960; Krischner and Chambers, 1970) that sea urchin eggs contain more than one population of yolk platelets. Acid nitrophenyl phosphatase and cY-L-fucosidase appear to be evenly distributed within all the yolk platelets, while N-acetyl glucosaminidase and N-acetyl galactosaminidase appear to be preferentially distributed within the slower sedimenting subpopulation of yolk platelets. These conclusions are supported by morphological observations which showed that the center of the acid phosphatase peak contained virtually a pure suspension of yolk platelets. Thus, no particle appeared to be present in sufficient numbers in this region of the gradient to account for the observed high levels of hexosaminidase activities, ot.her than the yolk platelets. A second band of hexosaminidase activity was found associated with particles which sedimented slightly slower, coincident. with the mitochondria. Yolk platelets are also observed at this level of the gradient (Schuel et al., 1969), which may reflect the presence of yet another sub-population of yolk platelets. However, insufficient data are curestablish to clearly rently available whether this slower sedimenting band of hexosaminidase activities is associated with the yolk platelets, mitochondria, or some other small cytoplasmic particle. In addition the number of enzymes which may be responsible for the observed N-
VOLUME 46, 1975
acetyl hexosaminidase activities has not yet been determined. Additional work is required to answer these questions. The presence of acid nitrophenyl phosphatase and several typical “lysosomal” glycosidases in yolk platelets of unfertilized sea urchin eggs provides additional support for the hypothesis (Bluemink. 1970’ Pasteels, 1973) that these organelles are “lysosome-like” storage particles. Lysosomes in adult cells actually are a specialized group of secretory organelles which function in intra-cellular digestion (DeDuve, 1969; Weissmann, 1973). They normally fuse with and discharge their hydrolases into the lumen of phagocytic or other membrane limited cgtoplasmic vacuoles. Yolk platelets are formed during oogenesis by fusion of Golgi derived vesicles with maccontaining endocyt ic vacuoles romolecules to be stored as nutrients for embryogenesis (Norrevang, 1968; Wallace and Bergink, 1974). However, degradation of these stored metabolites begins after fertilization (Kavanau. 1954). Similarly, since the cortical granules discharge their contents extra-cellularly. they are more akin to typical secretory granules in adult cells, ie., zymogen granules in exocrine cells, hormone-storage granules in endocrine cells, heparin granules in mast cells, etc. (Long0 and Schuel. 1973: Schuel et al.. 1973). The biological function of the cortical granule b-1,3 glucanase is uncertain at present, although Epel (197.5) has proposed that it might act upon the hyaline layer protein in the perivitelline space after fertilization. The yolk platelet appears to be a complex and highly structured organelle. A variety of glycoproteins, mucopolysaccharides, and glycolipids are known to be stored in yolk platelets of sea urchins and other organisms (Dauwalder et al., 1972; Immers, 1960; Kondo, 1972; Perlman et al., 1959; Schjeide and Urist, 1959). The results of the present study indicate that glycosidases capable of degrading these substrates are also constituents of sea ur-
SCHUEL et al.
Lysosomal
Hvdrolase
in Yolk Platelets
411
BEAMS. H. W., and KESSEL. R. G. (1968). The Golgi apparatus: structure and function. Int. ReLl. Cxtol. 23, 209-276. BLUEWICK. J. G. i1970). Are yolk granules related to lysosomes? Zeiss Info. 73, 95-99. BRYAN. ,J. (1970,. The isolation of a major structural element ofthe sea urchin fertilization membrane. J. Ce!l Eiol. 15, 606-614. BULL, A. L., and CHESTERS, C. G. C. (1966). The biochemistry of laminarin and the nature of laminarinase. Ado. Enzymol. 28, 325-364. CARDABIS. C.. SCHCIEL. H.. and HERMAN. L. (197-I) Ultrastructural localization of calcium in unfertilized sea urchin eggs. J. Cell Biol. 63, -I9a. CLARKE, A. E. (19651. Hydrolytic enzymes of human mlymorphonuclear leukocytes and rat monocytes. Amt. J. Exp. Bid. Med. Sci. 43, 201-“12. CLINK, G. B., and RYEL. R. B. (19711. Zonal centrifugation. Methods Enzvmol. 22. 168-XL4. COSTELLO. D. P. i 19.19,. The volumes occupied by the formed cytoplasmic components in marine eggs. PhMiOl. Zool. 12. 13-20. DAI.IVAI.DER. M.. ~\‘HAI.EI.. LV. G., and KEPHART, .J. E. (1972). Functional aspects of the Golgi apparatus. Sub-Cell. Biochem. 1, 225-275. DED~IVE. C. (19691. The Iysowme in retrospect. In. “Lysosomes in Biology and Patholog?.” ~Dingle. d. T. and Fell, H. B., eds.). Vol. I., pp. Z-42, U:iley. N. Y. DORE. D.. and COCMNEA[I, G. H. (1967). .4cid phosphatase analysis in sea urchin eggs and blastulae. Exp. Cell Res. 18, 179-18‘1. EPEI.. D. ( 1975). The program of and mechanisms 01 fertilization in the Echinoderm egg. Am. Zoo/., in press. EPEL, D.. I~EALER. A. hf.. hfVCHMORE. A. \r.. and SCHMKF. R. T. (1969). P-l.3 glucanase of sea urchin eggs:release from particles at fertilization. REFERENCES Science 163, 294-296. GROSS, P. R. (19541. On the mechanism of the ~~LLlsON. .A. c. 119691. Lysosomes and cancer. In. yolk-lysis reaction. Protoplasma 43, 416-428. “Lysosomes in Biology and Patholoe” (Dingle. .J. GROSS. P. R.. PHILPOTT, D. E.. and NASS, S. (1960). T. and Fell. H. B.. eds.). \Tol. II. pp. 1X-206. Electron microscopy of the centrifuged sea urchin iViley. N. Y. ANDERSON, E. I 19681. Oocyte differentiation in the sea egg with a note on the structure of the ground cytoplasm. J. f!io&vs. Biochem. C,vtol. 7. 1:X-1-12. urchin, Arbacia punctulata, with particular referGWATXIY, R. B. L.. WILLI.AMS. D. T.. HARTMAN. J. F.. ence to the cortical granules and their participation and KNIAZ~:K, bl. (19731. The zona reaction of in the cortical reaction. J. Cell Biul. 37, 511-539. hamater and mouse eggs: production in L’itro by a ANDERSON, N. G.. BARHINCER, H. P., B.USEI.AY,E. F.. trypsin-like protease from cortical granules. J. Re NKNI.EY. C. E.. BARTRLX hl. J., FfsfiER. W. D.. and prod. Fert. 32, 259-f65. RANKIN. C. T. 119661. The development of 101~. speed “A” series zonal rotors. Nat. Cancer Inst. HARVEY, E. N. (1932). Physical and chemical conMonogr. 21, 113-136. stants of the egg of the sea urchin. Arbacia puntBACGIOLINI, M. ( 19721. The enzymes of’ the granules ot tulata. Biol. Bd/. 62, 141-15.4. polymorphonuclear leukocytes and their functions HEILBRC~NN.L. V. (1956). “The Dynamics of Living Enqvme 13, 132-160. Protoplasm.” i\cademic Press, N. Y, BARREL‘T.A. d. I 19Z i. Lysosomal enzymes. In “LysoHULTIN. T. (1950). On the acid formation. breakdown somes. .4 Laboratory Handbook.” (Dingle. d. T.. of cytoplasmic inclusions, and increased viscosity in ed.i. pp. 16-135. Amer. Elsevier Publ. Co.. N. Y. Paracentrotus egg homogenates after the addition
chin yolk platelets. A similar situation has been described for the latent storage of phosphoprotein phosphatase and its subwithin amphibian yolk strate, vitellin. platelets (Nass. 1956). These observations provide the basis for posing additional questions. First. what mechanism prevents the hydrolases from attacking their substrates while packaged within the same storage granule for an extended period of time? Second. what mechanism activates hydrolysis and mobilization of stored yolk materials after fertilization? Similar questions could be asked about the storage of p-1.3 glucanase and its postulated substrate. hyaline. which are both packaged within the cortical (secretory) granules of unfertilized sea urchin eggs (Epel. 1975: Schuel et al.. 1972bi. Yolk mobilization and other metabolic processes may be activated at fertilization by an increase in the concentration of free calcium ions in the cytoplasm of the egg (Epel, 1975; 1956; Hultin. Gross, 1954; Heilbrunn, 1950; Mazia, 1937; Nass, 19561, resulting from the displacement of calcium ions from intra-cellular binding sites (Steinhardt and Epel. 1974 located in the plasma membrane as well as the membranes of the yolk platelets and cortical granules (Cardasis et al.. 1974).
412
DEVELOPMENTAL BIOLOI;Y
of calcium. Is’xp. Cell Res. 1, 272-283. IMMERS, J. (1960). Studies on cytoplasmic components of sea urchin eggs stratified by centrifugation. Exp. Ceil Res. 19, 499-514. KA~ANAU, J. L. (1954). Amino acid metabolism in the early development of the sea urchin Paracentrotus lividus. Exp. CelI Res. 7, 530-557. KONDO, H. (1972). Properties of glycoprotein particles and their changes during development of sea urchin eggs and embryos. Exp. Cell Res. 72, 51953’2. KRISCHNER, K. N., and CHAMBERS. E. L. (1970). Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization. J. Cell. Physiol. 76, 23-36. LONCO, F. J., and SCHUEL, H. (1973). An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata. Develop. Biol. 34. 187-199. LONCO, F. J.. SCHUEL, H.. and WILSON, W. L. (1974). Mechanism of soybean trypsin-inhibitor induced polyspermy as determined by an analysis of refertilized sea urchin (Arbacia punctulata) eggs. Develop. Biol. 41, 193-201. MAZIA, D. (1937). The release of calcium in .4rbacia eggs on fertilization. J. Cell. Comp. Phvsiol. 10, B-304. NASS, S. (19561. Phosphoprotein phosphatase of amphibian yolk. Trans. N. Y. Acad. Sci. 19, 118-129. NORREVANG.A. (1968). Electron microscopic morphology of oogenesis. Int. Rev. Cytol. 23, 114-186. PASTEELS.J. J. (1973). Yolk and lysosomes. In. “Lysosomes in Biology and Pathology,” (Dingle. J. T., ed.1. Vol. III, pp. 216-234, Wiley, N. Y. PERLMAN. P., BOSTROM, H., and VESTERMARK, A. (1959). Sialic acids in the gametes of the sea urchin. Exp. Cell Res. 17, 439-446. RUNNSTROM. J. (1966). The vitelline membrane and cortical particles in sea urchin eggs and their function in maturation and fertilization. Adi’. Morphopen. 5, 221-325. SCHJEIDE. 0. A.. and URIST, M. R. (1959). Proteins and calcium in egg yolk. Exp. Cell Res. 17, 84-94. SCHUEL, H.. and ANDERSON, N. G. (1964). Studies on isolated cell components. XVI. The distribution of acid phenyl phosphatase activities in rat liver brei fractionated in the zonal ultracentrifuge. J. Cell Bio[. 21, 309-323. SCHUEL, H., TIPTON, S. R., and ANDERSON, N. G. (1964). Studies on isolated cell components. XVII. The distribution of cytochrome oxidase activity in rat liver brei fractionated in the zonal ultracentri-
VOLL’ME 46, 1975 fuge. J. Cell Biol. 22, 317-326. SCHUEL, H.. SCHUEL. R., and UNAKAR, N. J. (19681. Separation of rat liver lysosomes and mitochondria in the A-XII zonal centrifuge. Anal. Biochem. 25, 146-163. SCHUEL, H., WILSON, W. L., WILSON. J. R.. and SCHUEL. R. (1969). Separation of intact cortical granules from homogenate of unfertilized sea urchin eggs by zonal centrifugation. J. Histochem. C,~to&em. 17, 703-713. SCHUEL, H.. LORAND. L.. CHEN. K., WILSON, IV. L., BRESSLER,R. S., and WILSON, J. R. (1972a). Distribution of acid hydrolases in unfertilized sea urchin eggs. J. Cell Biol. 55, 232a. SCHUEL, H., WIISON. W. L., BRESSLER,R. S.. KELLY, .I. W.. and WILSON. J. R. (1972b). Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation. Decelop.
Biol. 29, 307-320. SCHUEL, H., WILSON, W. L.. CHEN. K.. and LORAND. L. (1973). A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization. Deuelop. Rio/. 34, 175-186. SCHUEL. H.. KELLY, J. I%‘., BERGER. E. R., and WILSON, W. L. (1974). Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization. Exp. CelI Res. 88, 24-30. STEINHARDT, R. A.. and EPEL. D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sri. 71, 1915-1919. TAPPEL. A. L. (1972). Automated methods for measuring lysosomal enzymes. In, “Lysosomes, A Laboratory Handbook,” (Dingle, J. T., ed.). pp. 136-149, Amer. Elseiver Publ. Co., N. Y. TYLER A., and TYI.ER, B. S. (1966). The gametes; some procedures and properties. In, “Physiology of Echinodermata,” (R. A. Boolootian. ed.). pp. 639-682. Interscience. N. Y. VACQUIER, V. D., TECNER, M. d., and EPEL. D. (1973). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80, 111-119. WALLACE, R. A., and BERGINK, E. W. (1974). Amphibian vitellogenin: properties, hormonal regulation of hepatic synthesis and ovarian uptake, and conversion to yolk proteins. Amer. Zoo!. 14, 1159-1177. WEISSMANN, G. (1974). Crystals, lysosomes, and gout. Adv. ht. Med. 19, 239-257.
BIOLOGY
46, 404-412 (1975)
Heterogeneous Distribution of “Lysosomal” Hydrolases in Yolk Platelets Isolated from Unfertilized Sea Urchin Eggs by Zonal Centrifugation l HERBERT
SCHUEL.
WALTER
L. WILSON.
JEAN
R. WILSON.
AND ROBERT
S. BRESSLER
Department of Biochemists, Downstate Medical Center, SLTNY, Brooklyn. N. Y. 11203; Biology Department. Oakland University, Rochester, Michigan, Pathology Laboratory, Pontiac General Hospital, Pontiac, Michigan; Department of Anatomy, Mt. Sinai School of Medicine, CUNY, New York, N. Y.; and the Marine Biological Laboratory, Woods Hole, Massachusetts Accepted
May 20. 1975
Three typical “lysosomal” glycosidases, a-L-fucosidase. N-acetyl glucosaminidase and Nacetyl galactosaminidase. were localized within the yolk platelets of unfertilized Strongylocentrotus purpuratus eggs. Homogenates of eggs were fractionated by rate-zonal centrifugation, and the isolated particles were subjected to integrated biochemical and morphological (electron microscopic) analysis. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase). and cortical granules (B-l.3 glucanase) in the sucrose density gradient. Yolk platelets were isolated in a high state ot purity, with contamination by mitochondria and cortical granules at trace levels. Enzymatic heterogeneity exists within the yolk platelet population. Acid nitrophenyl phosphatase and a-L-fucosidase activities appear to be uniformly distributed within all the yolk platelets. while N-acetyl glucosaminidase and galactosaminidase activities appear to be preferentially distributed within the slower sedimenting sub-population of yolk platelets. Another band of hexosaminidase containing particles sedimented slightly slower than the bulk of the yolk platelets. coincident with the mitochondria. The acid hydrolases packaged in the yolk platelets may participate in the mobilization of yolk material after fertilization. The yolk platelet thus appears to be a highly complex and structured “lysosome-like” storage organelle. INTRODUCTION
from eggs by the discharge (exocytosis) of the cortical granules at fertilization promotes the establishment of the block to polyspermy (Epel. 1975; Gwatkin et al., 1973; Longo and Schuel, 1973; Longo et a/., 1974; Schuel et al.. 19’73; Vacquier et al.. 1973). The cortical granules and yolk platelets in eggs exhibit many structural and functional characteristics usually associated with lysosomes in adult tissues, and the! have been characterized as “lysosomelike” organelles for this reason (Allison. 1969; Bluemink, 1970; Dore and Cousineau, 196T: Pasteels, 19’i3: Schuel et al., 1969 and 19’iZb). Mucopolysaccharides and other glycoproteins are known to be biochemical constituents of the yolk platelets. cortical granules. and the extra-cellular investments of the eggs of sea urchins and other organisms (Dauwalder et al.. 1972:
Mature unfertilized eggs contain two distinct species of sub-cellular organelles which are assembled from derivatives of the Golgi apparatus during oogenesis, the yolk platelets and the cortical granules (Anderson, 1968; Beams and Kessel. 1968; Dauwalder et al., 1972; Norrevang, 1968). The yolk platelets are storage granules for metabolites required during embryogenesis (Kavanau, 1954). The cortical granules are true secretory organelles (Schuel et al., 19’72b). The secretory products released ‘This investigation was supported in part by grants from the American Cancer Society (No. P-616) and the SUNY Research Foundation (No. 7125A). and the Population Council (No. MT4.034Cl by a research contract from the National Institute of Child Health and Human Development (No. 69-2202). and by a Research Career Development Award (No. 5K4CA-8606) from the National Cancer Institute to H. Schuel. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
SCHUEL et al.
L.wosomal
Epel, 1975; Immers. 1960; Runnstrom, 1966; Schuel et al., 1974; Tyler and Tyler, 1966). Lysosomes in adult tissues contain a variety of glycosidic enzymes which hydrolyze mucopolysaccharides (Baggiolini, 1972; Barrett, 1972; Clarke, 19651. Previous studies have shown that the typical lysosoma1 enzyme acid phosphatase is localized within the yolk platelets of unfertilized sea urchin eggs, while p-1.3 glucanase is found in the cortical granules (Schuel et al., 1972bl. The experiments described in the present communication were conducted to elucidate the enzymatic properties of the cortical granules and yolk platelets in order to understand the functions of these organelles at fertilization, by determining the subcellular distribution of several typical glycosidases capable of de“lysosomal” (Barrett, grading mucopolysaccharides 1972) in unfertilized sea urchin eggs. A preliminary report of part of this study has been presented previously (Schuel et al.. 1972aJ. METHODS
Eggs obtained from the sea urchin Strong>,locentrotus purpuratus were used in this study. The animals were supplied by the Pacific Bio-Marine Supply Co., Venice, CA. Homogenates of unfertilized eggs were prepared and then fractionated in the type A-XII zonal centrifuge (Anderson et al., 1966: Cline and Ryel, 1971) as described previously (Schuel et al., 1972b3. The total force applied to the subcellular particles during centrifugation was monitored in terms of the time integral of the rotor’s angular velocity (w* .t) using the I. E. C. model 637lA electronic digital speed squared integrator (Cline and Ryel. 1971). The fractions recovered from the zonal centrifuge were analyzed biochemically. Automatic procedures described previously (Schuel et al., 1964; 1968; 1969; 1972b) were used to determine the distribution of subcellular particles (U. V. absorbance at 254 nm), protein, mitochondria (enzymatic marker:cytochrome oxidase), yolk platelets
Hvdrolases
in Yolk Platelets
405
(enzymatic marker:acid nitrophenyl phosphatase), and cortical granules (enzymatic marker: p-1.3 glucanasel. The sucrose concentration in the isolated fractions was measured with a refractometer (Schuel et al., 1969). Automated procedures. originally employed to study rat liver lysosomes (Schuel et al., 1968; Tappel. 19721. were adapted to measure the distribution of several typical “lysosomal” hydrolases (Barrett, 1972) in the fractions isolated from sea urchin eggs (Fig. 1 and Table 1). The following enzymes were assayed using nitrophenpl ester substrates: acid phosphatase, CI-L-fucosidase, N-acetyl glucosaminidase, and Nacetyl galactosaminidase. The non-ionic detergent Turgitol NPX (Schuel and Anderson, 1964) was included in the enzyme incubation media as indicated in order to measure the total hydrolase activities present in the egg homogenates and the isolated subcellular fractions. The morphology of the particles in the fractions isolated by zonal centrifugation was examined under the electron microscope as described previously (Schuel et al., 1969 and 1972b). RESULTS
The results of a typical experiment involving fractionation of unfertilized sea urchin eggs by zonal centrifugation is shown in Fig. 2. The sample zone in fraction No. 3 was sharply indicated in terms of the high concentration of protein which did not sediment into the gradient (Fig. 2al. Mitochondria (enzymatic marker:cytochrome oxidasel were distributed in the gradient just beyond the area occupied by the slowly sedimenting microsomal particles, with the center of the peak in cytochrome oxidase activity located in fraction No. 10. Yolk platelets (enzymatic marker:acid phosphatase) were distributed primarily in the middle region of the sucrose gradient, with the center of this symmetrical peak in fraction No. 14. Pure preparations of cortical granules (en-
406
DEVELOPMENTAL Bro~oc\
zymatic marker: p-1.3 glucanasei were present in the most rapidly sedimenting band of particles, which accumulated at the interface between the heavy end of the gradient and the cushion of 55(r, sucrose P_OPORTIONING PUMP
FIG. 1. Schematic diagram of automated assay system for lysosomal hydrolases utilizing modules of the Technicon Autoanalyzer and Beckman DB spectrophotometer. Flow rates are based on specifications provided by Technicon Instruments Corp. for the manifold tubing on the proportioning pump module. Details of the conditions for the enzyme assays using nitrophenyl ester substrates are presented in Table 1.
VOLCIME 16. 1975
(Schuel et al., 19Eb). Another peak of p-1,3 glucanase activity was also associated with the slowly sedimenting microsomal particles. The lysosomal glgcosidases were distributed in the middle region of the gradient occupied by the yolk platelets (Fig. 2bl. The distribution pattern for a-L-fucosidase was very similar to that observed for acid nitrophenyl phosphatase. N-acetyl glucosaminidase and galactosaminidase activities were distributed primarily within the slower sediment ing population of yolk platelets, peaks in fraction No. 12. Moreover, the hexosaminidase activities did not show a normal Gaussian distribution, since a shoulder on the curve indicated the presence of another slightly slower sedimenting band of particles containing these glycosidases in the gradient between the microsomal and main yolk regions, coincident with the mitochondria. This bimodal distribution pattern for hexoaminidase activities is even more striking when the data are
TABLE ASSAY CONDITIONS Enzyme
Phosphatase
a-r.-fucosidase
N-acetyl-glucosaminidase
N-acetyl-galactosaminidase
1
FOR AUTOMATED DETERMINATION OF ACID HVDROL~SE ACTKITIEL FRACTIONS LISINC. NITROPHENI L ESTER SLWSTRATE~
Substrate
Reagent linesb buffer-detergent
Color de\reloper
Buffer
PH
Trigitol NPX (5 V/V)
p-Nitrophenyl phosphate ( 10 mM)
Na-Acetate
5.0
0.1
p-Nitrophenyl a+fucoside (5 mh4
Na-Citrate
p-Nitrophenyl N-acetyl-Doglucosaminide (5 mM)
Na-Citrate
p-nitrophenyl N-acetyl-fir)galactosaminide (5 mM)
Na-citrate
(0.1 M)
(0.2Ml
3.0
0.5
’ 4.0
0.5
(0.2 M 1.0
IN SEA URCHIN
0.5
(0.2 Ml
a Assay conditions used with automated manifold shown in Fig. 1. DConcentration of reagents shown for lines entering automated assay system.
NaOH
Etc.
Bath (coil)
1J-Length
(0.2 Ml
Single
NH,OH
Full-length
(2.0 M)
Double
NH,OH
Full-length
(2.0 M)
Double
NH,OH
Full-length
(2.0 Ml
Double
SCHL~ELet al.
L.w~somal
407
H.vdrolases in Yolk Platelets
a IF 1
iSd -
FR>CTlShlS
i’.
b
,wm,,
Fro. 2. Fractionation of homogenate of unfertilized sea urchin (S. purpuratus) eggs by zonal centrif’ugation in type A-XII rotor at 3500 rpm for 60 min at 5°C’. (w”.t = 5.2069 x lOa * 0.0985 8 10*1. Sucrose density gradient constructed in 0.5 A4 KCI. 10m3 M EDTA at pH 7.0. A. Distribution of subcellular components (absorbance at 254 nm). protein, cytochrome oxidase. and 8-1.3 glucanase activities in gradient. B. Distribution of acid nitrophenyl phosphatase, o-L-fucosidase, N-acetyl glucosaminidase, and N-acetyl galactosaminidase activities in gradient. C. Distribution of specific activities for acid nitrophenyl phosphatase, a-t.-fucosidase, N-acetyl glucosaminidase. and N-acetyl palactosaminidase activities in gradient.
presented in terms of specific activity (Fig. 2~). The peaks for specific hexosaminidase activities were found in fractions No. 9-10, with a prominent shoulder also present in the main yolk region of the gradient. The specific activities for acid nitrophengl phosphatase and a-L-fucosidase, on the other hand, both showed simple normal distribution patterns in the middle region of the gradient occupied by the yolk platelets. An estimate was also made of the distribution of the biochemical constituents in the egg homogenate in various regions of the gradient (Table 2). For the purpose oi this calculation, the gradient was divided on the basis of the inflection points in protein concentration between the peaks in distribution of subcellular components (Schuel et al.. 1973). Approximately 65% of
the total egg protein was associated with particles that sedimented beyond the starting sample zone in the gradient, while 85-95Y of all the hydrolase activities studied were sedimentable under the low-speed centrifugation conditions employed. The yolk platelets contained about 30’+ of the total egg protein, while about 70-80% of the acid phosphatase and lysosomal glycosidases were in the yolk region of the gradient. The cortical granule fractions contained about 7%’ of the egg protein, and about 300; of the total p-1,3 glucanase activity. Approximately 5O’“r of the total p-l,3 glucanase activity was distributed between the yolk and cortical granule fract ions. In these experiments essentiall> 100%’ of all these biochemical constituents in the egg homogenate originally introduced into the zonal rotor was recovered in
408
DEVELOPMENTAL BIOLOGY TABLE
VOLUME 46, 1975 2
DISTRIBL~TIONAND RECOLEHY OF BIOCHEMICAL CoNsrrr~%vrs IN HOMOGENITES OF UNFERTILIZED SEA URCHIN EGGS (S. purpuratus) FRACTIONATED B\ ZONAL CENTRIFI.GI\TION” Percent ot total in gradient
Biochemical constituent
Recovery (percent)
Non-Sed. (soluble,
Sedimentable
Yolk
Cortical granules
Protein I14 exps)
35.3 * 4.5
64.; i 4.5
31.5 * 3.5
7.1 * 2.9
98.7 zt 19.8
Acid phosphatase (14 exps)
7.8 I 1.9
92.” * 1.9
71.1 * 2.7
3.4 * 1.1
99.2 * 18.5
a-L-fucosidase exps)
16
5.2 + 2.2
94.8 * 2.2
83.0 3: 2.3
3.0 3: 1.7
105.7 =t 5.9
N-acetgl-glucosaminidase (6 exps I
9.2 + 1.6
90.8 rt 1.5
68.6 * 3.6
3.7
3.3
104.5 Lt 11.1
N-acetylgalactosaminidase (6 exps)
8.6 * 2.1
90.9 * 2.7
69.9 * 3.9
3.1 * 2.2
104.6 + 7.7
j3-1,3 glucanase (13 exps)
15.1 * 1.7
84.9 k 1.7
15.8 :t 3.7
33.1 & 2.7
98.2 * 18.2
l
DConsult text for details concerning the division of the gradient into specified regions. Data calculated show mean (kfJ values with standard deviation I +-SD) obtained in series of experiments.
the gradient after centrifugal fractionation. Subcellular organelles separated by zonal centrifugation were examined in the electron microscope. A sample taken from fraction 14 (see Fig. 2, above) contained an almost completely pure suspension of yolk platelets, with a trace level of contamination by mitochondria and cortical granules (Fig. 3). The distribution and ultrastructure of organelles which sedimented slower vesicles, mito( ribosomes, microsomal chondria, etc.) or more rapidly (cortical granules) than the yolk platelets were similar to that reported in previous studies, and the mitochondrial region of the gradient was contaminated by yolk platelets and microsomal vesicles (Schuel et al., 1969; 1972b). These observations also showed that yolk platelets were ubiquitously distributed through the entire sucrose density gradient, and even contaminated the cortical granule fractions at trace levels. DISCUSSION
The observations described above provide direct biochemical evidence for the
to
distribution of a-L-fucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase activities within the yolk platelets of unfertilized sea urchin eggs. Furthermore, since these glycosidases are not constituents of the cortical granules. it is unlikely that they act upon the acid mucopolysaccharides present in these secretory granules (Schuel et al., 1974) or in the extra-cellular investments of the egg (Immers, 1960; Runnstrom, 1966: Tyler and Tyler, 1966) during fertilization. Ultra-structural observations indicated that yolk platelets were isolated from homogenates of unfertilized sea urchin eggs in a high state of purity by rate zonal centrifugation. The center of the yolk peak was contaminated by mitochondria and cortical granules at trace levels. These morphological observations thus probably account for the low levels of cytochrome oxidase (enzymatic marker for mitochondria) as well as B-1,3 glucanase and tryptic protease (enzymatic markers for cortical granules) activities detected within this region of the density gradient (Schuel et
al., 19’i”b and 2973). However. significant numbers of yolk platelets were also distributed through the entire gradient, and were present at trace levels only in the center of the cortical granule peak rSchuel et al.. 1969; LS’i’rb 1. The quantitative biochemical data oh-
FIG. :$. Electrm~ experiment depicted level contamination
that tained in the present study indicate the lrolk platelets contain about 30$ of’ the total egg protein. while the cortical granules contain ahout iC; The former is consistent with morphometric estimates of the percent of the egg volume occupied b\y yolk (Costello. J.%EJ; Harye!.. I932), while the
micrvgraph ot’ pellet obtained from the center ot the yolk peak ~haction 141 [tom the in Fig. 2. This trarticm contains a very puresu~pension ot intact yolk platelets. with a trace by mitochondria (arrow) and cortical granules tarrorr’ head). Bar, 1 bm x 14,4~0.
410
DEVELOPMENTAL BIOLOGY
later (Schuel et al., 1972b) is consistent with estimates of the percent of total egg protein released into the ambient sea water following discharge (exocytosis) of the cortical granules (Bryan, 1970). Furthermore, all the hydrolases studied appeared to be associated with sedimentable particles. The low levels of hydrolase activities remaining in the startmg sample zone (soluble phase) following centrifugation suggest that these cytoplasmic organelles were not damaged during the homogenization and fractionation procedures. The data also support suggestions made by previous investigators (Gross et al., 1960; Krischner and Chambers, 1970) that sea urchin eggs contain more than one population of yolk platelets. Acid nitrophenyl phosphatase and cY-L-fucosidase appear to be evenly distributed within all the yolk platelets, while N-acetyl glucosaminidase and N-acetyl galactosaminidase appear to be preferentially distributed within the slower sedimenting subpopulation of yolk platelets. These conclusions are supported by morphological observations which showed that the center of the acid phosphatase peak contained virtually a pure suspension of yolk platelets. Thus, no particle appeared to be present in sufficient numbers in this region of the gradient to account for the observed high levels of hexosaminidase activities, ot.her than the yolk platelets. A second band of hexosaminidase activity was found associated with particles which sedimented slightly slower, coincident. with the mitochondria. Yolk platelets are also observed at this level of the gradient (Schuel et al., 1969), which may reflect the presence of yet another sub-population of yolk platelets. However, insufficient data are curestablish to clearly rently available whether this slower sedimenting band of hexosaminidase activities is associated with the yolk platelets, mitochondria, or some other small cytoplasmic particle. In addition the number of enzymes which may be responsible for the observed N-
VOLUME 46, 1975
acetyl hexosaminidase activities has not yet been determined. Additional work is required to answer these questions. The presence of acid nitrophenyl phosphatase and several typical “lysosomal” glycosidases in yolk platelets of unfertilized sea urchin eggs provides additional support for the hypothesis (Bluemink. 1970’ Pasteels, 1973) that these organelles are “lysosome-like” storage particles. Lysosomes in adult cells actually are a specialized group of secretory organelles which function in intra-cellular digestion (DeDuve, 1969; Weissmann, 1973). They normally fuse with and discharge their hydrolases into the lumen of phagocytic or other membrane limited cgtoplasmic vacuoles. Yolk platelets are formed during oogenesis by fusion of Golgi derived vesicles with maccontaining endocyt ic vacuoles romolecules to be stored as nutrients for embryogenesis (Norrevang, 1968; Wallace and Bergink, 1974). However, degradation of these stored metabolites begins after fertilization (Kavanau. 1954). Similarly, since the cortical granules discharge their contents extra-cellularly. they are more akin to typical secretory granules in adult cells, ie., zymogen granules in exocrine cells, hormone-storage granules in endocrine cells, heparin granules in mast cells, etc. (Long0 and Schuel. 1973: Schuel et al.. 1973). The biological function of the cortical granule b-1,3 glucanase is uncertain at present, although Epel (197.5) has proposed that it might act upon the hyaline layer protein in the perivitelline space after fertilization. The yolk platelet appears to be a complex and highly structured organelle. A variety of glycoproteins, mucopolysaccharides, and glycolipids are known to be stored in yolk platelets of sea urchins and other organisms (Dauwalder et al., 1972; Immers, 1960; Kondo, 1972; Perlman et al., 1959; Schjeide and Urist, 1959). The results of the present study indicate that glycosidases capable of degrading these substrates are also constituents of sea ur-
SCHUEL et al.
Lysosomal
Hvdrolase
in Yolk Platelets
411
BEAMS. H. W., and KESSEL. R. G. (1968). The Golgi apparatus: structure and function. Int. ReLl. Cxtol. 23, 209-276. BLUEWICK. J. G. i1970). Are yolk granules related to lysosomes? Zeiss Info. 73, 95-99. BRYAN. ,J. (1970,. The isolation of a major structural element ofthe sea urchin fertilization membrane. J. Ce!l Eiol. 15, 606-614. BULL, A. L., and CHESTERS, C. G. C. (1966). The biochemistry of laminarin and the nature of laminarinase. Ado. Enzymol. 28, 325-364. CARDABIS. C.. SCHCIEL. H.. and HERMAN. L. (197-I) Ultrastructural localization of calcium in unfertilized sea urchin eggs. J. Cell Biol. 63, -I9a. CLARKE, A. E. (19651. Hydrolytic enzymes of human mlymorphonuclear leukocytes and rat monocytes. Amt. J. Exp. Bid. Med. Sci. 43, 201-“12. CLINK, G. B., and RYEL. R. B. (19711. Zonal centrifugation. Methods Enzvmol. 22. 168-XL4. COSTELLO. D. P. i 19.19,. The volumes occupied by the formed cytoplasmic components in marine eggs. PhMiOl. Zool. 12. 13-20. DAI.IVAI.DER. M.. ~\‘HAI.EI.. LV. G., and KEPHART, .J. E. (1972). Functional aspects of the Golgi apparatus. Sub-Cell. Biochem. 1, 225-275. DED~IVE. C. (19691. The Iysowme in retrospect. In. “Lysosomes in Biology and Patholog?.” ~Dingle. d. T. and Fell, H. B., eds.). Vol. I., pp. Z-42, U:iley. N. Y. DORE. D.. and COCMNEA[I, G. H. (1967). .4cid phosphatase analysis in sea urchin eggs and blastulae. Exp. Cell Res. 18, 179-18‘1. EPEI.. D. ( 1975). The program of and mechanisms 01 fertilization in the Echinoderm egg. Am. Zoo/., in press. EPEL, D.. I~EALER. A. hf.. hfVCHMORE. A. \r.. and SCHMKF. R. T. (1969). P-l.3 glucanase of sea urchin eggs:release from particles at fertilization. REFERENCES Science 163, 294-296. GROSS, P. R. (19541. On the mechanism of the ~~LLlsON. .A. c. 119691. Lysosomes and cancer. In. yolk-lysis reaction. Protoplasma 43, 416-428. “Lysosomes in Biology and Patholoe” (Dingle. .J. GROSS. P. R.. PHILPOTT, D. E.. and NASS, S. (1960). T. and Fell. H. B.. eds.). \Tol. II. pp. 1X-206. Electron microscopy of the centrifuged sea urchin iViley. N. Y. ANDERSON, E. I 19681. Oocyte differentiation in the sea egg with a note on the structure of the ground cytoplasm. J. f!io&vs. Biochem. C,vtol. 7. 1:X-1-12. urchin, Arbacia punctulata, with particular referGWATXIY, R. B. L.. WILLI.AMS. D. T.. HARTMAN. J. F.. ence to the cortical granules and their participation and KNIAZ~:K, bl. (19731. The zona reaction of in the cortical reaction. J. Cell Biul. 37, 511-539. hamater and mouse eggs: production in L’itro by a ANDERSON, N. G.. BARHINCER, H. P., B.USEI.AY,E. F.. trypsin-like protease from cortical granules. J. Re NKNI.EY. C. E.. BARTRLX hl. J., FfsfiER. W. D.. and prod. Fert. 32, 259-f65. RANKIN. C. T. 119661. The development of 101~. speed “A” series zonal rotors. Nat. Cancer Inst. HARVEY, E. N. (1932). Physical and chemical conMonogr. 21, 113-136. stants of the egg of the sea urchin. Arbacia puntBACGIOLINI, M. ( 19721. The enzymes of’ the granules ot tulata. Biol. Bd/. 62, 141-15.4. polymorphonuclear leukocytes and their functions HEILBRC~NN.L. V. (1956). “The Dynamics of Living Enqvme 13, 132-160. Protoplasm.” i\cademic Press, N. Y, BARREL‘T.A. d. I 19Z i. Lysosomal enzymes. In “LysoHULTIN. T. (1950). On the acid formation. breakdown somes. .4 Laboratory Handbook.” (Dingle. d. T.. of cytoplasmic inclusions, and increased viscosity in ed.i. pp. 16-135. Amer. Elsevier Publ. Co.. N. Y. Paracentrotus egg homogenates after the addition
chin yolk platelets. A similar situation has been described for the latent storage of phosphoprotein phosphatase and its subwithin amphibian yolk strate, vitellin. platelets (Nass. 1956). These observations provide the basis for posing additional questions. First. what mechanism prevents the hydrolases from attacking their substrates while packaged within the same storage granule for an extended period of time? Second. what mechanism activates hydrolysis and mobilization of stored yolk materials after fertilization? Similar questions could be asked about the storage of p-1.3 glucanase and its postulated substrate. hyaline. which are both packaged within the cortical (secretory) granules of unfertilized sea urchin eggs (Epel. 1975: Schuel et al.. 1972bi. Yolk mobilization and other metabolic processes may be activated at fertilization by an increase in the concentration of free calcium ions in the cytoplasm of the egg (Epel, 1975; 1956; Hultin. Gross, 1954; Heilbrunn, 1950; Mazia, 1937; Nass, 19561, resulting from the displacement of calcium ions from intra-cellular binding sites (Steinhardt and Epel. 1974 located in the plasma membrane as well as the membranes of the yolk platelets and cortical granules (Cardasis et al.. 1974).
412
DEVELOPMENTAL BIOLOI;Y
of calcium. Is’xp. Cell Res. 1, 272-283. IMMERS, J. (1960). Studies on cytoplasmic components of sea urchin eggs stratified by centrifugation. Exp. Ceil Res. 19, 499-514. KA~ANAU, J. L. (1954). Amino acid metabolism in the early development of the sea urchin Paracentrotus lividus. Exp. CelI Res. 7, 530-557. KONDO, H. (1972). Properties of glycoprotein particles and their changes during development of sea urchin eggs and embryos. Exp. Cell Res. 72, 51953’2. KRISCHNER, K. N., and CHAMBERS. E. L. (1970). Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization. J. Cell. Physiol. 76, 23-36. LONCO, F. J., and SCHUEL, H. (1973). An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata. Develop. Biol. 34. 187-199. LONCO, F. J.. SCHUEL, H.. and WILSON, W. L. (1974). Mechanism of soybean trypsin-inhibitor induced polyspermy as determined by an analysis of refertilized sea urchin (Arbacia punctulata) eggs. Develop. Biol. 41, 193-201. MAZIA, D. (1937). The release of calcium in .4rbacia eggs on fertilization. J. Cell. Comp. Phvsiol. 10, B-304. NASS, S. (19561. Phosphoprotein phosphatase of amphibian yolk. Trans. N. Y. Acad. Sci. 19, 118-129. NORREVANG.A. (1968). Electron microscopic morphology of oogenesis. Int. Rev. Cytol. 23, 114-186. PASTEELS.J. J. (1973). Yolk and lysosomes. In. “Lysosomes in Biology and Pathology,” (Dingle. J. T., ed.1. Vol. III, pp. 216-234, Wiley, N. Y. PERLMAN. P., BOSTROM, H., and VESTERMARK, A. (1959). Sialic acids in the gametes of the sea urchin. Exp. Cell Res. 17, 439-446. RUNNSTROM. J. (1966). The vitelline membrane and cortical particles in sea urchin eggs and their function in maturation and fertilization. Adi’. Morphopen. 5, 221-325. SCHJEIDE. 0. A.. and URIST, M. R. (1959). Proteins and calcium in egg yolk. Exp. Cell Res. 17, 84-94. SCHUEL, H.. and ANDERSON, N. G. (1964). Studies on isolated cell components. XVI. The distribution of acid phenyl phosphatase activities in rat liver brei fractionated in the zonal ultracentrifuge. J. Cell Bio[. 21, 309-323. SCHUEL, H., TIPTON, S. R., and ANDERSON, N. G. (1964). Studies on isolated cell components. XVII. The distribution of cytochrome oxidase activity in rat liver brei fractionated in the zonal ultracentri-
VOLL’ME 46, 1975 fuge. J. Cell Biol. 22, 317-326. SCHUEL, H.. SCHUEL. R., and UNAKAR, N. J. (19681. Separation of rat liver lysosomes and mitochondria in the A-XII zonal centrifuge. Anal. Biochem. 25, 146-163. SCHUEL, H., WILSON, W. L., WILSON. J. R.. and SCHUEL. R. (1969). Separation of intact cortical granules from homogenate of unfertilized sea urchin eggs by zonal centrifugation. J. Histochem. C,~to&em. 17, 703-713. SCHUEL, H.. LORAND. L.. CHEN. K., WILSON, IV. L., BRESSLER,R. S., and WILSON, J. R. (1972a). Distribution of acid hydrolases in unfertilized sea urchin eggs. J. Cell Biol. 55, 232a. SCHUEL, H., WIISON. W. L., BRESSLER,R. S.. KELLY, .I. W.. and WILSON. J. R. (1972b). Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation. Decelop.
Biol. 29, 307-320. SCHUEL, H., WILSON, W. L.. CHEN. K.. and LORAND. L. (1973). A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization. Deuelop. Rio/. 34, 175-186. SCHUEL. H.. KELLY, J. I%‘., BERGER. E. R., and WILSON, W. L. (1974). Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization. Exp. CelI Res. 88, 24-30. STEINHARDT, R. A.. and EPEL. D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sri. 71, 1915-1919. TAPPEL. A. L. (1972). Automated methods for measuring lysosomal enzymes. In, “Lysosomes, A Laboratory Handbook,” (Dingle, J. T., ed.). pp. 136-149, Amer. Elseiver Publ. Co., N. Y. TYLER A., and TYI.ER, B. S. (1966). The gametes; some procedures and properties. In, “Physiology of Echinodermata,” (R. A. Boolootian. ed.). pp. 639-682. Interscience. N. Y. VACQUIER, V. D., TECNER, M. d., and EPEL. D. (1973). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80, 111-119. WALLACE, R. A., and BERGINK, E. W. (1974). Amphibian vitellogenin: properties, hormonal regulation of hepatic synthesis and ovarian uptake, and conversion to yolk proteins. Amer. Zoo!. 14, 1159-1177. WEISSMANN, G. (1974). Crystals, lysosomes, and gout. Adv. ht. Med. 19, 239-257.
