442

Prelirninaty

notes

Whether the structures identified by this method are the same as those demonstrated by whole-mount electron microscopy [3, 5, 71 or by the squashing method in light microscopy [ 1l] needs further study. It is expected that further information will be obtained about chromosomal structure by the combined use of this immunoperoxidase technique with a more refined technique of chromosomal preparation. References 1. Barnett, R I, MacKinnon, E A & Romero-Sierra,

C. Cvtobios 11 (1974) 115. 2. Emmerich, G, Scharrer, S, Stengel-Rutkowski, S & Zang, K D, Humangenetik 19 (1973) 227. 3. DuPraw, E J, DNA and chromosomes, chap. 9, 11. Holt. Rinehart &Winston. New York (1970). 4. Ford, E’H R, Thurley, K & Woollam, D H M, J anat 103 (1968) 143. 5. Golomb, H M & Bahr, G F, Exp cell res 84 (1974) 79. 6. Kanayama, Y, Yamakami, K, Kato, N, Horiguchi, T, Inoue, T, Maeda, Y & Shiota, K, Osaka city med j 2 1 (1975) 27. 7. Lampert, F, Bahr, G F & DuPraw, E J, Cancer 24 (1969) 367. 8. Lubit, B W, Schreck, RR, Miller, 0 J & Erlanger, B F, Exp cell res 89 (1974) 426. 9. Nakane, P K & Pierce, G B, J cell biol 33 (1967) 307. 10. Schreck, R R, Warburton, D, Miller, 0 J, Beiser, S M & Erlaneer. B F. Proc natl acad sci US 70 (1973) 804. 11. Takayama, S, Exp cell res 91 (1975) 408. 12. Tan, E M & Kunkel, H G, J immunol 96 (1966) 464.

granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was comphetely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to orevent subsequently added sperm from penetrating (fertilizing) -ionophore-activated eggs, co-incident with the elevation of the fertilization membrane.These processes were retarded by inhibitors of the cortical eranule nrotease in ionoohore-activated eggs, just as they are when eggs are-initially stimulated bv snerm at fertilization. A23187-activated eggs did noidibide unless they had been secondarily fe%lized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles recmired to organize the mitotic apparatus. The centrioles are normally provided by the-sperm during fertilization. A23187 mav also be toxic to the eggs. Furthermore, since cortical granules are secretoryorganelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.

An increased concentration of free calcium ions in the cytoplasm appears to be a general feature of the mechanism for the coupling of excitation to the initiation of cellular response processes [14], such as activation of sea urchin eggs at fertilization [8]. The calcium may be derived in part by influx from the external sea water [25] and, or disReceived August 19, 1976 Accepted September 15, 1976 placement from intracellular binding sites [8], localized to certain cytoplasmic organelles [4]. Ionophore A23187, an antibiotic Physiological responses of sea urchin that selectively increases the free conceneggs to stimulation by calcium ionophore tration of calcium within cells [27], has been A23187 analysed with protease inhibitors used to parthenogenetically activate sea H. SCHUEL, W. TROLL and L. LORAND, Deparf- urchin eggs [3, 5, 15, 331. These studies inmerit of Biochemistry, Downstate Medical Center, dicated that ionophore A23 187 triggers sevSUflY, &ooklyn. NY 11203, Department of Environeral physiological responses in the eggs mental Medicine, New York University Medical Center, New York, NY 10016, Department of Biowhich are normally associated with the prochemistry, Northwestern University, Evanston, IL cess of fertilization, including: ion fluxes 60201, and Marine Biological Laboratory, Woods Hole, MA 02543, USA and alterations in membrane potential, cortical granule discharge, elevation of the Summary. Protease inhibitors were used to study certain physiological responses (secretion of the cortical fertilization membrane, increased respiraExp CellRes 103 (1976)

Preliminary

Table 1. Secretion of protease activity

by Arbacia eggs at fertilization or parthenogenetic activation by ionophore A23187

Secretory product

Activity trypsin equivalents (w/ml)

Unfertilized eggs Fertilized eggs Ionophore-activated eggs

0.0 9.3 8.3

Secretory product: supernatant collected from 0.2 ml packed eggs (per 4 ml total vol) 5 min following insemination or A23187 (37.5 PM) activation. Microscopic examination revealed that 100% of these eggs had elevated fertilization membranes within l-2 min after insemination or exposure to ionophore. Supernatant collected from unfertilized (control) eggs incubated for 5 min under similar conditions. Protease activity expressed in terms of free amino groups liberated from succinylated protamine (0.2%) substrate by bovine pancreatic trypsin at pH 8.0 (0.05 M phosphate buffer).

tion, and the initiation of protein and DNA biosynthesis. However, ionophore stimulated eggs do not undergo cell division or embryonic development. The experimental results described in the present communication show that sea urchin eggs will divide and develop into normal larvae provided they are penetrated (fertilized) by sperm after being parthenogenetically activated by calcium ionophore A23187. The ionophore also triggers the secretion of the cortical granule protease which promotes the elevation of the fertilization membrane to establish a barrier to penetration (fertilization) of the eggs by subsequently added sperm. These reactions also mimic physiological processes elicited when eggs are stimulated by sperm during fertilization [8, 13, 17, 18, 29, 32). A preliminary account of part of this study has been presented previously [36]. Materials

and Methods

Gametes were collected from mature Arbacia pnnctulatn (at Woods Hole during the summer) and Srron-

noles

443

purpurnrus (obtained from Pacific BioMarine Supply Co.. Venice, Calif., during the winter) as previously described [29]. The former were maintained in fresh-running sea water, while the latter were maintained in a marine aquarium (Aquarium Systems Inc., Wickliffe, Ohio). Sperm were collected “dry” and stored at 0°C. Working sperm suspensions were prepared by diluting the dry semen with sea water just prior to use. The eggs were filtered through cheese cloth to remove debris. Calcium ionophore ‘423187 [27] was kindly provided bv Dr R. Hoslev of Eli Lillv and Co.. Indianauolis. Ind. It was dissolved and stored in dimethyl sulfdaide: and diluted with sea water iust nrior to beine used to parthenogenetically activaie unfertilized eigs [33]. Suitable controls established that the solvent alone had no effect upon the eggs. The vevtide aldehvde nrotease inhibitors, leupeptin and &pain [l], were k&dly provided by Dr T. Sugimura of the National Cancer Center Research Institute, Tokyo, Japan. Soybean trvvsin inhibitor (SBTI) was obtained from Sigma Chemical Co. (St Louis; MO). Benzamidine-HC1 was obtained from Aldrich Chemical Co. (hlilwaukee. Wis.). The secretory product discharged from eggs ar fertilization or parthenogenetic activation with ionophore A23 187 was obtained by collecting ihe ambient sea water from the cultures by means of gentle cemdfugation. Secretory product preparations were stored at o”C, since freezing destroyed the protease activity. Protease activity was assayed using succinylated protamine as substrate [35j. In physiological experiments, eggs were partkenogenetically activated by A23187 in the presence or absence of protease inhibitors. Sperm were subsequently added to the eggs at various internals following initiation of ionopkore activation. The eggs were allowed to develop, fixed, and counted as describeti. previously [295.

gylocentrotus

Results

Protease activity was secreted by Arbacia eggs at fertilization or upon parthenogenetic activation by ionophore A23 187 (table 1). In both cases, the protease in the secretory product was completely inactivated when the enzyme assays were performed in the presence of either SBTI (5~ IF6 M), or antipain and leupeptin (2x lO+ M). These substances retard the establishment of the block to polyspermy at fertilization [S, 17, 18,29.32], and are known to be competitive inhibitors of serine proteases such as thrombin, trypsin. etc. [l, 111.Unfertilized eggs did not release detectable quantities of protease activity.

444

Preliminary notes

‘““[.-.,

Fig. I. Abscissa: activation time (set); ordinate: fertilized eggs (%). Receptivity of Arbacia eggs to sperm penetration (fertilization) after parthenogenetic activation by ionophore A23 187(25 PM). Unfertilized eggs (2 drops) were placed in 5 ml of sea water containing ionophore, and subsequently inseminated in this solution at indicated intervals by addition of one drop 6% sperm (3 drops “dry semen”/5 ml sea water). The cultures were incubated at room temperature (25”C), and fixed by addition of 2 drops 50 % alutaraldehvde at the time of fust cleavage incontrol>ggs. Eggs that had been fertilized (penetrated) by sperm after initiation of ionophore activation either divided or contained normal mitotic figures. Unfertilized ionophore-activated eggs in the cultures had not divided, and contained large monasters. Each data point represents a count of 100 eggs/dish.

Physiological experiments were conducted to evaluate the receptivity of ionophoreactivated eggs to sperm. Eggs that had been penetrated (fertilized) by sperm after initiation of ionophore activation either divided or contained normal biopolar mitotic figures when fixed at the time of normal first cleavage of control eggs inseminated in sea water. However, unfertilized ionophore-activated eggs did not divide and contained large monasters at this time, as reported by previous investigators [S, 331. The block to sperm penetration in Arbacia eggs was completed within 60-90 set after exposure to A23187 (25 PM), which corresponds to the period for the elevation of the fertilization membrane as observed under the light microscope (fig. I). Similar results were obtained with Strongylocentrotus eggs. The elevation of the fertilization membrane was inhibited when Arbacia eggs were parthenogenetically activated by Exp CellRes 103 (1976)

A23187 in the presence of SBTI, antipain, leupeptin, and also benzamidine. The latter is a rather weak inhibitor of the cortical granule protease [8]. The fertilization membrane in inhibitor-treated eggs had a rosette or scalloped appearance, as observed under the light microscope. This suggests that the fertilization membrane had only elevated from large numbers of localized regions instead of from the entire surface of the egg. Similar phenomena have been observed when eggs are stimulated by sperm in the presence of these inhibitors [8, 17, 18, 29, 321. Eggs were also inseminated at 120 and 300 set after initiation of ionophore activation in the presence of inhibitors of the cortical granule protease (fig. 2). Sperm could not penetrate eggs activated by A23187 in

fertilized eggs (%), n , 120; q d,300 sec. SBTI, 10m5M; Ap, Lp, 5X10-* M; Benz A, lO-2 M. Effect of protease inhibitors on receptivity of Arbaciu eggs to soerm uenetration (fertilization) following -6arthendgenetic activation by ionophore A23187 (25 uM). Eaas (2 dross) were ore-incubated for 5 min in ‘2.5 ‘ml ~~inhibito;, and then activated by addition of equal vol ionophore. Final cont. for ionophore and inhibitors are shown. The eggs were inseminated in these solutions by addition of one drop 6% sperm at 120 and 300 set after initiation of ionophore activation. Control eggs were treated in the same manner in the absence of protease inhibitors. The cultures were incubated, fixed and counted as described in fig. 1. Fig. 3. Ordinate: polyspermic eggs (%). Control, SW; SBTI, 1OP M; Ap, Lp, 5X lo-” M; benz A, lo-’ M. Incidence of nolvsoermv in Arbaciu eees fertilized in the presenceAof*protease inhibitors. T\o drops of eggs were incubated for 5 min in 5 ml of inhibitors, a&d then inseminated in these solutions by addition of one drop 6% sperm. The cultures were incubated, fixed, and counted as described in fig. I. Fig. 2. Ordinate:

Prditnitzmy tiotrs the absence of protease inhibitors at these times, whereas they readily penetrated (fertilized) the protease inhibitor-treated eggs. Furthermore, many of these eggs were polyspermic. The vulnerability of eggs to sperm penetration (fertilization) was reduced by approx. 50% at 300 sec. Qualitatively similar results have also been obtained with Strongylocentrofus eggs. Without prior A23 187 activation, protease inhibitor-treated eggs were highly vulnerable to polyspermic fertilization upon insemination with the same concentration of sperm (fig. 3). Essentially 100% normal monospermic fertilization was obtained with control eggs inseminated in sea water. A23 187-activated Arbacia eggs developed only as far as the streak stage, and they began to cytolyze and die within a few hours, as described by previous investigators [S, 331. Similar results were obtained when the ionophore was removed by extensive washing 10 min after initiation of parthenogenetic activation. However, normal development into mature larvae (pluteus stage) was obtained when the eggs were fertilized after activation in the presence of SBTI (as described above), provided the ionophore was removed by washing 10 min later. Unfertilized ionophore-activated eggs in these SBTI-treated cultures did not divide or develop.

445

under conditions where one sperm enters the egg), provided the ionophore is removed by washing. Two conclusions may be reached from these observations: (1) Prolonged exposure to the ionophore produced cytotoxicity in sea urchin eggs. (2) The sperm contributes a factor required for cell division, presumably centrioles [22, 341 which organize the poles of the mitotic apparatus [23], since mature unfertilized ses. urchin eggs do not contain centrioles [7]~ Sperm thus appear to participate in fertilization by stimulating the egg, and regrrlate subsequent developmental processes by both contributing parental genes and centrioles. Calcium ionophore A23187 may be an incomplete parthenogenetic agent be-. cause it cannot by itself stimulate the egg to assemble centrioles. A secondary parthenogenetic treatment with hypertonic sea water may substitute for this missing sperm funstion [3,7, 161. Ionophore-activated eggs were found to secrete a serine protease which participated in the elevation of the fertilization membrane to form a barrier that prevents penetration by subsequently added sperm. These functions were retarded by spec$c inhibitors of the cortical granule protease in lionophore-activated eggs. just as they are when eggs are stimulated by sperm at fertilization [29,32]. These observations suggest that the protease inhibitors act by a corn.Discussion mon mode of action in both situations, and The results described above further eluci- that sperm enter the eggs at sires (cortical date the physiological responses of sea ur- projections) where the viteiline layer rechin eggs to parthenogenetic activation by mains attached to the plasma membrane= [17, 18, 32, 341. These regions may correcalcium ionophore, and explain the difficulspond to the location of the sperm receptor ties encountered by previous investigators in obtaining cell division and embryogene- (penetration) sites in the unfertilized egg sis [3, 5, 15, 331. The eggs will divide and [32]. Hence, the ionophore-activated eggs develop into normal embryos if they are could be used as a model system to study fertilized shortly after exposure to A23187 alterations in sperm receptivity w place during fertilization. (or in the presence of protease inhibitors

446

Preliminary notes

Cortical granules in sea urchin eggs are secretory organelles [17, 28-32, 371. Calcium ionophores can substitute for normal physiological stimuli and trigger release of secretory products from adult somatic cells [lo] as well as eggs [S]. Calcium ions are believed to perform several essential functions in stimulus-secretion coupling [26]. Another possible role for calcium in the process is suggested by this and other recent studies of the secretory function of cortical granules in sea urchin eggs, namely the activation of a serine protease [9, 121 required for exocytosis [17, 19,29,31]. The mechanism by which calcium activates the protease stored within the cortical granules [29] has not yet been elucidated, but may involve reaction with carboxylated glutamic acid residues as in the conversion of prothrombin to thrombin [21] or reaction with sulfated acid mucopolysaccharides [30]. Evidence for the participation of proteases in the discharge of secretory granules has also been reported in other types of cells [2, 20, 24, 371, which suggests that this might be a general biological phenomenon [ 17,29,3 11.As components of the secretory product, serine proteases also perform a variety of highly specialized extracellular functions [29]. Protease secretion by eggs to establish the block to polyspermy is but one specific example of this general phenomenon. These factors may explain, in part, the effects of protease inhibitors on normal and pathological processes mediated by cell secretion, i.e., implantation of the blastocyst in the uterine mucosa [6], inflammation PI, carcinogenesis [35], and metastasis [38], etc.

We wish to thank Mrs Helen Kesner for her technical assistance This work was supported in pan by grants from the NSF (BMS 75-16126), Population Council (M 74.034), Exp Cell Res 103 (1976)

ACS (P-616) and SUNY Research Foundation (127125A) to H. Schuel, and NIH grants (CA-15315 and ES-00260) to W. Troll, and (5K6-HL-03512, HL-02212 and HL-16345) to L. Lorand.

References 1. Aoyagi, T & Umezawa, H, Cold Spring Harbor conf cell prolif 2 (1975) 429. 2. Back, M K, J theor biol45 (1974) 131. 3. Brandriff. B. Hineaardner. R T & Steinhardt, R A, J exb zoo1 19271975) i3. 4. Cardasis, C, Schuel, H & Herman, L, J cell biol63 (1974) 49a. 5. Chambers, E L, Pressman, B C & Rose, B, Biothem biophys res commun 60 (1974) 126. 6. Dabich, D & Andary, T, J fert steril 25 (1974) 954. 7. Dirkson, E R, J biochem biophys cytol 11 (1961) 244. 8. Epel, D, Am zoo1 15 (1975) 507. 9. Fodor, E J B, Ako, H & Walsh, K A, Biochemistry 14 (1975) 4923. 10. Foreman, J C, Mongar, J L & Gomperts, B D, Nature 245 (1973) 249. 11. Green, N M, J biol them 205 (1953) 535. 12. Gross, P R, Biol bull 107 (1954) 364. 13. Grossman, A, Levy, M, Troll, W & Weissmann, G, Nature new biol243 (1973) 277. 14. Heilbrunn, L V, Dynamics of living protoplasm. Academic Press, New York (1956). 15. Lallier, R, Exp cell res 89 (1974) 425. 16. Loeb, J, Artificial parthenogenesis and fertilization. University of Chicago Press, Chicago, Ill (1913). 17. Longo, F J & Schuel, H, Dev biol34 (1973) 187. 18. Longo, F J, Schuel, H & Wilson, W L, Dev biol 41(1973) 193. 19. Lonning, S, Sarsia 30 (1967) 107. 20. Lynn, J W & Clark, W H, J cell biol 67 (1975) 251 a. 21. Magnusson, S, Petersen, T E, Sottrup-Hensen, L & Claeys, H, Cold Spring Harbor conf cell prolif 2 (1975) 123. 22. Mailer, J, Poccia, D, Nishioka, D, Kidd, P, Gerhart, J & Hartman, H, Exp cell res 99 (1976) 285. 23. Mazia, D, The cell (ed J Brachet & A E Mirsky) vol. 3, p. 77. Academic Press, New York (1961). 24. Meizel, S & Lui, C W, J exp zoo1 195(1976)137. 25. Nakazawa, T, Asami, K, Shoger, R, Fujiwara, A & Yasumasu, I, Exp cell res 63 (1970) 143. 26. Poste, G & Allison, A C, Biochim biophys acta 300 (1973) 421. 27. Pressman, B C, Fed proc 32 (1973) 1698. 28. Schuel, H, Wilson, W L, Bressler, R S, Kelly, J W & Wilson, J R, Dev bio129 (1972) 307. ze. Schuel, H, Wilson, w L, Chen, K & Lorand, L, Dev bio134 (1973) 175. 30. Schuel, H, Kelly, J W, Berger, E R & Wilson W &, Exp cell res 88 (1974) 24. 31. Schuel, H & Longo, F J, J cell biol 70 (1976) 89~. 32. Schuel, H, Longo, F J, Wilson, W L & Troll, W, Dev bio149 (1976) 178.

Preliminary

notes

447

33. Steinhardt, R A & Epel, D, Proc natl acad sci mum can be “set-back” in their progress us 71 (1974) 1915. towards division. The extent of set-back in 34. Summers, R G, Hylander, B L, Colwin, L H & Colwin, A L, Am zoo1 15 (1975) 523. the fission yeast Schizosaccharomyces 35. Troll, W, Rossman, T, Katz, J, Levitz, M & Sugimura. T, Cold Spring Harbor conf cell prolif 2 pombe, the protozoan Tetrahymena pyri(1975) 977. formis, and in mouse fibroblasts is a func36. Troll, W, Schuel. H & Lorand, L, Biol bull 149 tion of the stage in the cell cycle at which (1975) 449. 37. Vacquier, V D, Dev bio143 (1975) 62. the heat shock is applied [I]. The sensitivity 38. Yamamoto, R S. Umezawa, H, Takeuchi, T, of synchronized cultures of ~amrnal~a~ Matsushima, T. Hara, K & Sugimura, T, Proc Am assoc cancer res 16 (1975) 69. cells to thermal death has been shown to Received August 24. 1976 Accepted August 26, 1976

Effect of heat on the viability

of 972hgrowing in synchronous cultures SchizQsaccharQmyces

pombe

J. G. BULLOCK and W. T. COAKLEY, Depavtmenr of Microbiology, University College, Card& Wales Swzma~y. The viability of synchronous cultures of the fission yeast Schizosaccharomyces pombe 972h- has been examined after exposure to temperatures of 49°C. Synchronous cultures were established by continuous flow size selection. Samnles were taken at 20 min intervals over two cell cycles and heat shocked for I5 min. The cells showed different sensitivities to heat treatment during the cell cycle. The sensitive srage lasted from nuclear division to a point in early 62. The position in the cell cycle and duration of the heat sensitive stage of S. pombe are similar to those reported for the response of this organism to ultraviolet light, y radiation. and to suicide labelling with SZP.

The growth rate of asynchronous cultures of microorganisms exposed to temperatures above the optimum growth temperature decreases to zero as the temperature is increased. At still higher temperatures the viability of the cells decreases and a death rate may be measured. In many cases the viability of the cell population appears to decrease exponentially with time and the death rate is therefore constant. The death rate increases as the temperature increases. In addition to the lethal effect of heat, cells exposed to temperatures above their opti-

vary with different stages of the cell cycle P, 3, 41.

In the present work we have examined the cell killing in suspensions of selection synchronized cultures of the eukaryotpombe ic microbe Schizosaccharomyces strain 972h- during the ceil cycle. During nuclear division, at 0.75 of the way throu the cell cycle, the nuclear membrane remains intact and an ordered array of microtubules becomes visible. Unlike the situation in most animal cells the nuclear division cycle and the cell division cycle are out of phase in this cell [5]. Nuclear division is followed by a short G1 and a shopr .S phase which coincides with cell division. Materials

and Methods

The yeast strain used in these experiments, Schizosaccharomvces Dombe 972h- was supplied by Dr D. Lloyd. Thk ceils were grown with -forced aeration (1 litre air/litre mediumlmin) at 30°C in 4-litre flasks containing 2.5 litres of defined medium [6j. For exaeriments with asvnchronouslv rtrowina cells, the culture was grown to-mid-log phase (ipproxy loj cells/ml). Samples of this culture (15 ml) were then placed in 100 ml flasks, in a shaking water bath, at different temperatures in the range of 45 to 52°C. Cell samples were removed at 5 min intervals, diluted and immediately spread on plates of the above medium solidified with 1.5% purified agar (Oxoid). Viability was determined by counting colony formation after incubation at 30°C for 60 h. For experiments with synchronous cultures, midlog phase cells were subjected to size selection centrifugation using a continuous flow centrifuge 171. The centrifugation was carried out at 3O”C, and the inflowing culture was maintained at 30°C using a water bath. The centrifuge (M.S.E. Superspeed 18) was nm at 3 000 rpm and the flow rate into the rotor was adjusted so that between 8 and 12% of the cells were collected in the outflowing culture. This method of

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors.

442 Prelirninaty notes Whether the structures identified by this method are the same as those demonstrated by whole-mount electron microscopy [3, 5...
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