Relation of intracellular pH and pronuclear development in the sea urchin, Arbacia punctulata,

DEVELOPMENTAL

BIOLOGY

79,478-487 (1980)

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NOTES

Relation of Intracellular pH and Pronuclear Development the Sea Urchin, Arbacia punctulata A Fine Structural

in

Analysis

CHRISTOPHERP. CARRONAND FRANK J. LONGO Department of Anatomy, University of Iowa, Iowa City, Iowa 52242, and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543 ReceivedAugust 23, 1979; accepted in revised form March IO, 1980 Zygotes, treated with sodium-free artificial seawater (Na-free ASW) in order to inhibit the elevation of intracellular pH at fertilization, were examined by light and electron microscopy. Although such specimens elevated fertilization membranes, male pronuclear development and sperm aster formation were suppressed. Inhibition of these events was reversed when zygotes were resuspended in seawater or in Na-free ASW containing ammonia. These results indicate that alkalinixation following insemination or processes accompanying this alteration induce a pervasive change within the zygote which supports events of fertilization. INTRODUCTION

Fertilization of sea urchin eggs triggers a sodium-dependent hydrogen efflux resulting in the elevation of intracellular pH and is associated with the initiation of activation including an increase in protein synthesis, DNA replication, pronuclear development, sperm aster formation, and pronuclear fusion (Epel et al., 1974; Johnson et al., 1976). The Na+-H+ exchange can be circumvented by the addition of agents to sodium-free seawater which induce an increase in intracellular pH (Shen and Steinhardt, 1978). Such studies indicate that the initiation of the aforementioned events does not require sodium per se, but depends upon the resultant increase in intracellular pH or events accompanying this change. Knowledge of the relation of the Na’-H+ exchange and the concomitant elevation of intracellular pH to pronuclear development and fusion has been limited, for precise lesions and the extent of inhibition have not been identified (Chambers, 1975; Johnson et al., 1976). In order to more fully understand the relation of the pH change during activation to pronuclear morpho-

genesis, fertilized sea urchin (Arbacia pun&data) eggs, incubated in sodium-free seawater, have been examined at the ultrastructural level. MATERIALS

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

METHODS

Arbacia punctulata were acquired from either the Marine Biological Laboratory, Woods Hole, Massachusetts, or the Gulf Specimen Company, Inc., Panacea, Florida. Eggs and sperm were obtained according to procedures previously described (Costello et al., 1957). At 30 set postinsemination fertilized ova were transferred to sodiumfree, choline chloride-substituted artificial seawater (Na-free ASW, MBL Formulae VI, p. 67; sodium chloride replaced by the molar equivalent of choline chloride) and incubated for 22 min. Samples were taken at periodic intervals throughout a 22-min period of incubation in Na-free ASW and prepared for light and electron microscopy (Long0 and Anderson, 1972). At 22 min postinsemination the remaining zygotes were transferred to seawater and sampled periodically during an additional incubation of 20 min. To delimit the period of

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cubation in Na-free ASW. Initially, incorporated sperm nuclei were located along the egg periphery, however, by 20 min postinsemination they were distributed randomly throughout the ooplasm. Occasionally, sperm nuclei were seen closely associated with the female pronucleus (Fig. 3). Throughout the period of incubation in Na-free ASW (0.5-22 min postinsemination), sperm nuclei, lacking a nuclear envelope, were composed predominately of a central core of coarsely aggregated chromatin surrounded by a periphery of filamentous material (Fig. 4). Occasionally sperm nuclei, largely decondensed and filamentous in appearance, were observed in some specimens (Fig. 6). Completely dispersed sperm nuclei, as observed in control preparations 10 min postinsemination, were not found in samples taken throughout the RESULTS period of incubation in Na-free ASW. Within individual polyspermic zygotes, Effects of Na-free ASW on unfertilized and fertilized eggs. Female pronuclei of sperm nuclei exhibited similar morphologic fertilized and unfertilized eggs became ir- features. regular in outline during the incubation in Vesicles, possibly derived from the enNa-free ASW. Unfertilized eggs incubated doplasmic reticulum and the former sperm in Na-free ASW did not activate; a cortical nuclear envelope, were observed along the granule reaction was not evident and the periphery of partially dispersed chromatin. female pronucleus did not migrate centrad. Segments of flattened cisternae, apparently However, by 60 min of incubation, greater derived from the fusion of these vesicles than 50% of the ova examined had degen- and interrupted by cisternae-free areas, enerated. circled the partially decondensed chromaFertilization cones in eggs examined at 5 tin. The development of cisternae did not min postinsemination contained fascicles of appear to have a direct relation to the demicrofilaments, some cisternae, and ribo- gree of chromatin dispersion or to the time somes; frequently sperm axonemes were of sampling. The cisternae remained as inpresent (Fig. 1). Microvilli adjacent to fer- dependent structures in almost all cases; tilization cones were approximately three they did not coalesce to form a continuous times longer (1.0-1.5 vs 0.5 pm, respec- pronuclear envelope. They were, however, tively) than their counterparts from unfer- distinguished by the presence of armuli, tilized eggs and displayed prominent cores structurally similar to nuclear pores (Fig. of microfilaments (Fig. 2). Although elon- 8). Sperm chromatin surrounded by a congate microvilli were present over the re- tinuous nuclear envelope was rarely obmainder of the fertilized egg .surface, few served in zygotes incubated in Na-free were observed to contain microfilaments. ASW. Pronuclear fusion was not observed Fertilized eggs contained a variable num- in Na-free ASW-treated zygotes. ber of sperm (two to six) and had elevated The sperm mitochondrion, axonemal fertilization membranes that progressively complex, and centrioles remained in assobecame disrupted during the period of in- ciation with incorporated sperm nuclei sodium dependence, eggs fertilized in seawater were placed at 3-min intervals in Nafree ASW and incubated for a total of 25 min. Samples were then prepared for light and electron microscopy and examined for the extent of pronuclear development and fusion. Unfertilized eggs, incubated in Nafree ASW for periods of up to 1 hr, were also examined. Eggs fertilized in seawater were transferred 1 min postinsemination to Na-free ASW containing 10 mM NH&l (pH 8.0). Eggs were also fertilized in seawater containing 10 mMNH&l (pH 8.0). Unfertilized eggs were incubated in seawater or Na-free ASW, both containing 10 mM NH&l (pH 8.0). Samples of these preparations were taken at periodic intervals and prepared for microscopic examination.

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throughout the period of incubation in Nafree ASW. Development of a sperm aster was not observed. In addition, there was an aggregation of mitochondria, yolk bodies, and pigment granules into reticulated clusters (Fig. 3). Migration of pigment granules to the zygote cortex did not occur in specimens incubated in Na-free ASW. Eggs fertilized in seawater and subsequently transferred to Na-free ASW at varying intervals thereafter demonstrated the existence of a sodium-dependent period for pronuclear development of less than 6 min in duration. Pronuclear development in zygotes transferred to Na-free ASW at 30 set postinsemination was inhibited, whereas eggs transferred at 3 min postinsemination demonstrated variations in their capacity for pronuclear development. Formation of male pronuclei, sperm asters, and zygote nuclei (pronuclear fusion) was consistently observed in eggs transferred to Na-free ASW 6 min postinsemination. Transfer of Na-free ASW-treated zygotes to seawater. The crenulated female pronucleus of eggs and zygotes incubated in Na-free ASW became more regular in outline when transferred to seawater, indicating that the substitution of choline chloride for sodium was responsible for this morphological alteration. Dispersion of sperm nuclei was completed 6 min after transferal of Na-free ASW-treated zygotes to seawater. All of the paternally derived chromatin pos-

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sessed a fine-textured appearance similar to that observed in untreated specimens. Six to eight minutes after transferal to seawater, morphogenesis of the male pronuclear envelope was complete (Fig. 9). Male pronuclei underwent a progressive enlargement throughout the period prior to their fusion with the female pronucleus. Developing sperm asters were initially observed 6-8 min following replacement of Na-free ASW with seawater. Segments of microtubules, annulate lamellae, and accumulations of endoplasmic reticulum appeared in the vicinity of the sperm centrioles. Over the next 2 min a rapid accumulation of these cytoplasmic elements contributed to the enlargement of the aster. Fusion between male and female pronuclei, as previously described (Long0 and Anderson, 1968), was observed as early as 6 min following the transfer of Na-free ASW-treated zygotes to seawater. This was due presumably to the close association that developed between some female pronuclei and sperm nuclei while zygotes were incubated in Na-free ASW (Fig. 3). Pronuclear fusion was observed in polyspermic zygotes up to 40 min postinsemination (Figs. 9-11). Fusion of male pronuclei with one another was observed throughout the same interval (Fig. 12). Effects of seawater and Na-free ASW containing NH&l on unfertilized and fertilized eggs. Although unfertilized eggs in-

FIG. 1. Zygote incubated in Na-free ASW demonstrating a portion of its fertilization cone, approximately 5 min postinsemination. The egg was fertilized in seawater and subsequently transferred to Na-free ASW at 0.5 min postinsemination. The fertilization cone contains fascicles of microfiiaments (MF), ribosomes, and axonemal complexes (AC). x 46,500. FIG. 2. Microvillus observed in the area adjacent to a fertilization cone of a zygote incubated in Na-free ASW for 5 min demonstrating a core of microfdaments (MF). Microvilli containing microfilaments were less numerous over the remainder of the egg surface. x 35,000. FIG. 3. Photomicrograph of two sperm nuclei (SN) adjacent to the female pronucleus (FPN) at 20 min postinsemination. Eggs were fertilized and transferred to Na-free ASW. Note the aggregations of cytoplasmic organelles (arrows) which were observed throughout the period of incubation in Na-free ASW. The aggregations consisted primarily of yolk bodies and mitochondria. x 500. FIG. 4. Sperm nucleus exhibiting minimal decondensation of its condensed chromatin (specimen prepared 10 min postinsemination; 9.5 min in Na-free ASW). Note the vesicles (V) and cisternae (CN) located along the periphery of the dispersing chromatin (DC). YB, yolk bodies; CC, condensed chromatin; M, mitochondria; AC, axonemal complex; CF, centriolar fossa. x 27,000.

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F .

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cubated in seawater and Na-free ASW, both containing 10 mM NH&l (pH 8.0), did not elevate fertilization membranes, they did exhibit nuclear activation in that the female pronucleus moved centrad and chromosome condensation was evident after 70 min of incubation. The cortex of eggs examined after 10 min of incubation appeared similar to that of untreated ova and exhibited short microvilli lacking microfilaments [cf. Mazia et al. (1975) and Spiegel and Spiegel (1977) for additional information on microvillar elongation in sea urchin eggs under similar conditions]. Female pronuclei of unfertilized and fertilized eggs became crenulated during the incubation in Na-free ASW containing 10 mM NH&l (pH 8.0). Eggs incubated in seawater containing 10 mM NH&l (pH 8.0) and mixed with sperm underwent a cortical granule reaction followed by pronuclear formation and fusion as observed in control specimens. Samples examined at 40 and 70 min postinsemination underwent chromosome condensation. Although mitotic figures were present in zygotes fixed at 70 min postinsemination, greater than 90% of the living specimens examined 12-15 hr later had failed to cleave and were degenerate. Eggs fertilized in seawater and transferred to Na-free ASW containing 10 mM NH&l (pH 8.0) at 1 min postinsemination exhibited the same pattern of morphogenesis

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as ova fertilized in seawater containing 10 mM NH&l (pH 8.0). DISCUSSION

Results of experiments reported here demonstrate that sperm aster formation, pronuclear development, and fusion are inhibited when zygotes are incubated in Nafree ASW. Investigations utilizing Na-free ASW containing ammonia indicate that these are not sodium dependent events per se but are due to an inhibition of cytoplasmic alkalinization or events normally accompanying the change in intracellular pH at fertilization (cf. Chambers, 1976; Johnson et al., 1976; Shen and Steinhardt, 1978; Winkler and Grainger, 1978). We are unable to reconcile all aspects of the inhibition of pronuclear development and sperm aster formation with the absence of cytoplasmic alkalinization. Consequently, inhibition of these events of fertilization may be due to an alteration of processes occurring concomitantly with cytoplasmic alkalinization. There is little information as to what parameters other than sodium and pH (e.g., ionic; cf. Nakamaru and Schwartz, 1972) are affected in zygotes incubated in Na-free ASW. Cytoplasmic alterations in zygotes incubated in Na-free ASW. Based on the

observations of Begg and Rebhun (1978) suggesting that the pH increase accompa-

FIG. 5. Sperm nucleus from a zygote fixed 5 min postinsemination (incubated in Na-free ASW for 4.5 min). The cistemae indicated by the arrows incompletely surround the partially dispersed chromatin (DC). A discontinuity in the layer of cistemae is indicated by an asterisk. Most sperm nuclei examined throughout the period of incubation in Na-free ASW had the appearance depicted here. CC, condensed chromatin. x 22,500. FIG. 6. Sperm nucleus from a zygote fixed 10 min postinsemination incubated in Na-free ASW for 9.5 min) demonstrating the maximal extent of decondensation observed throughout the period of incubation in Na-free ASW. Compare with Figs. 4 and 5. Note the array of cistemae (CN) delimiting the dispersing chromatin (DC). CF, centriolar fossa; C, centriole. x 12,009. FIG. 7. Sperm nucleus observed ln a zygote fmed 18 min postinsemination (incubated in Na-free ASW for 17.5 min) surrounded by an apparently complete envelope (NE). Most specimens examined did not show as extensive development of a bilayered envelope. SM, sperm mitochondria, DC, dispersed chromatin; CC, condensed chromatin. x 23,000. FIG. 8. Sperm nucleus from a zygote fixed 18 min postinsemination (incubated in Na-free ASW for 17.5 min) demonstrating pores (arrows) within the cistemae (CN) that partially surround its periphery. A discontinuity in the cistemal layer is indicated by an asterisk. SNE, apical portion of the former sperm nuclear envelope; DC, dispersed chromatin; CC, condensed cbromatin. x 9000.

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FIG. 9. Zygote previously incubated in Na-free ASW for 21.5 min and transferred to fresh seawater (incubated for 6 min in seawater). The egg was fertilized in seawater and subsequently transferred to Na-free ASW at 0.5 min postinsemination. The male pronucleus (MPN) has completed the dispersion of its chromatin and pronuclear envelope formation. The arrows indicate the site of fusion between the male and the female pronuclei (FPN). MT, microtubules. x 35,000. FIG. 10. Photomicrograph depicting fused male and female pronuclei (MPN and FPN) in a sample fmed 14 min after a Na-free ASW-treated zygote (21.5 min) was transferred to seawater. Eggs were fertilized in seawater and subsequently transferred to Na-free ASW at 0.5 min postinsemination. X 550. FIGS. 11 AND 12.. Photomicrographs of male pronuclei (MPN) fusing with a female pronucleus (Fig. 11) and with each other (arrows, Fig. 12). Zygotes were treated in Na-free ASW for 21.5 min and transferred to seawater (incubated in seawater for 12 min). Eggs were fertilized in seawater and transferred to Na-free ASW at 0.5 min postinsemination. X 550.

nying ization soned would

fertilization results in actin polymerand microvillar elongation, we reathat inhibition of the pH increase prevent formation of microfilaments

and, hence, elongation of microvilli. However, the results presented here demonstrate microfilaments within the fertilization cone and adjacent microvilli of eggs

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panying this change) and not sodium per se incubated in Na-free ASW. The appearance of microfilaments at the site of sperm in- is responsible for aster development comes from experiments in which fertilized eggs corporation may be due to a restricted Na+H+ exchange before the inseminated eggs were incubated in seawater and Na-free ASW, both containing 10 mM NH&l (pH were transferred to Na-free ASW. Additionally, since echinoderm sperm appear to 8.0). In both cases pronuclear development and aster formation were similar to those undergo an acid efflux upon activation (Tilney et al., 1978), a localized increase in the of untreated specimens. Pronuclear development in zygotes intracellular pH of the egg may be brought treated with Na-free ASW. Unlike the inabout by the fusion of a relatively alkaline hibition of sperm aster development, male sperm with the egg. We recognize that various technical as- pronuclear morphogenesis was not compects of our preparations may have induced pletely prevented, i.e., some aspects of proan apparent dichotomy with respect to the nuclear development were evident (e.g., presence or absence of filaments within mi- sperm nuclear envelope breakdown). The crovilli of zygotes incubated in Na-free rapidity with which dissolution of the ASW. Nevertheless, the appearance of sperm nuclear envelope occurred suggests elongate microvilli lacking microfilaments that its breakdown took place while fertilized eggs were suspended in seawater or in specimens incubated in Na-free ASW raises questions concerning the functional shortly after transferal to Na-free ASW. In relation of microvillar elongation and filaeither case, sperm nuclear envelope breakment production (Harris, 1968; Burgess and down occurred before the increase in intraSchroeder, 1977; Spudich and Amos, 1979; cellular pH, i.e., at pH values of the unfertilized egg. Begg and Rebhun, 1978). That the replacement of Na-free ASW Sperm aster formation began approximately 6-8 min after the replacement of with seawater allowed resumption of chromatin dispersion leads to the question of Na-free ASW with seawater. If the moment pH affect of replacement is considered as time zero, how variations in intracellular chromatin organization. Dispersion of the then the 6- to 8-min period for aster development is comparable to that reported for sperm nucleus is believed to be due to sperm aster formation in untreated specichanges in the nucleoprotein content of the mens (Longo, 1973). The similarity of the paternally derived chromatin (Ecklund and time interval in both cases indicates the Levine, 1975; Kopecny and Pavlok, 1975; sensitivity of an early or rate-limiting Zirkin and Chang, 1977; Kunkle et al., event(s) of sperm aster formation to the 1978). Enzymatic processes have been imrise in intracellular pH (or accompanying plicated in the modification or removal of changes). A similar statement concerning histones from nuclei of sperm and other the temporal alteration of male pronuclear cells (Bradbury et al., 1974a,b; Marushige envelope formation and sperm chromatin and Marushige, 1975). One possible expladispersion cannot be made due to the varination to account for the inhibition of chroable extent of inhibition of these processes matin dispersion in zygotes incubated in in zygotes treated with Na-free ASW. Na-free ASW is that processes normally The role of pH in sperm aster formation resulting from a shift toward the pH optima is unknown. It has been suggested that of a rate-limiting reaction fail to occur in microtubule assembly may be regulated via the absence of an increase in intracellular modulation of intracellular pH (Sakai, PH. 1978). Additional evidence that an increase Our observations suggest that the forin intracellular pH (or a process(es) accom- mation of the male pronuclear envelope is

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sensitive to the increase in pH that occurs at fertilization (or to a process(es) accompanying this alteration). Obara et al. (1973) have provided evidence that the formation and disruption of the nuclear envelope in Don cells are sensitive to the environmental pH. They also suggeked that the structural integrity of the nuclear envelope might be regulated by fluctuations in the intracellular pH (Obara et al., 1973, 1974). Incomplete inhibition of chromatin dispersion and pronuclear envelope formation may be due to a limited increase in cytoplasmic pH localized to the site of spermegg fusion. Completion of pronuclear development, however, may depend upon a continued increase, as obtained when Na-free ASW-incubated zygotes are transferred to seawater. Although our observations do not establish the mechanism(s) whereby elevation of intracellular pH or processes accompanying this alteration regulate specific developmental processes, they demonstrate lesions of pronuclear development associated with an inhibition of the pH increase at fertilization. These observations reinforce the idea that the pH increase at fertilization (or a process accompanying this alteration) induces a single, pervasive change which activates a series of independent events leading to the development of the male pronucleus and the sperm aster (Epel et al., 1974). Portions of this investigation were supported by funds from the National Science Foundation. REFERENCES BEGG, D. A., and REBHUN, L. I. (1978). pH induced changes in actin associated with the sea urchin egg cortex. J. Cell Biol. 79, 276a. BRADBURY,E. M., IN~LIS, R. J., and MATHEWS, H. R. (1974a). Control of cell division by very lysine rich histone. Nature (London) 247,257-261. BRADBURY, E. M., INGLIS, R. J., MATTHEWS, H. R., and LANGAN, T. A. (1974b). Molecular basis of central mitotic cell division in eukaryotes. Nature (London) 249.553-555. BURGESS,D. R., and SCHROEDER,T. E. (1977). Polarized bundles of actin Naments within microvilli of

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nuclear membrane formation in interphase-metaphase cells. J. Cell Biol. 58, W-617. OBARA, Y., CHAI, L. S., WEINFELD, H., and SANDBERG, A. A. (1974). Prophasing of interphase nuclei and induction of nuclear envelopes around metaphase chromosomes in HeLa and Chinese hamster homo- and heterokaryons. J. Cell Biol. 62,104-113. SAKAI, H. (1978). The isolated mitotic apparatus and chromosome motion. Znt. Reu. Cytol. 55.23-48. SHEN, S. S., and STEINHARDT, R. A. (1978). Direct measurement of intracellular pH during metabolic derepression of the sea urchin egg. Nature (London) 272,253-254. SPIEGEL, E., and SPIEGEL, M. (1977). Microvilli in sea urchin eggs, differences in their formation and type. Exp. Cell Res. 109.462-465.

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actin filament bundles from microvilli of sea urchin eggs. J. Mol. Biol. 129,319-331. TILNEY, L. G., KIEHART, D. P., SARDET, C., and TILNEY, M. (1978). Polymerization of actin. IV. Role of Ca*’ and H’ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm. J. Cell Biol. 71.536-550. WINIUER, M. M., and GRAINCER, J. L. (1978). Mechanism of action of NH&l and other weak bases in the activation of sea urchin eggs. Nature (London) 273,536-538. ZIRKIN, B. R., and WANG, T. S. K. (1977). Involve-

ment of endogenous proteolytic activity in thiol induced release of DNA template restrictions in rabbit sperm nuclei. Biol. Reprod. 17,131-137.