Cell, Vol. 65, 569-576,

May 17, 1991, Copyright

0 1991 by Cell Press

Sperm Decondensation in Xenopus Egg Cytoplasm Is Mediated by Nucleoplasmin Anna Philpott,’ Gregory H. Lena; and Ronald A. Laskey’ Cancer Research Campaign Molecular Embryology Group Department of Zoology University of Cambridge Downing Street Cambridge CB2 3EJ England

Summary At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensatlon of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs. Introduction Decondensation of sperm chromatin to form the male pronucleus is an essential step in fertilization. Upon entering egg cytoplasm, the sperm nucleus undergoes membrane breakdown followed by an initial rapid decondensation of the highly compact chromatin. A new envelope reforms and a second stage of membrane-dependent nuclear swelling occurs (for review see Longo and Kunkle, 1978). Subsequently, the male pronucleus migrates centrally to associate with the female pronucleus. Early experiments used microinjection of a variety of nuclei into oocytes and eggs of the frog Xenopus laevis to study the decondensation abilities of cytoplasm (Graham et al., 1966; Gurdon, 1968, 1976). Substantial swelling of a wide variety of nuclei was seen when the nuclei were injected into the oocyte nucleus or egg cytoplasm. In contrast, studies involving microinjection of sperm into enucleated oocytes showed that only very limited decondensation can occur in an oocyte cytoplasmic environment. However, when oocyte nuclear material was reintroduced

Present address: WellcomelCRC Institute, University of bridge, Tennis Court Road, Cambridge CB2 IQR, England. l

Cam-

by microinjection, these sperm nuclei regained their ability to decondense completely (Lohka and Masui, 1983a). Similarly, mouse sperm injected into rat oocytes failed to decondense until oocyte nuclear envelope breakdown had occurred (Thandani, 1979). Thus, oocyte nuclei contain decondensation factors that remain at high concentration in egg cytoplasm after oocyte nuclear breakdown. Nucleolin, a major acidic phosphoprotein of the nucleolus, is able to partially decondense chromatin (Erard et al., 1988). However, this is unlikely to be present at the high concentrations required for decondensation in the oocyte nucleus or egg cytoplasm. Nucleoplasmin is a highly acidic, thermostable protein, known to bind to histones in Xenopus egg extracts (Laskey et al., 1978; Earnshaw et al., 1980). It is the most abundant protein in theoocyte nucleus, where it specificallyaccumulates, and after germinal vesicle breakdown, it remains in the egg cytoplasm at high concentation (Mills et al., 1980; Krohne and Franke, 1980). lmmunodepletion and fractionation experiments (Dilworth et al., 1987; Kleinschmidt et al., 19881990) haveshownitsinvolvement innucleosome assembly in Xenopus egg extracts, specifically by binding and transferring histones H2A and H2B to chromatin. In addition, nucleoplasmin is able to bind to all four core histones (Earnshaw et al., 1980), and also to histone Hl (J. 0. Thomas, W. C. Earnshaw, and R. A. Laskey, unpublished data). In light of these properties, we haveexamined whether nucleoplasmin may have a role in nuclear decondensation. One of the first activities of amphibian eggs to be reconstituted in vitro was the decondensation of chromatin (Barry and Merriam, 1972; Lokha and Masui, 1983a, 1983b, 1984). Using extracts of the eggs of Xenopus laevis, we have investigated in detail the first stage of nuclear decondensation (stage l), which occurs very rapidly after template addition and in high speed supernatants that are devoid of membranes. The second stage of swelling of sperm (stage 2) leading to pronuclear formation is dependent on formation of a nuclear envelope, occurring only in a low speed egg supernatant, and has not been studied here. We report the effects of immunodepleting nucleoplasmin. Depleted extracts are unable to decondense sperm nuclei at the rate seen in mock-depleted controls, while readdition of nucleoplasmin restores the ability of depleted extracts to bring about decondensation. A physiological concentration of purified egg nucleoplasmin is able to bring about stage 1 sperm decondensation with approximately the same efficiency as whole extract. Decondensation of other nuclear templates is restricted in nucleoplasmin-depleted extracts. Moreover, these templates will decondense in purified nucleoplasmin alone. Thus, here we show that the nucleosome assembly factor nucleoplasmin is both necessary and sufficient for stage 1 decondensation of sperm and also for the decondensation of somatic nuclear templates.

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Figure

1. Time Course

of Sperm

Decondensation

in Egg Extract

Demembranated Xenopus sperm nuclei were (a) taken fresh, 1 min, (c) 5 min, (d) 10 min, (e) 30 min or (9 60 min and prepared Procedures. Scale bar is 50 urn

or (b-f) incubated at 100 nglul DNA in egg extract. Samples were removed at (b) for photography with the DNA stain propidium iodide, as described in Experimental

Results Demembranated Xenopus sperm nuclei decondense rapidly upon incubation in Xenopus egg high speed supernatants (Figure 1). DNA staining shows that nuclear length increases rapidly with a more modest increase in width. With increasing decondensation, nuclei also lose their cork screw-like three-dimensional structure. Length is the most reliable parameter with which to judge the extent of sperm decondensation; photographed width is not a reliable parameter as condensed sperm fail to stick to the slide, but move during photography. This, together with out-of-focus flare, makes photographed nuclei look artifactually wide. The greater length of the sperm nucleus is less distorted by these effects and can be used more reliably to judge the extent of decondensation. Sperm nuclei decondense considerably from coiled, filamentous structures into elongated, snake-like forms, increasing3-fold in length in 1 hr (Figure 1). This initial stage of decondensation is very rapid, most decondensation occurring between 1 and 10 min (compare Figures la-ld). Decondensation continues after this point but more slowly (Figures Id-lf). If a membrane fraction is added, nuclei continue to swell to form rounded, pronucleus-like structures as nuclear proteins are imported (Lokha and Masui, 1984; Blow and Laskey, 1986). However, the extracts used for the studies

described here have been cleared of the membrane fraction by high speed centrifugation, and so only the first stage (stage 1) of sperm decondensation can occur. The morphological changes of sperm nuclei occurring within the first 10 min in this high speed supernatant have been studied further. On the basis of its high negative charge, its abundance in oocyte nuclei and egg cytoplasm, and its ability to bind histones, the nuclear protein nucleoplasmin was considered to be a good candidate for a role in nuclear decondensation. Nuceoplasmin can be immunodepleted from Xenopus egg extract using monoclonal antibodies and protein A (Dilworth et al., 1987). This technique has been used to define the role of nucleoplasmin in nucleosome assembly, and we have used it to investigate a possible role of egg nucleoplasmin in stage 1 decondensation of sperm nuclei. Nuclear decondensation does not appear to result from nonspecific protease activities in the extract bringing about partial nuclear protein degradation and so allowing release of packaged chromatin. Similar results were obtained in extracts prepared in the presence or absence of the protease inhibitors leupeptin, pepstatin A, and aprotinin at 10 uglml (data not shown). Histones act as a sensitive assay for proteases, undergoing degradation when even low levels of activity are present. Histone H5, purified from chicken erythrocyte nuclei, was labeled with 3H by reductive methylation (Lambert and Thomas, 1986) and

Nucleoplasmin 571

Decondenses

Sperm

Nuclei

tl* c “@i

Npl,

1234567 Figure

2. Western

Blot Analysis

of Nucleoplasmin

Depletion

Lane 1, total extract. Lanes 2-4, nucleoplasmin depletion: lane 2. pellet 1; lane 3, pellet 2; lane 4, supernatant. Lanes 5-7, mock depletion: lane 5, pellet 1; lane 6, pellet 2; lane 7, supernatant from mock depletion. Proteins from 1 pl packed volume of PAS pellets, along with 1 ul of mock- and nucleoplasmindepleted supernatants, were separated on a 15% SDS-polyacrylamide gel. Pellet-bound material represents the amount of protein removed from 1 ul of depleted extract. Proteins were transferred to nitrocellulose, and the blot was probed for nucleoplasmin as described in Experimental Procedures. The positions of nucleoplasmin and immunoglobulin heavy and light chains (Npl, H, and L, respectively) are indicated.

incubated in egg extract for 4 hr. Proteins sequently separated by SDS-polyacrylamide phoresis and processed for autoradiography. degradation was seen, even after a month-long graphic exposure (data not shown), indicating tracts used here do not contain significant protease activities.

were subgel electroNo histone autoradiothat the exanti-histone

Nucleopiasmin is Necessary for Sperm Decondensstion Nucieoplasmin-specific monoclonal antibodies were incubated in extract for 45 min and removed by addition and subsequent pelleting of protein A-Sepharose (PAS). After repeating the procedure, the depleted extract was used to study the effect of nucleoplasmin removal on nuclear decondensation. To determine the efficiency and selectivity of immunodepletion, the proteins of depleted and mock-depleted extracts were separated by polyacrylamide gel electrophoresis together with proteins bound to the PAS used to remove depleting antibodies. Proteins were transferred to nitrocellulose and Western blots probed with the anti-nucleoplasmin monoclonal antibody PA3C5 (Figure 2). Each lane represents loading of proteins present in or removed from an equivalent volume of extract. Virtually all the nucleoplasmin has been removed from the antibody-depleted extract, the majority bound to antibodies on the first PAS pellet (see lanes 2 and 4). In contrast, mock-depleted extract still retains nearly all its nucleoplasmin. The small amount of nucleoplasmin seen in pellets from mock depletions represents whole-extract contamination of the unwashed beads. The prominent bands at approximately 50 kd and 25 kd in lanes 2 and 3 are immunoglobulin heavy and light chains, respectively,

of the monocional antibody used for depletion. These bands are visualized due to the anti-mouse antibody binding function of the secondary antibody used for detection. Decondensation in depleted extract was compared with that seen in a mock-depleted control where buffer had been added in place of antibody during the depletion procedure. Sperm nuclei incubated in egg extract depleted of nucleopiasmin decondense more slowly than those incubated in a mock-depleted extract, viewed at parallel times (Figures 3a and 3b). The nuclei in depleted extract appear shorter and more coiled than their counterparts from mock-depleted controls, and indeed, very little changed from the input fresh nuclei (Figure 3d). An identical control depletion performed with antibodies against another Xenopus protein, TFIIIA (Figure 3c), did not lead to a decrease in the extent of decondensation of sperm nuclei, indicating that decrease in decondensation after nucleoplasmin depletion is an effect specific to the removal of nucleoplasmin and not due to the depletion process. These results indicate that nucleoplasmin is necessary for the decondensation of sperm nuclei in the egg extract. Purified Nucleoplasmin Rescues immunodepieted Extracts and Is Sufficient for Sperm Nuclear Decondensation Readdition of purified nucleoplasmin at an estimated physiological concentration of 700 nglpi (Mills et al., 1980) to a depleted extract restores its ability to bring about stage 1 decondensation of sperm nuclei (Figure 4) confirming that nucleoplasmin is necessary for sperm decondensation in egg extracts. Readdition of purified egg nucleoplasmin at physiological concentration actually causes more extensive decondensation than that seen in the mock-depleted extract (compare Figure 4c with 4a). At present, the reason for this effect is unclear. Preparation of immunopurified nucleoplasmin has allowed us to investigate whether incubation in egg nucieoplasmin alone is sufficient to bring about sperm nuclear decondensation. Purified egg nucleoplasmin at physiological concentration is able to bring about stage 1 sperm decondensation (Figures 5a and 5b). Sperm swell rapidly from the input condensed, coiled form to more flattened, snake-like structures. An approximately equivalent extent of sperm decondensation occurs in purified egg nucleoplasmin as in egg extract at parallel times (compare Figures Id and 5b). Thus, pure egg nucleoplasmin brings about sperm decondensation with the efficiency of total extract, confirming the role of this protein as a major sperm decondensation factor. Polyanions, such as heparin and polyglutamic acid, have been shown to bring about extensive chromatin decondensation at high concentrations (Barry and Merriam, 1972). They are also able to participate in nucleosome assembly (Stein et al., 1979). We were interested in determing whether polyanions could mimic the ability of egg nucleoplasmin at the same concentration to bring about sperm decondensation, even though these molecules are not present in egg extract. We incubated sperm nuclei in polyglutamic acid at 700 nglul for 10 min. There was no observable change in the extent of decondensation of

Cell 572

- NPL

MOCK

-TFIHA Figure

3. Effect of Nucleoplasmin

FRESH lmmunodepletion

on Sperm

Decondensation,

Compared

with Control

Depletions

Sperm nuclei were incubated at 100 nglr.4 DNA in egg extract that had been (a) mock depleted, (b) depleted of nucleoplasmin, or (c) depleted of TFIIIA. The unincubated sperm preparation is shown in(d). After 10 min, samples were prepared for photography, as described. Note that movement during photography causes some nuclei to have an artifactually great width, not seen when viewing the samples fresh. Scale bar is 50 sm.

these compared with the input nuclei (Figures 5a and 5~). This supports the idea that nucleoplasmin has a specific role in bringing about sperm decondensation. Figures 3 and 4 illustrate a clear difference between the ability of extract depleted of nucleoplasmin and of mockdepleted extract to bring about sperm decondensation; virtually no decondensation of sperm in nucleoplasmindepleted extract was observed over 10 min, while considerable decondensation occurred in mock-depleted extract. Xenopus oocyte nuclear contents and egg cytoplasm have been shown to bring about the decondensation of a wide variety of nuclear templates (Gurdon, 1966, 1976; Barry and Merriam, 1972). We have seen that egg extracts are able to bring about considerable decondensation of demembranated mouse myeloma nuclei (Figure 6) and chicken erythrocyte nuclei (data not shown), albeit over a longer time than that taken to decondense sperm nuclei. As one round of depletion was seen to be sufficient to remove virtually all nucleoplasmin (Figure 2), only a single round of antibody incubation and removal was used when looking at the effect of the removal of nucleoplasmin on myeloma nuclear decondensation. Figure 6 shows that decondensation of mouse myeloma nuclei is significantly decreased in egg extract that has been depleted of nucleo-

plasmin compared with those in a mock-depleted extract. Reconstitution of extract using purified nucleoplasmin to approximately physiological concentration restored the ability of extract to decondense these nuclei (Figure 6). The volume increase of myeloma nuclei on decondensation in egg cytoplasm is less dramatic than the change seen with sperm nuclei, but myeloma nuclei are roughly spherical, sovolumecan be easily calculated from an average diameter of each nucleus, allowing quantitation of the relative extents of decondensation under these differing conditions. Despite the fact that myeloma nuclei begin partially decondensed due to metabolic activity, there is a clear difference in volume between nuclei incubated in an extract depleted of nucleoplasmin, in a mock-depleted extract, and in a depleted extract where purified egg nucleoplasmin has been added back to physiological concentration (Figures 6a’-6d’). To quantitate this difference, 110 nuclei in each incubation were photographed and the diameter of each measured (Figures 6a-6d). In contrast to sperm nuclei, myeloma nuclei added to extract depleted of nucleoplasmin decondense approximately 2.5fold compared with the size of input nuclei (Figure 6a, 6a’, 6c, 6~‘) but to a much lesser extent than nuclei incubated in a mock-depleted extract, which attain approximately 5.3-

Nucleoplasmin 573

Decondenses

Sperm

Nuclei

MOCK

+NPL Figure

4. Effect on Sperm

Decondensation

of Adding

Back Nucleoplasmin

to a Depleted

Extract

Sperm nuclei were incubated at 100 ng/ul extract in (a) mock-depleted, (b) nucleoplasmindepleted, purified egg nucleoplasmin reconstituted to 700 nglpl extract before sperm addition. The unincubated min, samples were prepared for photography as described. Scale bar is 50 urn.

fold the starting size and P.l-fold the size of nuclei incubated in depleted extract (Figures 6b, 6b’). Purified nucleoplasmin restores the ability of depleted extract to bring about nuclear decondensation to the extent seen in the

Figure

5. Effect

of Purified

Egg Nucleoplasmin

and Polyglutamic

or (c) nucleoplasmindepleted sperm preparation is shown

extract with in (d). After 10

mock-depleted control with mean volumes approximately equal (Figures 6d, 6d’). Mouse myeloma nuclei, incubated in purified egg nucleoplasmin alone at physiological concentration, un-

Acid on Sperm

Decondensation

Sperm nuclei were added to (a) buffer D alone, (b) buffer D with purified egg nucleoplasmin 700 ng/ul, as described in Experimental Procedures. After 10 min, samples were prepared blurred objects are a result of movement on photography. Scale bar is 50 urn.

at 700 nglul, or (c) buffer D with polyglutamic acid at for photography as described. Note that spherical and

Cell 574

70 -l 60 -

50-

Fresh

a

mean=327

40 30 20 10 O=

Figure 7. Effect densation

b

60 -

Mock

50-

mean=1749

of Purified

Egg Nucleoplasmin

on Myeloma

Decon-

Mouse myeloma nuclei were incubated at 100 nglpl for 4 hr in (a) buffer D alone or (b) buffer D with purified egg nucleoplasmin at 700 nglpl and prepared for photography as described. Scale bar is 25 pm.

40 30-

dergo decondensation (Figure 7), the extent of which is approximately equivalent to that found upon incubating nuclei in whole egg extract (compare Figures 6 and 7). Thus, nucleoplasmin is necessary and sufficient for stage 1 decondensation of the physiological template sperm and also for decondensation of nuclei from somatic cells. It takes several hours for mouse myeloma nuclei to undergo extensive decondensation, while sperm nuclei undergo the majority of their stage 1 decondensation within 10 min of addition to the extract. This difference in rate of decondensation may reflect the different histone content of the templates (see Discussion), but in spite of this difference, the observation that nucleoplasmin is necessary and sufficient for the initial decondensation of both templates implies that the same mechanism may operate.

20 10 0’ 60

Depleted

50

mean=821

C

40

Addback

d Discussion

mean=1670

Figure 6. Effect of Nucleoplasmin eloma Nuclear Decondensation

Depletion

and Readdition

on My-

Mouse myeloma nuclei were (a, a’) taken fresh or (b and b’ lo d and d’) incubated at 100 nglpl extract for 7 hr in (b, b’) mock-depleted, (c, c? nucleoplasmin-depleted. or (d, d’) nucleoplasmindepleted extract reconstituted with purified nucleoplasmin at 700 nglpl extract before nuclear addition. Samples were prepared for photography as described; representative fields are shown in (a’+. Scale bar is 25 pm. Measurements of average radius from 110 nuclei under each incubation condition were used to calculate volume, in arbitrary units. These data were used to plot frequency histograms of volume of nuclei

(a-d), and the mean volume is shown for each incubation condition.

Extracts of Xenopus eggs are able to decondense Xenopus sperm nuclei, mimicking the situation in the fertilized egg, where the incoming sperm must decondense to form the male pronucleus, which can then fuse with the female pronucleus. The extract from a single egg can decondense up to 30,000 sperm nuclei, but beyond this level, the decondensation factor(s) becomes limiting (data not shown). The broad function of nuclear decondensation factors in bringing about nuclear swelling accompanied by chromatin remodeling has been known for many years (for review see Longo and Kunkle, 1978), although the identity of such factors has remained elusive. We have identified nucleoplasmin as a major nuclear decondensation factor in the Xenopus egg extract. Chromatin decondensation logically complements its known function in chromatin assembly. The time course shown in Figure 1 indicates that most of the first stage of sperm decondensation in egg extracts occurs within the first 10 min of sperm addition. Although immunodepletion of nucleoplasmin from extract causes inhibition of sperm decondensation over the first critical 10 min period, this does not rule out the possibility that other factors present are able to bring about decondensation. Indeed, sperm nuclei added to an extract depleted of nucleoplasmin are eventually able to decondense to the size of nuclei incubated in

Nucleoplasmin &condenses

Sperm

Nuclei

575

the mock-depleted control extract, but they do so much more slowly. While other factors are able to bring about decondensation, only nucleoplasmin is able to produce this effect at a physiological rate. Pure nucleoplasmin alone can bring about the decondensation of sperm and other nuclei at approximately the same rate as egg extract, and at a considerably greater rate than egg extract immunodepleted of nucleoplasmin. Thus, nucleoplasmin appears to be acting as the major nuclear decondensation factor of egg extracts. The mechanism nucleoplasmin uses to bring about nuclear decondensation has not been investigated here. However, it is likely that it acts via its ability to bind histones (Earnshaw et al., 1980). The nucleoplasmin molecule is pentameric, and the C-terminal half of each subunit contains a cluster of negatively charged amino acids, including a long polyglutamic acid tract. It has been suggested that binding of nucleoplasmin to histones is mediated by these negatively charged regions acting together as a “five-fingered grab” (Dingwall et al., 1987). Other polyanions, including heparin and the acidic nucleolar protein nucleolin, have been reported to decondense chromatin (Barry and Merriam, 1972; Erard et al., 1988). but they are not present at effective concentrations in the egg. Our results suggest that nucleoplasmin is not simply acting as a nonspecific polyanion. Unlike nucleoplasmin, polyglutamic acid in similar concentrations failed to produce a detectable change in nuclear size over a similar 10 min incubation (Figure 5~). Sperm decondensation was detectable when higher concentrations of polyglutamic acid and longer incubation times were used, but the clear difference is that nucleoplasmin is active over the physiological time course at a physiological concentration. Furthermore, after nucleoplasmin immunodepletion, there remains no protein in the egg extract that can bring about this decondensation at a physiological rate. Upon oocyte maturation, the large stores of nucleoplasmin become massively phosphorylated (S. M. Taylor and R. A. Laskey, unpublished data; Laskey, 1983; Sealy et al., 1988), greatly increasing the overall negative charge of the protein. It would be interesting to know whether such a modification is involved in sperm decondensation. A second nuclear protein, Nl , has been shown to perform a similar function as nucleoplasmin in nucleosome assembly. While nucleoplasmin binds and transfers histones H2A and H2B, Nl binds and transfers H3 and H4. We have attempted to determine the effect of immunodepleting Nl on sperm decondensation . We have observed no effects, but we have not been able to remove all of the Nl , due to poor binding of the antibodies to protein A, so these results should be interpreted cautiously. Nevertheless, the results of nucleoplasmin immunodepletions indicate that neither Nl nor any other protein can fully assume nucleoplasmin’s role after nucleoplasmin has been removed. Other members of the nucleoplasmin family have been identified; N038, a nucleolar protein found both in Xenopus oocytes and somatic tissues from a variety of species (Schmidt-Zachmann et al., 1987) has significant sequence homology with nucleoplasmin and shares the

presence of acidic tracts. In addition, mammalian chromatin assembly factors have been identified (Ishimi et al., 1984; Stillman, 1988; Cotten and Chalkley, 1987). Decondensation factors are present in limiting amounts in the eggs of other species, including mammals (Long0 and Kunkle, 1978; Hunter, 1987). It would be interesting to know whether these or related proteins could be mediating decondensation of nuclei in the cytoplasm of mammalian eggs or, indeed, of nuclei in heterokaryons. Many heterokaryon studies have investigated the effect of fusion of mammalian sperm with somatic cells. While some studies show little sign of obvious morphological change of the sperm head (Sawicki and Koprowski, 1971) others show rapid enlargement of the sperm nuclei in somatic cell cytoplasm (Johnson et al., 1970; Gledhill et al., 1972). Such nuclear enlargement seems to involve disaggregation of condensed chromatin (Zelenin et al., 1974). Disulfide bridges play an important role in stabilizing the sperm of eutherian mammals and must be broken before decondensation can occur, but this does not seem to be the case in lower vertebrates (Calvin and Bedford, 1971; Bedford and Calvin, 1974). Changes in divalent cation concentration have also been reported to influence the degree of sperm condensation in buffers (Leake et al., 1972; Barry and Merriam, 1972), but we have found no effect of magnesium on decondensation with nucleoplasmin in buffer. We have been unable to quantitate the extent of sperm decondensation accurately. Their complex spiral shape makes volume measurements difficult. Instead we attempted to assess the degree of chromatin decondensation by rate of digestion with micrococcal nuclease. Although initial digestion rates of sperm incubated in extract appeared over 10 times faster than those of sperm incubated in buffer, the kinetics were complex as rapid nucleosome assembly in extract protected the DNA from digestion. This resulted in an extract-dependent limit digestion product and therefore an ambiguous assay for decondensation. Quantitation by volume measurement is possible with myeloma nuclei, which are approximately spherical (Figure 8) but while the inactive sperm nuclei are added to extract in a highly condensed form and undergo extensive decondensation in egg cytoplasm, nuclei from myeloma cells that are metabolically active contain less tightly packed chromatin and so undergo less dramatic decondensation when added to the extract. In addition, these myeloma nuclei take considerably longer to decondense than sperm, possibly reflecting the different basic protein composition of the two templates. Xenopus sperm nuclei contain little histone H2A and H2B and very little Hl (unpublished data; Wolffe, 1989). Also, they contain avariety of other protein species that migrate close to the core histones on one-dimensional polyacrylamide gels and that cross-react with an anti-Xenopus histone monoclonal antibody, J2B2 (Dilworth et al., 1987; data not shown). In contrast, mouse myeloma nuclei contain the four core histones and histone Hl in roughly stoichiometric amounts. A decondensation effect brought about by binding or removal of specific histones could thus occur at quite different rates on templates with such different basic protein compositions.

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The exceptionally dense chromatin packing in sperm nuclei compared with that in nuclei from somatic cells has two consequences. Sperm-specific basic proteins may allow very tight packing of the male DNA (Pogany et al., 1981), to give increased motility. In addition, it is possible that chromatin structure is adapted to allow rapid stage 1 decondensation of the male nucleus upon fertilization. This would be reflected in the ease of Xenopus sperm nuclear decondensation in egg extracts compared with somatic nuclear templates. Recently, a system using extracts from Drosophila embryos has been described that allows nuclear decondensation and assembly in vitro (Ulitzur and Gruenbaum, 1989; Berrios and Avilion, 1990). These extracts are able to bring about decondensation of both Xenopus and rooster sperm in two stages that seem to be analogous to stage 1 and stage 2 decondensation of sperm in Xenopus egg extracts. Indeed, morphologically, the swelling of Xenopus sperm to the elongated stage 1 nuclei and rounded stage 2 nuclei with intact membranes seems strikingly similar to that seen in Xenopus egg cytoplasm. It will be interesting to determine if a similar protein is used. Nucleoplasmin is the most abundant protein in the oocyte nucleus, remaining at high concentration in the egg cytoplasm. The immunodepletion experiments described here have shown that it is necessary for the efficient decondensation of both sperm and myeloma nuclei. In addition, purified egg nucleoplasmin at physiological concentration is sufficient to bring about the decondensation of both templates. These observations, together with the abundance of the protein in the egg, indicate that nucleoplasmin does indeed act as a physiological decondensation factor in Xenopus, bringing about sperm decondensation rapidly after fertilization. Experimental

Procedures

Production of Extract Activated eggs of Xenopus laevis were used to produce a high speed supernatant devoid of membrane vesicles, as described by Sheehan et al. (1988). Extracts used for nucleoplasmin purification had protease inhibitors (20 PM PMSF, 8 PM phenylanthroline, 20 VM para-aminobenzamide, 1% aprotinin) added after the first spin. Preparation of Nuclei Sperm Nuclei Male X. laevis frogs were injected with 50 U of chorionic gonadotrophin (Chorulon. Intervet) a week prior lo testes removal. Testes were homogenized in 5 ml of buffer XN (50 mM HEPES-KOH [pH 7.0],250 mM sucrose, 75 mM NaCI, 0.5 mM spermidine, 0.15 mM spermine), and debris was removed by centrifugation at 1000 rpm at 4’C. Sperm were pelleted from this supernatant by centrifugation at 4500 rpm in an HB4 rotor for 5 min, resuspended avoiding contaminant erythrocytes, and washed three times in XN. Washed sperm were resuspended in 500 PI of XN. To demembranate sperm, 100 PI of 2 mglml lysolecithin (Sigma) in buffer XN was added followed by incubation on ice for 10 min. Permeabilization was stopped by adding 1 ml of 3% bovine serum albumin in buffer XN. Sperm nuclei were washed three times in buffer XN, then finally resuspended in buffer XN, 50% glycerol and stored at -8OOC. DNA concentration was determined by counting nuclei with a hemocytometer. Mouse Myeloma Nuclel Approximately 1 x 108 mouse myeloma SP2/0 cells were pelleted, washed in PBS, and resuspended in 1 ml of low salt lysis buffer (20 mM HEPES-KOH [pH 6.81, 5 mM KCI, 5 mM MgC12, 0.5% Nonidet P-40,0.1% sodium deoxycholate, 1% aprotinin, 0.1 mM PMSF)(Evan

and Hancock, 1985) on ice. Cells were pelleted at 2900 x g at 4OC and rinsed twice in lysis buffer, cell permeabilization being monitored by the uptake of trypan blue. Demembranated nuclei were resuspended in 400 ~1 of buffer XN, 50% glycerol. DNA concentration was determined by counting nuclei with a hemocytometer. Monoclonal Antibody Production and Purification Monoclonal antibodies were obtained from clones isolated by Dilworth et al. (1987) and production and purification were conducted as described by these authors. The final precipitate formed during the purification of monoclonal antibodies was redissolved at concentrations between 5 and 15 mglml and extensively dialyzed against 20 mM HEPES-KOH (pH 7.8), 50 mM potassium acetate. lmmunoaffinity Purification of Nucleoplasmin lmmunoaffinity columns were prepared essentially as described in Harlow and Lane (1988). In detail, 1 ml packed volume of PAS was washed two times in 0.1 M Na2HP0, (pH 8.2), then mixed with 200 ml of culture supernatant from hybridoma line PA3C5, producing antinucleoplasmin monoclonal antibodies at approximately 2-5 pglml culture supernatant (Dilworth et al., 1987). After a 1 hr incubation at room temperature with rocking, PAS beads were washed twice in 0.2 M Na2B407 (pH 9.0), then resuspended in 10 ml of sodium borate. Solid protein cross-linker dimethylpimilimidate (Pierce and Warriner) was added to a final concentration of 20 mM, and the mix was incubated at room temperature for 30 min. To inactivate the cross-linker, beads were washed and then incubated in 0.2 M ethanolamine (pH 8.0) for 2 hr at room temperature. Beads were washed two times in PBS and stored in PBS, 0.1% merthiolate (Sigma). Two to three milliliters of high speed supernatant was diluted to 10 ml with PBS, then antibody-coupled PAS was added and incubated with rocking for 4 hr at 4OC. The PAS beads were extensively washed in NET buffer (150 mM NaCI. 5 mM EDTA, 50 mM Tris [pH 7.51 and poured as a 1 ml column. The column was extensively washed with NETand then eluted with half-column volumesof 25 mM sodium citrate (pH 2.9) into 100 ~1 of 1 M HEPES-KOH (pH 8.0). Column fractions containing nucleoplasmin, as determined by analyzing fractions with SDS-PAGE, were pooled and extensively dialyzed against 20 mM ammonium bicarbonate solution. Protein concentration was determined by amino acid analysis. Afler lyophilization, nucleoplasmin was dissolved in 20 mM HEPES-KOH (pH 7.8) at 5 mglml. lmmunodepletion The high speed supernatant was rapidly thawed and supplemented with 1120 volume energy regenerating system (final concentrations 60 mM phosphocreatine, 150 pglml creatine phosphokinase; Sigma). Monoclonal antibodies PA3C5 (anti-nucleoplasmin; Dilworth et al., 1987) or 06C5 (anti-TFIIIA) were incubated in extract for 45 min at 0°C. PAS (2 ~11~1 extract) slurry (1 :l in 20 mM HEPES-KOH [pH 7.81, 50 mM KOAc) (Bioprocessing; 23 mglml IgG binding) was spun down and the buffer was removed and extract added, with the beads resuspended by gentle mixing. After incubation for 15 min at O°C with gentle agitation, PAS was spun out and the depletion repeated on the isolated supernatant. Volumes were kept constant between depleted extracts and controls by adding antibody buffer alone where appropriate. Similarly, in nucleoplasmin readdition experiments, buffer alone was added in place of nucleoplasmin solution in controls. Decondensation Studies After rapid thawing or after depletion, extract was supplemented with energy regenerator (150 rc.g/ml creatine phosphate. 80 mM phosphocreatine). Sperm nuclei were added at 100 nglvl and mouse myeloma nuclei at 100 nglvl original volume of whole extract, i.e., the volume of extract used before dilution on depletion. Where appropriate, purified nucleoplasmin solution was added lo a final concentration of 700 ngl ul, while an equal volume of buffer alone was added to controls. Alternatively, nuclei were incubated in buffer D (50 mM HEPES-KOH [pH 7.81, 75 mM potassium acetate. 0.5 mM spermidine, 0.15 mM spermine) with or without purified nucleoplasmin or with polyglutamic acid (Sigma) each at 700 nglpl. Samples were mixed gently, and at each time point, l-2 pl aliquots were removed to slides, mixed with the DNA stain propidium iodide at

Nucleoplasmin 577

Decondenses

Sperm

Nuclei

a final concentration of 5 uglml, and rapidly viewed fluorescence using the Nikon Microphot microscope.

Gurdon, J. 8. (1968). growing and maturing 26401-414.

by rhodamine

Western Blotting Proteins were separated by SDS-PAGE and transferred to nitrocellulose (Towbin et al., 1979). Blots were incubated in anti-nucleoplasmin PA3C5 monoclonal antibody at 2 uglml, rinsed, and incubated in horseradish peroxidase-conjugated anti-mouse antibody, diluted l:lOC@. Sites of antibody binding were visualized using the ECL Western blotting detection system (Amersham). Acknowledgments We are particularly grateful to Dr. Steve Dilworth for his generous gift of the anti-nucleoplasmin clone PA3C5 and the anti-Xenopus histone clone J2B2, and for the TFIIIA monoclonal antibodies. We also appreciate his valuable advice concerning the immunopurification of nucleoplasmin and immunodepletion experiments, and for critically reading this manuscript. We are also grateful to Dr. Jean Thomas for providing us with chicken erythrocytes and purifed histone H5. A. P. is supported by a Wellcome Trust Studentship. This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

19, 1990; revised

March

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