(Received for publication, January 4, 1996; and in revised form, February 7, 1996)
From the
Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus.
Oocyte maturation and fertilization are accompanied by major changes in chromatin structure. In Xenopus, metaphase egg cytoplasm induces chromosome condensation by the action of the cyclin-dependent kinase cdc2. In contrast, activated interphase egg extract rapidly decondenses the exceptionally compact chromatin of sperm nuclei, and it also decondenses the chromatin of introduced somatic cell nuclei(1, 2) . Embryonic nuclei continue to have unusually decondensed chromatin until the midblastula transition, which occurs at 4,000 cells. Oocyte nucleoplasm is intermediate between the extreme states of metaphase and interphase egg cytoplasm. It will slowly induce limited chromatin decondensation of injected nuclei whereas oocyte cytoplasm does not decondense injected nuclei at all(3) . We have investigated the molecular basis of these differences.
Recently, the acidic protein
nucleoplasmin has been identified as the chromatin decondensation
factor responsible for the rapid first stage of sperm decondensation in Xenopus laevis(4, 5) . It promotes sperm
decondensation by binding and removing sperm basic proteins and
replacing them by histones H2A and H2B, resulting in the formation of
nucleosome cores(5, 6) . In this light, we have
investigated the possibility that the change in chromatin
decondensation activity observed between oocytes and eggs is mediated
by changes in the biochemical properties of nucleoplasmin. A major
change occurs to nucleoplasmin when Xenopus oocytes undergo
meiosis. It becomes very highly phosphorylated, gaining 14-20
phosphates/polypeptide chain and, therefore, 70-100
phosphates/nucleoplasmin pentamer(7, 8, 9) .
Since nucleoplasmin is the most abundant protein in the Xenopus oocyte nucleus, occurring at 5-8 mg/ml and representing 10%
of nuclear protein (10, 11, 12) , the final
level of phosphorylation of this single nuclear protein exceeds the
amount of phosphate in oocyte DNA by a factor of
10(7) .
We have compared the efficiency of sperm chromatin decondensation in oocyte versus egg extracts and in purified nucleoplasmin from oocytes and eggs. We observe that egg extracts and purified nucleoplasmin from eggs decondense sperm nuclei much faster than oocyte extracts or oocyte nucleoplasmin. We also observe that the remodeling of sperm chromatin, associated with decondensation, occurs in egg extracts and egg nucleoplasmin but not oocyte extracts or oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows decondensation and the remodeling of chromatin, indicating that nucleoplasmin phosphorylation is responsible for this difference in decondensation capacity between egg and oocyte nucleoplasmin.
Demembranated sperm nuclei were incubated at 100 ng of DNA/µl of extract in egg (13) and oocyte (14) HSSs. Samples were removed, stained with Hoechst 33258, and observed after various incubation times. After 5 min, decondensation was extensive in egg extract but minimal in oocyte extract. This difference persists but becomes less pronounced at 20 min, indicating that oocyte extracts are eventually able to decondense sperm chromatin but not at the physiological rate.
Fig. 1shows that egg extract is much more efficient at removal of sperm-specific basic proteins x and y than oocyte extract. Here, as has been shown previously(5, 6) , on decondensation in egg extract (Fig. 1A), removal of proteins x and y is accompanied by their replacement with histones H2A and H2B, finally giving approximately stoichiometric levels with histones H3 and H4 already present on the sperm chromatin. By contrast, when sperm nuclei are incubated in oocyte extract (Fig. 1B), proteins x and y are not removed nor are histones H2A and H2B assembled onto the DNA, consistent with the limited extent of decondensation observed with this extract.
Figure 1: Sperm basic proteins are removed more efficiently by egg extracts than by oocyte extracts. Demembranated Xenopus sperm nuclei were incubated for 10 min at 23 °C in egg high speed supernatant (A) or oocyte high speed supernatant (B) and then removed by centrifugation, rinsed, and analyzed by TAU SDS two-dimensional PAGE as described previously (6) . Note that the egg extract has removed sperm basic proteins x and y and increased the amount of histones H2A and H2B, whereas the oocyte extract has not. (The positions of the four core histones are indicated as 2a (H2A), 2b (H2B), 3 (H3), and 4 (H4) in A and by arrows alone in B.)
Similar differences in decondensation (Fig. 2) and chromatin remodeling (see Fig. 4, A and B) were observed when sperm nuclei were incubated with purified egg or oocyte nucleoplasmin. In egg nucleoplasmin, extensive decondensation of sperm nuclei was observed within 1 min of incubation (A) while nuclei incubated in oocyte nucleoplasmin for the same period of time showed little or no decondensation (C). Even after 10 min in purified oocyte nucleoplasmin (D), the extent of decondensation was still less than that observed in egg nucleoplasmin after 1 min (compare Fig. 2D with 2A).
Figure 2: Egg nucleoplasmin decondenses sperm nuclei more efficiently than oocyte nucleoplasmin. Demembranated Xenopus sperm nuclei were incubated for 1 min (A and C) or 10 min (B and D) in buffer containing 700 ng/µl nucleoplasmin from either eggs (egg npl, A and B) or oocytes (oocyte npl, C and D). An aliquot was then removed from each sample at the indicated time, diluted with Hoechst 33258 to label DNA, and photographed unfixed immediately. Scale bar, 25 µm.
Figure 4:
Phosphatase treatment of egg nucleoplasmin
decreases its chromatin remodeling activity. Xenopus sperm
nuclei were incubated and viewed as described in Fig. 3. The
remaining samples were then centrifuged, rinsed, and analyzed by TAU
SDS two-dimensional PAGE. Note that mock-treated egg nucleoplasmin has
removed most of the sperm basic proteins x and y (Egg NPL, A)
whereas untreated oocyte nucleoplasmin (Oocyte NPL, B) and
phosphatase-treated egg nucleoplasmin (Egg NPL +
P'ase, C) have not. (The positions of the four core histones
are indicated as 2a (H2A), 2b (H2B), 3 (H3),
and 4 (H4) in A and by arrows alone in B and C.)
Figure 3:
Phosphatase treatment of egg
nucleoplasmin decreases its chromatin decondensation activity. Egg
nucleoplasmin was treated with phosphatase or mock treated as
described under ``Experimental Procedures.'' Demembranated
sperm nuclei were then incubated in buffer D (BUFFER, A),
oocyte nucleoplasmin (OOCYTE NPL, B), mock-treated egg
nucleoplasmin (EGG NPL, C), or phosphatase-treated egg
nucleoplasmin (EGG NPL +
P'ASE, D) for 10
min. An aliquot was then removed from each sample, diluted with
propidium iodide to label DNA, and photographed unfixed immediately.
Scale bar, 10 µm.
Consistent with the observed differences in the efficiency of decondensation (Fig. 3), oocyte nucleoplasmin was also less efficient than mock-treated egg nucleoplasmin at remodeling sperm chromatin (Fig. 4). While the sperm basic proteins x and y were not removed with oocyte nucleoplasmin (Fig. 4B), most of these proteins were removed with mock-treated egg nucleoplasmin (Fig. 4A). However, phosphatase-treated egg nucleoplasmin was unable to remove x and y with the same efficiency as the mock-treated control (Fig. 4, compare C with A). Similar levels of the core histones H2A and H2B were observed in all samples (Fig. 4, A-C), indicating similar efficiencies of chromatin recovery following incubation. Purified H2A and H2B were not added to these incubations, and therefore, the levels of these core histones would not be expected to change during remodeling in mock-treated egg nucleoplasmin (Fig. 4A). As with decondensation, the extent of remodeling with phosphatase-treated egg nucleoplasmin was similar to that observed with oocyte nucleoplasmin (Fig. 4, compare C with B).
When Xenopus oocytes mature into eggs their
cytoplasm acquires the ability to decondense chromatin
rapidly(1, 2) . This property is obviously relevant to
the requirement for rapid sperm chromatin decondensation at
fertilization. We have shown that different abilities of eggs and
oocytes to decondense chromatin rapidly are mimicked by extracts and
that this difference can be attributed to the state of the
nucleoplasmin they each contain ( Fig. 2and Fig. 3). Egg
nucleoplasmin decondenses sperm chromatin at the rate seen in
fertilization, whereas oocyte nucleoplasmin does not ( Fig. 2and Fig. 3). Oocyte nucleoplasmin can cause very slow decondensation
but far below the physiological rate. We have shown that this
difference in decondensation activity can be explained by the state of
phosphorylation of nucleoplasmin. Nucleoplasmin phosphorylation is
correlated with its ability to decondense sperm chromatin and to remove
the basic proteins x and y. Egg and oocyte nucleoplasmin differ sharply
in both properties ( Fig. 3and Fig. 4). Dephosphorylation
of egg nucleoplasmin by treatment with phosphatase decreases the
rate of sperm decondensation (Fig. 3) and the extent of
chromatin remodeling (Fig. 4). Dimitrov et al.(18) have shown that remodeling of Xenopus sperm
chromatin is accompanied by core histone phosphorylation. While this
may also contribute to subsequent decondensation events, it cannot be
contributing to the decondensation shown in Fig. 2Fig. 3Fig. 4. The data we present in these
three figures are obtained in the absence of both ATP and magnesium
ions and therefore in the absence of de novo protein
phosphorylation.
After phosphorylation of nucleoplasmin during oocyte maturation, the phosphate is not removed on entry into S phase. It persists until the midblastula transition, at which time the level of phosphorylation falls again (data not shown but see (19) ). Interestingly, this is the time that the cell cycle elongates and that the exceptionally rapid cell proliferation of the early embryo slows down. This raises the interesting possibility that nucleoplasmin phosphorylation is required to keep chromatin maximally extended to allow access of replication initiation factors at close intervals on all regions of the DNA. Such close access may be essential to allow S phases of only 20 min as seen before the midblastula transition.
The observations we describe explain several phenomena in the literature concerning Xenopus development. First, they explain why oocyte nuclei decondense injected nuclei slowly while oocyte cytoplasm does not(2, 3) . Second, they explain why eggs decondense nuclei much more rapidly than oocytes(1, 2) . Third, they identify a biological function of the massive phosphorylation of nucleoplasmin, which occurs during oocyte maturation to form an egg (7, 8, 9) . It will be interesting to determine if similar mechanisms are involved in fertilization of other species, particularly mammals.
Note Added in Proof-After this work was completed corroborating data that agree well with our findings were published by Ohsumi et al. (Ohsumi, K., Shimada, A., Okumura, E., Kishimoto, T., and Kitagiri, C.(1995) Dev. Growth Differ.37, 329-336).