FERTILIZATION AND DEVELOPMENTAL BIOLOGY 281

acetate, germinal vesicles disappeared, but spindles failed to followed by stabilization of pH,. Addition of NH,CI, pH 8.2, to
form, and therefore, this is not a true progression to M phase. a final 10 mM resulted in a rapid and substantial alkalinization
Next we treated oocytes with NH4Cl, pH 8.2, which directly of the cytoplasm. As shown above, this treatment did not elicit
increases pH, (9). However, concentrations of NH4Cl up to 10 GVBD in prophase-arrested oocytes and only weakly elicited
mM in artificial seawater failed to induce GVBD. At 20 mM polar body formation in metaphase-arrested oocytes.
NH4CI germinal vesicles disappeared, but, as with acetate- We conclude that the pH, changes little, if at all, during
buffered seawater, spindles failed to form. Third, we tested GVBD and after egg activation in this organism and that pH,
NH&Z1 on metaphase-arrested oocytes. Concentrations up to has no obligatory role in GVBD or egg activation.
0.5 mM had no effect. Higher concentrations caused elevation We thank Dr. Peter J. S. Smith for access to the Attofluor
of some fertilization envelopes in a dose-dependent manner. microscope, supported by NCRR (P4 1 RR0 1395). W.R.E. was
In Chaetoptetxs oocytes fertilization envelope elevation re- supported by grants from the NIH (GM080 16) and the Council
sults from cortical microvillar elongation (10). At 10 mM for Tobacco Research, USA, Inc. (#3378). F.D. was supported
NH4Cl, some oocytes became ameboid, indicating the onset by an NSERC-Canada grant.
of pseudocleavage, a sign of differentiation without cleavage;
at 20 mM, approximately 20% of the oocytes formed protru- Literature Cited
sions at the animal pole, which resembled attempts at polar 1. Ikegami, S., T. S. Okada, and S. S. Koide. 1976. Dev. Growth
body formation. By 90 min after activation, many oocytes in D&v. 18: 33-43.
2. Lillie, F. R. 1902. W’ilh~dm Roux Arch. Entw. Org. 14: 477-499.
20 mM NH4CI had polar bodies, and some were ameboid; a
3. Epel, D. 1990. Cell DiJJr. Dev. 29: l- 12.
few in 10 mMNH4CI also had polar bodies and were ameboid. 4. Jaffe, L. F. 1985. Pp. 127-165 in The Fertilization Response qf
However, at these high concentrations, NHICl raises the pH, the Egg. C. B. Metz and A. Monroy, eds. Academic Press, New
to unphysiological levels (Fig. I) and may have other effects as York.
well (1 l), so it would be unwise to attribute this egg activation 5. Dub&, F. 1988. Dev. Bid. 126: 233-241.
solely to increased pH,. 6. Dub&, F., and W. R. Eckberg. 1996. Bid. Bull. 191: 279-280.
We used the Attofluor digital microscope to perform experi- 1. Johnson, C. H., and D. Epel. 1982. Dev. Bid 92: 46 I-469.
8. Peaucellier, G., A. Picard, J. J. Robert, J. P. Capony, and J. C.
ments to examine whether the pH, changes during GVBD or
egg activation (Fig. I). pH, underwent a slow downward drift Labbh. 1988. Exp. Ceil Res. 174: 7 I-88.
9. Epel, D., and F. Dub& 1987. Pp. 363-393 in Control cfAnimaI
during GVBD. We do not believe that this represents acidifi- Ceil Prol@ration. A. L. Boynton and H. L. Leffert. eds. Academic
cation of the cytoplasm associated with GVBD, as a similar Press, New York.
drift was observed in oocytes not undergoing GVBD. Addition 10. Eckberg, W. R., and Y. H. Kang. 1981. D~~J~renriarion 19: 154-
of KC1 to a final 100 mA4 activated eggs as evidenced by polar 160.
body formation. This involved a brief, more rapid, decrease I I. Dub&, F., and D. Epel. 1986. E.vp. Cell. Res. 162: 19 I-204.

Reference: Bid BUN. 191: 28 l-282. (October, 1996)

Gradual Loss of a DNA-Inducible Protein Kinase Activity From the

Cytoplasmic Extracts of Avbaciu Embryos
Jyotshnuhalu Kunungo, Benjamin Culhoun, Yoshihiko Tukeda, John A. Hurdin, and Howard Rasmussen
(Institute ofMoleclrlur Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912)

The DNA-dependent protein kinase is a serine/threonine
protein kinase that requires nicked double-stranded (ds) DNA
for its activity (I). It plays a crucial role in DNA repair (2)
but its role in development is not clear. DNA-activated protein
phosphorylation has been reported in extracts from cultured
human cells and the oocytes or the embryos of several marine
invertebrates (3). Herein, we show that the cytoplasmic extracts
of sea urchin (Arbacia punctdata) eggs contain a kinase that is
inducible by exogenous dsDNA only after fertilization and is
not detectable in cytoplasmic extracts prepared from blastulae,
gastrulae, and plutei.
Homogenate-catalyzed DNA-dependent phosphorylation
assays performed in cytoplasmic extracts of eggs and embryos
demonstrate that unfertilized eggs lack the DNA-inducible ki-
nase activity (Fig. 1). However, the enzyme activity is seen in
extracts from fertilized eggs. A gradual decline in the cyto-
plasmic enzyme activity is observed through 50 (first cleavage)
and 103 (second cleavage) minutes post-fertilization. Cyto-
plasmic extracts prepared from the blastulae, gastrulae, and
plutei also lack the DNA-inducible phosphorylation activity.
Unfortunately, we could not perform a meaningful enzyme as-
say in whole cell extracts because cell sonication generates
nicked nuclear DNA. For this reason, the control reaction in
the absence of DNA could not be performed in these extracts
since a-casein is a non-specific substrate for DNA-dependent
protein kinase and can be phosphorylated by other kinases.
282 REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS
+ - + - + - + DNA - + - + - + - + - + - + DNA
L ’ .

Egg lo’ Post-Fert. Blastula Gestrule Pluteus

50’ Post-Fert. 103’ Post-Fert.

Figure 1. Autoradiogram shows variation in DNA-dependent protein phosphorylation activity during embryonic development. Cytoplasmic
extracts from eggs and embryos were prepared as already reported (4). A I5 ~1 reaction required 10 ~1 of cytoplasmic extract, 75 ng of sonicated calf
thymus DNA, 15 pg dephosphorylated cr-casein (Sigma) as substrate, 2 mM MgCl~, and 130 ti ATP. The reaction was carried out at IS-C, and
started by adding 10 pCi of [gamma-3ZP]ATP (6000 Ci/mmol) (NEN, Du Pont). The phosphorylation reaction (I pr) was analyzed on a 10%
polyacrylamidegel (SDS-PAGE). E-extract only (without any exogenous substrate); C-extract with exogenous cu-casein added as an exogenous
substrate. Arrows indicate the position ofphosphorylatedcasein. Absence andpresence ofdsDNA in the extract are indicated by - and +, respectively.

Therefore,a specificsubstrateisneededto assayfor the specific helpful discussions.

Grant support camefrom NIH (AR 32549
enzyme activity in wholecell extracts. to JAH), Mason Trust, the Arthritis Foundation.
Theseresultsdemonstratethe presenceofa DNA-dependent
Literature Cited
protein kinasein the cytoplasmof eggsand embryosof seaur-
chin. The rapid appearanceof activity in the cytoplasmic ex- 1. Carter, T., I. Vancurova, I. Sun, W. Lou, and S. DeLeon. 1990.
Mol. Cell. Biol. 10: 6460-6411.
tracts from fertilized eggscould be dueto either the synthesisof
2. Finnie, N. J., T. M. Gottlieb, T. Blunt, P. A. Jeggo, and S. P. Jack-
the enzyme itself or the activation of preexisting inactive en- son. 1995. Proc. Natl. Acad. Sci. USA 92: 320-324.
zyme by post-translationalmodification. However, we can not 3. Walker, A. I., T. Hunt, R. J. Jackson, and C. W. Anderson. 1985.
rule out the possibility that the enzyme is nuclear in unfertil- EMBO J. 4: 139-145.
ized eggsand isreleasedafter fertilization. 4. Ballinger, D. G., S. J. Bray, and T. Hunt. 1984. Dev. Biol. 101:
We thank Dr. Carl Anderson and Dr. William Dynan for 192-200.