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Compartmentalization of DNA polymerases, α and γ, in maturing oocytes of the loach (Misgurnus fossilis L.)
Cell Differentiation, 13 (1983) 87-91
87
Elsevier ScientificPublishers Ireland, Ltd.
Compartmentalization of DNA polymerases, a and T, in maturing oocytes of the loach ( Misgurnus fossilis L.) V.S. Mikhailov, A.A. K o s t o m a r o v a , D.B. G u l y a m o v a n d E.F. K n y a z e v a N.K. Koltzov Institute of Developmental Biology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R.
(Accepted 22 April 1983)
The nuclei (germinal vesicles - GV) were isolated from the maturing oocytes of the loach (Misgurnus fossills L.) by means of microsurgery. The activities of DNA polymerases, ct and V, in the extracts of GV and enucleated oocytes were determined by two techniques: from crude extracts and following ultracentrifugation of the extracts in glycerol gradients. Nearly 80% of the total ct-polymerase activity was shown to be associated with GV while the T-polymerase activity was revealed in the cytoplasm. The accumulation of non-histone nuclear proteins in the GV during oocyte maturation was demonstrated by means of autoradiography.
maturing oocytes; DNA polymerases; intracellular location
Introduction
Since the DNA polymerases were discovered in animal cells, a number of attempts have been made to describe the intracellular localization of the enzymes, and this knowledge of DNA polymerase compartmentalization seems of importance for the elucidation of the control mechanisms of DNA synthesis. However, the presence of different forms of DNA polymerases in the cell and differences in their extractability from cell compartments in the course of cell fractionation have made the interpretation of the data obtained, in some cases, ambiguous. Nevertheless, by means of several techniques, i.e. fractionation in organic solvents (Foster and Gurney, 1976), breakage of the cell into cyto- and caryoplasts in the presence of cytochalasin B (Herrick et al., 1976), and microsurgical enucleation of amphibian oocytes (Grippo et al., 1977; Fox et al., 1980), DNA polymerase t~ was shown to be localized mainly in the cell nucleus. This information was recently confirmed
by immunocytochemical determinations by using monoclonal antibodies specific for human DNA polymerase a (Bensch et al., 1982). Contrary to this, Brown et al. (1981) reported the bulk of a-polymerase in bovine fibroblasts to be located in the perinuclear region of the cytoplasm. The data concerning DNA polymerase T compartmentalization are contradictory as well. Several authors describe DNA polymerase T in mitochondria only (Tanaka and Koike, 1978; BaT~icalupo, 1979; Fox et al., 1980), whereas others suggest the presence of some portions of the enzyme in the cell nuclei (Bertazzoni et al., 1977; HObscher et al., 1977; Habara et al., 1979; Kalf et al., 1980). Hence the investigation of the intracellular localization of DNA polymerases remains noteworthy. The aim of the present study was to elucidate the distribution of DNA polymerases, a and T, between the nucleus and the cytoplasm in the oocytes of a teleost fish, the loach (Misgurnus fossilis L.). DNA polymerase fl compartmentalization was not determined due to an extremely low
0045-6039/83/$03.00 © 1983 ElsevierScientificPublishersIreland, Ltd.
88 activity of this enzyme in full-grown oocytes and mature eggs of the loach (Mikhailov et al., 1980). The data obtained have shown that in maturing oocytes of the loach, D N A polymerase a is localized mainly in the germinal vesicle (GV), while D N A polymerase "r is virtually absent from this cell compartment. On the contrary, DNA polymerase 7 is shown to be located in the oocyte cytoplasm.
Materials and Methods
Biochemistry The oocytes were obtained from the loach females after the injection of the gonadotropic hormone choriogonin (G. Richter) according to Neyfakh (1959). Maturing oocytes at the preovulation stage, i.e. just before the germinal vesicle breakdown (GVBD), were obtained at 16°C, 18-19 h after the hormone injection. The ovaries were r e m o v e d into m o d i f i e d , d o u b l e - s t r e n g t h Holtfreter's solution (Ca 2÷ replaced by Mg2+), rinsed, and cut into small pieces. Then the oocytes were carefully extracted from the ovary pieces with steel needles and rinsed several times with the same solution. The isolated oocytes were pierced with a needle in the animal pole region. By a slight pressure of the needles the GVs were extruded and then were immediately collected with a micropipette into an appropriate cold extraction buffer (0.8 ml at 0°C). The enucleated and intact oocytes were also transferred into the cold buffer. Burst or leaking oocytes were discarded. Each sample contained 100 intact oocytes or 100 enucleated oocytes (cytoplasmic fraction), or 100 germinal vesicles. All GVs, enucleated oocytes, and intact oocyte material in the experiment came from the ovaries of the same female. The buffer for DNA polymerase a extraction (buffer A) contained 200 mM K-phosphate, pH 7.5; 5% glycerol; 5 mM 2-mercaptoethanol; 1 mM EDTA; 0.25 mM phenylmethylsulfonyl fluoride (PMSF); 0.5% Triton X-100. The buffer for DNA polymerase "~ extraction (buffer B) contained the same components though KC1 was added to final
concentration 1 M, and K-phosphate was replaced by Tris-HC1 100 mM, pH 7.5. After homogenization in a 'Potter S' homogenizer (Braun) homogenates were sonicated for 10 s. Aliquots of 1, 2, and 4/~1 were diluted to 20/~1 with buffer A and taken for the DNA polymerase c~ assay. The reaction was performed in a sample of 100/~1 containing the following additional components: MgCI 2 8 mM; bovine serum albumin 200/~g/ml; activated calf thymus DNA 125 /~g/ml (Aposhian and Kornberg, 1962); spermidine 1 mM; dTTP, dCTP, d G T P each at 25 ~M; [3H]dATP 15 #M, 10 # C i / m l . Aliquots of 2.5 and 5/~1 diluted to 10 ~tl with buffer B were taken for DNA polymerase ~, assay. The reaction was performed in a sample of 50 #1 containing the additional components: MnCI 2 1.2 mM; bovine serum albumin 200 # g / m l ; poly(A) 2 U / m l ; olygo(dT) 0.1 U / m l ; [3H]dTTP 7.5/~M, 20/~Ci/ml. After the reaction was run for 60 min at 25°C the acid-insoluble radioactivity was estimated as described earlier (Mikhailov and Gulyamov, 1981a). The incorporation of 1 pmol of d N M P into DNA during 60 min at 25°C was taken as a unit of DNA polymerase activity. The concentration of protein was determined according to Lowry (Lowry et al., 1951).
A utoradiography 500 /~Ci of [3H]tryptophan (spec. act. 3.2 Ci/mmol) was injected into loach females simultaneously with choriogonin (200 MU) at 18°C. The oocytes were obtained from three females. Pieces of ovaries were fixed with Bouin's fluid at 20 and 25 h after injection and embedded in paraffin wax by the routine method. Serial sections 5-7 /~m thick were covered with liquid, radiosensitive emulsion (type 'M', NIIKHimFoto, Moscow) and incubated for 21 days at 4°C. Amidol was used as developer. After development the sections were stained with Carrachi haematoxylin. The number of silver grains was counted over equal nuclear and cytoplasmic sectors, for 50 sectors in 25 oocytes. The values obtained were recalculated for a nuclear and cytoplasmic area equal to 100/~m 2. The mean value a n d standard error were calculated for each observation point.
89
Results
Biochemical analysis The results of the estimation of D N A polymerase, a and y, activities are given in the table. The table shows that the GV contains nearly 80% of the oocyte D N A polymerase a activity. The quantity of protein in the GV is 4.0 #g, which accounts for 2.5% of the total protein content of the oocyte. As follows from the above data, DNA polymerase a specific activity in the GV is about 100 times greater than that in the cytoplasm. On the contrary, D N A polymerase 3, activity is revealed to be primarily in the cytoplasm (Table I). The data presented in Table I were obtained for crude extracts. However, the results obtained for crude extracts need to be proved by some other methods, since inhibitors or activators of D N A polymerases present in the extracts may change D N A polymerase activity. Therefore, special experiments were performed to analyse the extracts by glycerol gradient centrifugation as described earlier (Mikhailov et al., 1980). The data obtained (Fig. 1) show that the maturing oocytes reveal both a high activity of D N A polymerase a and the presence of D N A polymerase 3, activity. The GV was shown to contain D N A polymerase a but no D N A polymerase 3,, whereas in the cytoplasm only D N A polymerase 3, was revealed. Thus both kinds of experiments - glycerol density centrifugation (Fig. 1) and analysis of crude extracts (Table I) have revealed the nuclear localization of the main portion of DNA polymerase a and the cytoplasmic localization of DNA polymerase 3' in the maturing oocytes.
E
E
O.
O.
'" ;
.-
3
_ ?,D.n
A~
~
b
-
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0
0
0 L
0 ~
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~
-~ 2
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< Z 123
2
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bottom
~
10
20
FRACTION
NUMBER
Fig. 1. Distribution of D N A polymerase activity of extracts of (a) whole oocytes, (b) germinal vesicles, and (c) enucleated oocytes following 10-30% glycerol gradient centrifugation. D N A polymerase a activity was assayed on activated DNA, K-phosphate, 33 m M (1). D N A polymerase ~, activity was assayed on poly(A).oligo(dT) (2). Arrow shows bovine serum albumin in the gradient (4.3 s). The sedimentation coefficient for loach D N A polymerases is equal to: a = 6.8 s, "y = 6.3 s (Mikhallov and Gulyamov, 1981a).
1OO ~m
TABLE I Intracellular localization of the D N A polymerases, a and 3', in maturing oocytes of the loach (Misgurnua fossilis) Oocyte fraction
Protein (#g)
Activity of D N A polymerases (U) a
Intact oocyte Germinal vesicle Enucleated oocyte (cytoplasm)
1.6 x 102 4.0
5.4 X 102 4.2 x 102
1.5 x 102
0.9 x 102
"y 9.6 0.16 10.5
Fig. 2. Nuclear accumulation of non-histone proteins labeled by [3H]tryptophan in the course of loach oocyte maturation (25 h of labeling). 1 = oocyte membrane; 2 = yolk granules; 3 = caryoplasm; 4 = nucleoli.
90
Autoradiography The data presented above point to a high affinity of DNA polymerase a for the GV of loach oocytes. Several other fractions of nonhistone nuclear proteins may be accumulated by GV during the oocyte maturation. This process can be analyzed by means of autoradiography. The results of grain counts 20 h and 25 h after [3H]tryptophan injection into the loach females have shown that the grain densities per 100/~m 2 of nuclear area are 57 + 8 and 77 + 10, respectively. In the cytoplasm the corresponding values are 32 ___4 and 31 ___4, i.e. the grain density in the GV is 1.8-2.7 times higher than that in the cytoplasm (Fig. 2). The data obtained show that during oocyte maturation non-histone proteins are accumulated in the GV. Among the population of non-histone nuclear proteins molecules of DNA polymerase a are present.
Discussion
The intracellular location of different DNA polymerases is obviously related to their function in DNA synthesis. It was previously shown that in the early loach embryos DNA polymerase a took part in nuclear DNA replication (Mikhailov and Gulyamov, 1981b). The involvement of a-polymerase in nuclear DNA synthesis suggests nuclear localization of this enzyme in the cell. The latter was confirmed by the analysis of DNA polymerase a compartmentalization in the loach oocytes. A major portion of a-polymerase activity was revealed in the nucleus (GV) of maturing loach oocytes (Table I, Fig. 1). In somatic cells D N A polymerase a is generally suggested to be localized in the nucleus also (Lynch et al., 1975; Foster and Gurney, 1976; Herrick et al., 1976; Bensch et al., 1982). Concerning the localization of DNA polymerases in germ cells few data are available. In the large oocytes of Xenopus laeois about 90% of DNA polymerase a activity is localized in GV (Martini et al., 1976; Grippo et al., 1977; Fox et al., 1980). However, in sea urchin eggs a large portion of a-polymerase activity is stored in the cytoplasm (Shioda et al., 1977). The passage of
a-polymerase from the cytoplasmic compartment to the nuclear one occurs in rapidly dividing sea urchin embryonic cells (Shioda et al., 1982). Nearly all a-polymerase activity was found to be in the GVs of full-grown oocytes of the toad, Bufo bufo japonicus (Nagano et al., 1982). It has been reported that during oocyte maturation in the toad D N A polymerase a is released into the cytoplasm and binds to the endoplasmic reticulum at the time of GVBD (Nagano et al., 1982). As shown above, in maturing teleostean oocytes DNA polymerase a is mainly located in the GV as well. The accumulation of DNA polymerase a, as well as some other DNA-binding proteins by GV, occurs despite the fact that GV disintegrates during oocyte maturation. The nuclear components causing DNA polymerase a accumulation by the GV remain unknown. The capacity of GV chromatin is obviously insufficient for retention of the whole pool of DNA-binding proteins. Nevertheless, a large portion of both basic and acidic proteins which participate in nuclear DNA packing at the early stages of development are stored in GV (Bonner, 1975). It was suggested that the nuclear skeleton may be an appropriate structure for the retention of DNA polymerase a (Smith and Berezney, 1980; Mikhailov and Tsanev, 1983). An extremely low activity of DNA polymerase fl in the maturing loach oocytes (Mikhailov et al., 1980) does not permit us to elucidate the location of this enzyme. In amphibian or sea urchin oocytes most of fl-polymerase activity was observed in the cytoplasm (Shioda et al., 1977; Fox et al., 1980; Hobart and Infante, 1980). The mitochondrial location of "t-polymerase was revealed in mammalian cells (Bolden et al., 1977; Tanaka and Koike, 1978), as well as in amphibian oocytes (Bazzicalupo, 1979; Fox et al., 1980). The conclusion that, in the loach oocytes, DNA polymerase ~/ is entirely localized in the cytoplasm agrees with a possible mitochondrial location of this enzyme. The data obtained are consistent with the fact that in the loach embryonic cells the whole D N A polymerase 3, activity is associated with mitochondria (Mikhailov et al., 1983). Since both DNA polymerase a and DNA polymerase "t are synthesized in the cytoplasm, a selective transport of D N A polymerase a to the nuclei
91 a n d D N A p o l y m e r a s e 3' to m i t o c h o n d r i a m a y b e p r o v i d e d b y s o m e f a c t o r s . H o w e v e r , t h e n a t u r e of these presumed factors remains unknown.
Acknowledgements W e w i s h to t h a n k Prof. I.B. Z b a r s k y a n d D r . O . G . S t r o e v a for t h e i r i n t e r e s t in the p r e s e n t w o r k .
References Aposhian, H.V. and A. Kornberg: J. Biol. Chem. 237, 519-525 (1962). Bazzicalupo, P.: Biochem. Biophys. Res. Commun. 87, 1218-1225 (1979). Bensch, K.G., S. Tanaka, S.-Z. Hu, T.S.-F. Wang and D. Korn: J. Biol. Chem. 257, 8391-8396 (1982). Bertazzoni, U., A.I. Scovassi and G.M. Brun: Eur. J. Biochem. 81, 237-248 (1977). Boiden, A., G. Pedrali-Noy and A. Weissbach: J. Biol. Chem. 252, 3351-3356 (1977). Bonner, W.M.: J. Cell Biol. 64, 431-437 (1975). Brown, McK., F.J. Bollum and L.M.S. Chang: Proc. Natl. Acad. Sci. U.S.A. 78, 3049-3052 (1981). Foster, D.N. and T. Gurney Jr.: J. Biol. Chem. 251, 7893-7898 (1976). Fox, A.M., C.B. Breaux and R.M. Benbow: Dev. Biol. 80, 79-95 (1980). Grippo, P., C. Taddei, G. Locorotondo and A. Gambino-Giuffrida: Exp. Cell Res. 109, 247-252 (1977). Habara, A., H. Nagano and Y. Mano: Biochim. Biophys. Acta 561, 17-29 (1979).
Herrick, G., B.B. Spear and G. Veomett: Proc. Natl. Acad. Sci. U.S.A. 73, 1136-1139 (1976). Hobart, P.M. and A.A. Infante: Biochim. Biophys. Acta 607, 256-268 (1980). Hiibscher, U., C.C. Kuenzle and S. Spadari: Eur. J. Biochem. 81,249-258 (1977). Kalf, G.F., R.F. Maguire, R.M. Metrione and T.R. Koszalka: Dev. Biol. 77, 253-270 (1980). Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall: J. Biol. Chem. 193, 265-275 (1951). Lynch, W.E., S. Surrey and I. Liberman: J. Biol. Chem. 250, 8179-8183 (1975). Martini, G., F. Tato, D.G. Attardi and G.P. Tocchini-Valentini: Biochem. Biophys. Res. Commun. 72, 875-878 (1976). Mikhailov, V.S. and D.B. Gulyamov: Biochemistry (Russian) 46, 1539-1547 (1981a). Mikhailov, V.S. and D.B. Gulyamov: Dokl. Akad. Nauk SSSR (Russian) 257, 229-233 (1981b). Mikhailov, V.S. and R. Tsanev: Int. J. Biochem. 15, 855-859 (1983). Mikhailov, V.S., D.B. Gulyamov and I.IK Zbarsky: Dold. Akad. Nauk SSSR (Russian) 254, 503-506 (1980). Mikhailov, V.S., D.B. Gulyamov, A.V. Peskin and L.S. Filatova: Biochemistry (Russian) 48, 1012-1018 (1983). Nagano, H., K. Okano, S. Ikegami and C. Katagiri: Biochem. Biophys. Res. Commun. 106, 683-690 (1982). Neyfakh, A.A.: Zh. Obsch. Biol. (Russian) 20, 202-213 (1959). Shioda, M., H. Nagano and Y. Mano: Biochem. Biophys. Res. Commun. 78, 1362-1368 (1977). Shioda, M., H. Nagano and Y. Mano: Dev. Biol. 91, 111-120 (1982). Smith, H.C. and R. Berezney: Biochem. Biophys. Res. Commun, 97, 1541-1547 (1980). Tanaka, S. and K. Koike: Biochem. Biophys. Res. Commun. 81, 791-797 (1978).
87
Elsevier ScientificPublishers Ireland, Ltd.
Compartmentalization of DNA polymerases, a and T, in maturing oocytes of the loach ( Misgurnus fossilis L.) V.S. Mikhailov, A.A. K o s t o m a r o v a , D.B. G u l y a m o v a n d E.F. K n y a z e v a N.K. Koltzov Institute of Developmental Biology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R.
(Accepted 22 April 1983)
The nuclei (germinal vesicles - GV) were isolated from the maturing oocytes of the loach (Misgurnus fossills L.) by means of microsurgery. The activities of DNA polymerases, ct and V, in the extracts of GV and enucleated oocytes were determined by two techniques: from crude extracts and following ultracentrifugation of the extracts in glycerol gradients. Nearly 80% of the total ct-polymerase activity was shown to be associated with GV while the T-polymerase activity was revealed in the cytoplasm. The accumulation of non-histone nuclear proteins in the GV during oocyte maturation was demonstrated by means of autoradiography.
maturing oocytes; DNA polymerases; intracellular location
Introduction
Since the DNA polymerases were discovered in animal cells, a number of attempts have been made to describe the intracellular localization of the enzymes, and this knowledge of DNA polymerase compartmentalization seems of importance for the elucidation of the control mechanisms of DNA synthesis. However, the presence of different forms of DNA polymerases in the cell and differences in their extractability from cell compartments in the course of cell fractionation have made the interpretation of the data obtained, in some cases, ambiguous. Nevertheless, by means of several techniques, i.e. fractionation in organic solvents (Foster and Gurney, 1976), breakage of the cell into cyto- and caryoplasts in the presence of cytochalasin B (Herrick et al., 1976), and microsurgical enucleation of amphibian oocytes (Grippo et al., 1977; Fox et al., 1980), DNA polymerase t~ was shown to be localized mainly in the cell nucleus. This information was recently confirmed
by immunocytochemical determinations by using monoclonal antibodies specific for human DNA polymerase a (Bensch et al., 1982). Contrary to this, Brown et al. (1981) reported the bulk of a-polymerase in bovine fibroblasts to be located in the perinuclear region of the cytoplasm. The data concerning DNA polymerase T compartmentalization are contradictory as well. Several authors describe DNA polymerase T in mitochondria only (Tanaka and Koike, 1978; BaT~icalupo, 1979; Fox et al., 1980), whereas others suggest the presence of some portions of the enzyme in the cell nuclei (Bertazzoni et al., 1977; HObscher et al., 1977; Habara et al., 1979; Kalf et al., 1980). Hence the investigation of the intracellular localization of DNA polymerases remains noteworthy. The aim of the present study was to elucidate the distribution of DNA polymerases, a and T, between the nucleus and the cytoplasm in the oocytes of a teleost fish, the loach (Misgurnus fossilis L.). DNA polymerase fl compartmentalization was not determined due to an extremely low
0045-6039/83/$03.00 © 1983 ElsevierScientificPublishersIreland, Ltd.
88 activity of this enzyme in full-grown oocytes and mature eggs of the loach (Mikhailov et al., 1980). The data obtained have shown that in maturing oocytes of the loach, D N A polymerase a is localized mainly in the germinal vesicle (GV), while D N A polymerase "r is virtually absent from this cell compartment. On the contrary, DNA polymerase 7 is shown to be located in the oocyte cytoplasm.
Materials and Methods
Biochemistry The oocytes were obtained from the loach females after the injection of the gonadotropic hormone choriogonin (G. Richter) according to Neyfakh (1959). Maturing oocytes at the preovulation stage, i.e. just before the germinal vesicle breakdown (GVBD), were obtained at 16°C, 18-19 h after the hormone injection. The ovaries were r e m o v e d into m o d i f i e d , d o u b l e - s t r e n g t h Holtfreter's solution (Ca 2÷ replaced by Mg2+), rinsed, and cut into small pieces. Then the oocytes were carefully extracted from the ovary pieces with steel needles and rinsed several times with the same solution. The isolated oocytes were pierced with a needle in the animal pole region. By a slight pressure of the needles the GVs were extruded and then were immediately collected with a micropipette into an appropriate cold extraction buffer (0.8 ml at 0°C). The enucleated and intact oocytes were also transferred into the cold buffer. Burst or leaking oocytes were discarded. Each sample contained 100 intact oocytes or 100 enucleated oocytes (cytoplasmic fraction), or 100 germinal vesicles. All GVs, enucleated oocytes, and intact oocyte material in the experiment came from the ovaries of the same female. The buffer for DNA polymerase a extraction (buffer A) contained 200 mM K-phosphate, pH 7.5; 5% glycerol; 5 mM 2-mercaptoethanol; 1 mM EDTA; 0.25 mM phenylmethylsulfonyl fluoride (PMSF); 0.5% Triton X-100. The buffer for DNA polymerase "~ extraction (buffer B) contained the same components though KC1 was added to final
concentration 1 M, and K-phosphate was replaced by Tris-HC1 100 mM, pH 7.5. After homogenization in a 'Potter S' homogenizer (Braun) homogenates were sonicated for 10 s. Aliquots of 1, 2, and 4/~1 were diluted to 20/~1 with buffer A and taken for the DNA polymerase c~ assay. The reaction was performed in a sample of 100/~1 containing the following additional components: MgCI 2 8 mM; bovine serum albumin 200/~g/ml; activated calf thymus DNA 125 /~g/ml (Aposhian and Kornberg, 1962); spermidine 1 mM; dTTP, dCTP, d G T P each at 25 ~M; [3H]dATP 15 #M, 10 # C i / m l . Aliquots of 2.5 and 5/~1 diluted to 10 ~tl with buffer B were taken for DNA polymerase ~, assay. The reaction was performed in a sample of 50 #1 containing the additional components: MnCI 2 1.2 mM; bovine serum albumin 200 # g / m l ; poly(A) 2 U / m l ; olygo(dT) 0.1 U / m l ; [3H]dTTP 7.5/~M, 20/~Ci/ml. After the reaction was run for 60 min at 25°C the acid-insoluble radioactivity was estimated as described earlier (Mikhailov and Gulyamov, 1981a). The incorporation of 1 pmol of d N M P into DNA during 60 min at 25°C was taken as a unit of DNA polymerase activity. The concentration of protein was determined according to Lowry (Lowry et al., 1951).
A utoradiography 500 /~Ci of [3H]tryptophan (spec. act. 3.2 Ci/mmol) was injected into loach females simultaneously with choriogonin (200 MU) at 18°C. The oocytes were obtained from three females. Pieces of ovaries were fixed with Bouin's fluid at 20 and 25 h after injection and embedded in paraffin wax by the routine method. Serial sections 5-7 /~m thick were covered with liquid, radiosensitive emulsion (type 'M', NIIKHimFoto, Moscow) and incubated for 21 days at 4°C. Amidol was used as developer. After development the sections were stained with Carrachi haematoxylin. The number of silver grains was counted over equal nuclear and cytoplasmic sectors, for 50 sectors in 25 oocytes. The values obtained were recalculated for a nuclear and cytoplasmic area equal to 100/~m 2. The mean value a n d standard error were calculated for each observation point.
89
Results
Biochemical analysis The results of the estimation of D N A polymerase, a and y, activities are given in the table. The table shows that the GV contains nearly 80% of the oocyte D N A polymerase a activity. The quantity of protein in the GV is 4.0 #g, which accounts for 2.5% of the total protein content of the oocyte. As follows from the above data, DNA polymerase a specific activity in the GV is about 100 times greater than that in the cytoplasm. On the contrary, D N A polymerase 3, activity is revealed to be primarily in the cytoplasm (Table I). The data presented in Table I were obtained for crude extracts. However, the results obtained for crude extracts need to be proved by some other methods, since inhibitors or activators of D N A polymerases present in the extracts may change D N A polymerase activity. Therefore, special experiments were performed to analyse the extracts by glycerol gradient centrifugation as described earlier (Mikhailov et al., 1980). The data obtained (Fig. 1) show that the maturing oocytes reveal both a high activity of D N A polymerase a and the presence of D N A polymerase 3, activity. The GV was shown to contain D N A polymerase a but no D N A polymerase 3,, whereas in the cytoplasm only D N A polymerase 3, was revealed. Thus both kinds of experiments - glycerol density centrifugation (Fig. 1) and analysis of crude extracts (Table I) have revealed the nuclear localization of the main portion of DNA polymerase a and the cytoplasmic localization of DNA polymerase 3' in the maturing oocytes.
E
E
O.
O.
'" ;
.-
3
_ ?,D.n
A~
~
b
-
o
0
0
0 L
0 ~
E
E
~
-~ 2
< Z 121
< Z 123
2
C
L22_ 1
~__
bottom
~
10
20
FRACTION
NUMBER
Fig. 1. Distribution of D N A polymerase activity of extracts of (a) whole oocytes, (b) germinal vesicles, and (c) enucleated oocytes following 10-30% glycerol gradient centrifugation. D N A polymerase a activity was assayed on activated DNA, K-phosphate, 33 m M (1). D N A polymerase ~, activity was assayed on poly(A).oligo(dT) (2). Arrow shows bovine serum albumin in the gradient (4.3 s). The sedimentation coefficient for loach D N A polymerases is equal to: a = 6.8 s, "y = 6.3 s (Mikhallov and Gulyamov, 1981a).
1OO ~m
TABLE I Intracellular localization of the D N A polymerases, a and 3', in maturing oocytes of the loach (Misgurnua fossilis) Oocyte fraction
Protein (#g)
Activity of D N A polymerases (U) a
Intact oocyte Germinal vesicle Enucleated oocyte (cytoplasm)
1.6 x 102 4.0
5.4 X 102 4.2 x 102
1.5 x 102
0.9 x 102
"y 9.6 0.16 10.5
Fig. 2. Nuclear accumulation of non-histone proteins labeled by [3H]tryptophan in the course of loach oocyte maturation (25 h of labeling). 1 = oocyte membrane; 2 = yolk granules; 3 = caryoplasm; 4 = nucleoli.
90
Autoradiography The data presented above point to a high affinity of DNA polymerase a for the GV of loach oocytes. Several other fractions of nonhistone nuclear proteins may be accumulated by GV during the oocyte maturation. This process can be analyzed by means of autoradiography. The results of grain counts 20 h and 25 h after [3H]tryptophan injection into the loach females have shown that the grain densities per 100/~m 2 of nuclear area are 57 + 8 and 77 + 10, respectively. In the cytoplasm the corresponding values are 32 ___4 and 31 ___4, i.e. the grain density in the GV is 1.8-2.7 times higher than that in the cytoplasm (Fig. 2). The data obtained show that during oocyte maturation non-histone proteins are accumulated in the GV. Among the population of non-histone nuclear proteins molecules of DNA polymerase a are present.
Discussion
The intracellular location of different DNA polymerases is obviously related to their function in DNA synthesis. It was previously shown that in the early loach embryos DNA polymerase a took part in nuclear DNA replication (Mikhailov and Gulyamov, 1981b). The involvement of a-polymerase in nuclear DNA synthesis suggests nuclear localization of this enzyme in the cell. The latter was confirmed by the analysis of DNA polymerase a compartmentalization in the loach oocytes. A major portion of a-polymerase activity was revealed in the nucleus (GV) of maturing loach oocytes (Table I, Fig. 1). In somatic cells D N A polymerase a is generally suggested to be localized in the nucleus also (Lynch et al., 1975; Foster and Gurney, 1976; Herrick et al., 1976; Bensch et al., 1982). Concerning the localization of DNA polymerases in germ cells few data are available. In the large oocytes of Xenopus laeois about 90% of DNA polymerase a activity is localized in GV (Martini et al., 1976; Grippo et al., 1977; Fox et al., 1980). However, in sea urchin eggs a large portion of a-polymerase activity is stored in the cytoplasm (Shioda et al., 1977). The passage of
a-polymerase from the cytoplasmic compartment to the nuclear one occurs in rapidly dividing sea urchin embryonic cells (Shioda et al., 1982). Nearly all a-polymerase activity was found to be in the GVs of full-grown oocytes of the toad, Bufo bufo japonicus (Nagano et al., 1982). It has been reported that during oocyte maturation in the toad D N A polymerase a is released into the cytoplasm and binds to the endoplasmic reticulum at the time of GVBD (Nagano et al., 1982). As shown above, in maturing teleostean oocytes DNA polymerase a is mainly located in the GV as well. The accumulation of DNA polymerase a, as well as some other DNA-binding proteins by GV, occurs despite the fact that GV disintegrates during oocyte maturation. The nuclear components causing DNA polymerase a accumulation by the GV remain unknown. The capacity of GV chromatin is obviously insufficient for retention of the whole pool of DNA-binding proteins. Nevertheless, a large portion of both basic and acidic proteins which participate in nuclear DNA packing at the early stages of development are stored in GV (Bonner, 1975). It was suggested that the nuclear skeleton may be an appropriate structure for the retention of DNA polymerase a (Smith and Berezney, 1980; Mikhailov and Tsanev, 1983). An extremely low activity of DNA polymerase fl in the maturing loach oocytes (Mikhailov et al., 1980) does not permit us to elucidate the location of this enzyme. In amphibian or sea urchin oocytes most of fl-polymerase activity was observed in the cytoplasm (Shioda et al., 1977; Fox et al., 1980; Hobart and Infante, 1980). The mitochondrial location of "t-polymerase was revealed in mammalian cells (Bolden et al., 1977; Tanaka and Koike, 1978), as well as in amphibian oocytes (Bazzicalupo, 1979; Fox et al., 1980). The conclusion that, in the loach oocytes, DNA polymerase ~/ is entirely localized in the cytoplasm agrees with a possible mitochondrial location of this enzyme. The data obtained are consistent with the fact that in the loach embryonic cells the whole D N A polymerase 3, activity is associated with mitochondria (Mikhailov et al., 1983). Since both DNA polymerase a and DNA polymerase "t are synthesized in the cytoplasm, a selective transport of D N A polymerase a to the nuclei
91 a n d D N A p o l y m e r a s e 3' to m i t o c h o n d r i a m a y b e p r o v i d e d b y s o m e f a c t o r s . H o w e v e r , t h e n a t u r e of these presumed factors remains unknown.
Acknowledgements W e w i s h to t h a n k Prof. I.B. Z b a r s k y a n d D r . O . G . S t r o e v a for t h e i r i n t e r e s t in the p r e s e n t w o r k .
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