- Email: [email protected]
A comparison of RNA and protein synthesis in fertilized and unfertilized eggs of Urechis caupo
DEVELOPMENTAL
BIOLOGY
19,
A Comparison
482497
(1969)
of RNA
Fertilized
and
and
Unfertilized
n[EREDITH of Biological
in
Eggs
caupo’
of Urechis
Department
ProteinzSynthesis
~.GOULD'
Sciences, Stanford
University,
Accepted January
Stanford,
California
$4305
22, 1969
INTRODUCTION
In the preceding paper(Gould, 1969), some features of the RNA and protein synthesis which occur in the unfertilized eggs of UTechis caupo were described. One of the first questions to arise concerning the developmental significance of this activity was whether it continued unchanged in fertilized eggs, or was unique to the fully grown oocytes. The results of a comparison of patterns of RNA and protein synthesis in unfertilized and fertilized eggs are reported here. A second reason for examining patterns of synthesis during early development in Urechis has already been discussed (Gould, 1969). Very little is known about the biochemistry of early development of organisms in which meiosis occurs after fertilization, or in which cleavage is spiral. [For a review on the biochemistry of “mosaic” eggs, see Collier (1965).] Comparative data of this sort will be necessary before generalizations can be drawn correlating biochemical and developmental events in early development. The experiments reported here include a comparison of apparent rates of RNA and protein synthesis before and after fertilization, characterization of the RNA synthesized during early development by sucrose gradient sedimentation and autoradiography, and some observations on the fate of labeled unfertilized egg RNA after fertilization. MATERIALS
AND
METHODS
Methods for obtaining and incubating Urechis eggs, and the techniques employed to characterize RNA and protein synthesis have been de1 Supported by National Institutes of Health grants GM-10060 and 5-Fl-GM21,696. 2 Present address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada. 482
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
TABLE TIMETABLE
VS UNFERTILIZED
EGGS
483
1
OF NORMAL DEVELOPMENT
IN Urechis
caupo AT 17”Ca Time
stage Sperm addition Nucleolus breaks down Germinal vesicle breakdown completed 1st polar body 2nd polar body 2 cells 4 cells 16 cells 32 cells 40 cells 64 cells, ciliated blastula Gastrulation (148 cells at start)
0 l(t12 minutes 15 minutes 35 minutes 45 minutes 90 minutes 2 hours 23g hours 3% hours 5 hours 7 hours l(i-18 hours
Feedirlg larva Metamorphosis
About 40 hours GOdays
n From Gould (1967). scribed (Gould, 1967, 1969). Sperm was obtained from the storage sacs of worms by the same method used for eggs. One hundred percent fertilization, and normal synchronous development through gastrulation, were obtained routinely with eggs, from healthy females, incubated at 17°C in 100 volumes of seawater containing sperm diluted 1: 400,000. A time table of normal development is given in Table 1. Consult Newby (1932, 1940) for a thorough description of Urechis development, including cell lineage through early development. male
RESULTS
Protein
Synthesis
When rates of amino acid incorporation are compared in the con tinuous presence of external precursor, fertilized eggs appear considerably more active than unfertilized eggs (lower curves, D’ig. 1). However, the total uptake of radioactivity into fertilized eggs is also much greater (upper curves, lcig. l), which suggests that the greater incorporation in fertilized eggs might result simply from a higher proportion of isotope in the internal pool of amino acids available for protein synthesis. To avoid this complication of differential uptake, fertilized and un fertilized eggs with the same internal isotope content were compared in the absence of external precursor. This was accomplished by preincubating unfertilized eggs in 14C-labeled amino acids, washing them thoroughly
484
GOULD
.60
? 0 -
.40 20
* .F E ' 2 0
0
20
40
60
60
100
minutes
FIG. l.rAmino acid uptake and incorporation by fertilized and unfertilized eggs incubated with W-labeled algal hydrolyzate. Eggs (0.7 cc) were incubated in 46 ml of seawater with 9pC of 1%. Sperm and isotope were added at zero time. Sample size: 0.042 cc of eggs. Lower curve and left ordinate, TCA-insoluble counts per minute; upper curve and right ordinate, tot,al count,s per minute -O-e-, fertilized eggs; -O-O-, unfertilized eggs.
in unlabeled seawater, and fertilizing half of them. Pleasured in this manner, only a small (about two fold) increase in the rate of protein synthesis occurs after fertilization (Fig. 2 is typical of several experiments). The fact that incorporation does not reach a plateau, but continues to rise as shown by the final sample at 1446 hours, indicates that rates of incorporation were not limited by the rapid exhaustion of internal label available for protein synthesis. (Although values for total isotope content at 14% hours were not obtained, data from similar experiments indicate that essentially no loss of total isotope occurs during such long “chase” periods.) RNA Synthesis RNA synthesis is very low during the first 4-5 hours after fertilization (Fig. 3a). Measurable amounts of precursor are incorporated into RNA during early cleavage, but RNA synthesis between fertilization and completion of the meiotic divisions is virtually undetectable (Fig. 3b). When incorporation is compared in fertilized and unfertilized eggs preloaded with uridine-3H, the apparent rate of RNA synthesis actually decreases after fertilization (Fig. 4).
RNA
AND
PROTEIN
SYNTHESIS:
I
FERTILIZED
: : : : : 0 20 40
VS UNFERTILIZED
: : : : I'; :
minutes
60
60
EGGS
485,
J
14 ‘I ‘2 hours
FIG. 2. Amino acid uptake and incorporation by fert.ilized and unfertilized eggs preloaded with W-labeled amino acids. Eggs (1.1 CC) were incltbated in 25 ml of seawater with 1OrC ‘%-L-amino acid mixture. The eggs were labeled for 8 minutes, then washed repeatedly during the next, 40 minutes. Two equal portions were each resuspended in 50 ml of seawater, and one portion was fertilized at zero t,ime. Sample size: 0.044 cc of eggs. Untreated and TCA-extracted egg pel1et.s were both dissolved ill 0.5 ml of concentrat,ed formic acid and colmted at similar efficiencies. The data are plotted as described for Fig. 1. There are several differences bet\veen the RXA synthesized by unfertilized eggs and that synthesized after fertilization. IGg. 5 shows the sedimentation patterns of RNA synthesized by fertilized and unfertilized eggs during 39; hours of incubation. The more heterogeneous distribution of high molecular weight RNA label from fertilized eggs, and the proportionately greater synthesis of lower molecular weight RNA (1s S and less) are characteristic of stages from early cleavage through ciliated blastula (7 hours). So distinct peaks of radioactivity were associated with ribosomal RNA in preparations from fertilized eggs labeled with uridine-3H or 3”P04 at any stage through ciliation. Hobvever, by 30 hours (trochophore larvae), radioactivity and optical density are coincident in 28 S and 18 ‘S RXA (Fig. 6). To determine Avhether ribosomal RNA synthesis occurs earlier in development, eggs were labeled Tvith methionine-methyl-14C, which is incorporated preferentially into ribosomal and transfer RNA. In these preparations (Vig. 7) essentially no rapidly sedimenting RNA is labeled throuwhb 54$ hours, although considerable slowly sedimenting in-
486
GOULD
1
2
3
4
5
6
7
8
9
10 11 12
Hours FIG. 3. Incorporation of uridine-3H into RNA after (0.8 cc) were incubat)ed in 50 ml of seawater, with sperm zero time. Sample size: 0.048 cc of eggs. (b) Eggs (1.0 cc) of seawater with 50 pC 3H and sperm added at zero time. eggs.
fertilization. (a) Eggs and 15 #.Z’ 3H added at were incubated in 75 ml Sample size: 0.053 cc of
1200. IOOO/
.z 600.. .E z
I'
6oo-400t
PU
"--L-+0
__------
j I:'
&P-
I 200 t
e
minutes
F-J 5‘13 hours
FIG. 4. Uridine-3H incorporation in preloaded fertilized and unfertilized eggs. Eggs (0.8 cc) were incubated in 35 ml of seawater with 50 PC $H for 1% hours, washed, and resuspended in two equal portions of 40 ml each. Sperm addition at zero time. Sample size: 0.03 cc of eggs. -O-O--, fertilized eggs; -O-O--, unfertilized eggs.
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
487
.-IO0 .; -00 5 " 40 --1 Fraction
no
FIG. 5. RNA synthesis in fertilized and unfertilized eggs. Fertilized or unfertilized eggs (0.75 cc) were incubated in 72 ml of seawater with 100 FC uridine-3H for 335 hours. Isotope was added 15 minut,es after fertilization. RNA was extracted with phenol-SDS, including DNase digestion (see Ciould, 1969). --, ODxo; unfertilized eggs; -a-@-, counts per minute, . . ) counts per minute, fertilized eggs.
IO 0.0 0 d2 0.6
0% x 6 .; m
d 0.4
4z
0.2
2
5
IO
15 Fraction
20
25
30
no.
FIG. 6. RNA-V accumulated in 30-hour embryos labeled since early cleavage. Eggs (0.4 cc) were incubated in 40 ml of seawater wit,h 190 PC 3LP04 added at the &cell stage. --, ODzsO; -*-a-, counts per minute.
corporation is evident. A small amount of rapidly sedimenting incorporation of uncertain nature appears by 7% hours, and is not increased significantly through 11 hours (data not shown). By 16% hours (midgastrula) a distinct peak of 28-30 S RNA label is seen, indicating resump-
488
GOULD
r
2800 0.6
1
0
5
IO
15 Fraction
20
25
no
FIG. 7. Incorporation of L-methionine-methyl-% into RNA after fertilization. The 5x- and ig-hotlr radioactivity profiles are from an experiment in which 1.0 cc of eggs (0.33 cc per sample) were incubated in 90 ml of seawater with 10 PC l*C added 1 hour after fertilization. The 16?4 hollr radioactivity profile is from an experiment in which 0.6 cc eggs (0.3 cc per sample) were incubated in 65 ml seawater with 5 pC 14C added 13 minutes after fertilization. RNA extraction included pronase digestion. --, ODB~; . counts per minute at 5?$ hours; -O-O-, at 7% hours; -O-O--, at 1634 hours.
tion of ribosomal RNA synthesis. An earlier low level synthesis is of course not excluded by the data. In autoradiograms of fertilized eggs incubated continuously with uridine-3H. most of the RNA label is nuclear in stages through ciliation (Figs. 8 and 9), although some cytoplasmic label is detected as early as 4% hours after fertilization (3240 cells; Fig. 8). “Nucleoli” reappear soon after the 40-cell stage (5 hours), frequently overlain by a cluster of grains (Fig. 9) absent in ribonuclease-digested controls (not shown). Small dense bodies resembling nucleoli are also seen by phase contrast microscopy in living cells at this time. The Fate, after Fertilization,
of RNA Synthesized in Unfertilized Eggs
Although pulse-chase labeling conditions cannot ordinarily be achieved in Urechis eggs, the near cessation of RNA synthesis after fertilization
RNA
AND
PROTEIK
SYXTHESIH:
FERTILIZED
VS UNFERTILIZED
EGGS
489
Fro. 8. Aatoradiograms of 4% hour blastrdae labeled with uridine-W. Eggs (0.8 cc) were incrlbated in 80 ml of seawater with 140 PC :‘H added at fertilization. Autoradiograms were exposed 4 weeks. Hematoxylin stained. X 425. (a) DNasedigested section. (b) RNase-digested section. FIG. 9. Autoradiogram of ti$i hour blastula labeled with uridine-3H. Eggs (1.0 cc) were incubated in 75 ml of seawater with 50 PC “H added at) fertilization. Arrow points to nucleollls. Other det,ails as for Fig. 8.
allows uridine-3H-labeled RNA synthesized in unfertilized eggs to be traced at least through first cleavage. When labeled unfertilized egg RNA is traced through the completion of meiosis (45 minutes after fertilization), no significant changes are seen in the gradient profiles of phenol-SDS extracted RNA (Fig. 10) or of radioactive RNA from the
490
GOULD
-800 -700 0.6..
-600
0.5-
-500
0.4.-
-400
0.3-e
-300
0.2 --
-200
0. I .-
-100
.’
5
IO
15 Fraction
20
25
E 2 0
30
no.
FIG. 10. The fate, after fertilization, of RNA synthesized in unfertilized eggs: sedimentation profile of ext,racted RNA. Eggs (2.0 cc) were incubated in 100 ml of seawater with 500 rC 3zPOd for 15 hours. After washing, 0.3 cc of eggs each were resuspended in two flasks with 50 ml of seawater each, and one portion was fertilized. Both portions were harvested 40 minutes later. RNA from the fertilized eggs is shown; that from the unfertilized eggs sedimented identically. --, O&60; -@-•--, counts per minute.
15,000 g supernatant of egg homogenates (Fig. 11; compare also with Fig. Sa in Gould, 1969). Followed in autoradiograms, however, the intracellular distribution of the labeled unfertilized egg RSA does change after fertilization. These changes are summarized in the series of autoradiograms in Fig. 12. As the nucleolus breaks down prior to the meiotic divisions, RNA spreads throughout the germinal vesicle area. This spread is accompanied by a marked increase in RNA basophilia (by Azure B staining, not shown) in the nucleus. Much of the label remains associated with the more basophilic spindle-forming region during meiosis (Figs. 12b and c). By first cleavage label is spread rather evenly throughout the cytoplasm (12e). The bulk of the radioactive RnTA apparently does not reenter the interphase nuclei (12f). When unfertilized egg RNA labeled with methionine-met,hyl-14C is
RNA
AND
PROTEIK
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
I
1.6. 1.6
EGGS
-320
I1.4 .4.-
-200
1.2.. 1.2 I
--240 -200
1.0.. 1.01 00: : & 0.6.. & 0.6..
.-I60
i
66
0.6.. 0.6..
I
0.4..
o.2
491
c .E ;
--I 20 .- 60
Y
i
.. 40
pv-
”
5
IO
15
20
Fraction
25
30
no
FIG. 11. The fate, after fertilization, of RNA synthesized in unfertilized eggs: distribution in egg homogenate. Eggs (1.0 cc) were incubated in 100 ml of seawater with 170 PC uridineJH for 10 holux, then washed, divided into two equal portions, and resuspended in 25 ml of seawater each. Sperm was added to one portion, and egg both were homogenized (see Gould, 1969) 30 minutes lat,er. The fertilized preparation is shown; the unfertilized egg preparation was identical. p, ODz60; -@-a--, cormts per minute; , colmts per minute after IlNase (2 rg for 2 hours on ice prior to gradient, cent rifugation).
followed through early cleavage, the 28-30 S RNA appears to persist through 645 hours (Fig. 13; no 14C-labeled Z-30 S RNA was detected during this period in eggs labeled with methionine-14C after fertilization, Fig. 7). DISCUSSION
Protein Synthesis before and afte? Fehlization The differing uptake of amino acids by fertilized and unfertilized eggs complicates attempts to compare rates of protein synthesis. Nevertheless, data from preloading experiments (which rest on the assumption that internal amino acid pools do not change markedly after fertilization) indicate that the increase in amino acid incorporation after fertilization in Urechis (‘L-fold) is not as dramatic as that in the sea urchin (5 to 16-fold, as estimated by Epel, 1967). In a slightly different approach to the prob-
492
GOULD
FIG. 12. The fate, aft,er fertilization, of RNA synthesized in unfertilized eggs: autoradiograms. Eggs (0.4 cc) were incltbated in 12 ml of seawater with 50 PC uridine-3H for 2 hours, washed, and resuspended in 42 ml of seawater with 52 pg of unlabeled uridine, then fertilized (a-e; for f, preincnbation was for 12 hours, and reincubation was in the absence of excess urllabeled uridine). Autoradiograms a-e were exposed for 4 weeks; f, for 8 weeks. Hematoxylin stained. X 540. (a) Shortly before fertilization. (b) At 1555 minutes after fertilization. (c and d) At 20 minutes after fertilization; d is RNase-digested. Arrows point to metaphase (meiosis I) chromosomes. (e) At 88 minlltes. The cleavage spindle has formed. (f) At 2 hours, interphase of the 2-cell stage. Lighter central areas are nuclei.
lem of differential uptake, Tyler et al. (1966) estimated a lo-fold increase following fertilization in sea urchin eggs by dividing the total acidsoluble uptake into the amount of incorporation. Calculated in this manner from the data in Fig. 1, a 2-fold increase in Urechis eggs is again obtained. The data of Smith et al. (1966) and Ecker and Smith (1968), who showed that unfertilized Rcma pipiens eggs microinjected with 1eucineJH were synthesizing protein at the same rate as fertilized eggs, provide
RSA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS USFERTILIZED
EGGS
493
0.2..
5
IO
15
20
Fraction
25
30
35
no
Fro. 13. The fate after fert,ilization of RNA labeled with L-methionine-methyl14C in unfertilized eggs. Eggs (0.9 cc) were incubated in 100 ml of seawater with 30 PC W for 11 hours and washed; half were fertilized. RNA extraction included -a-@--, pronase digestion. e, OD260; . . , counts per minute, unfertilized; counts per minute GjJJ hours after fertilization.
another case where fertilization is not accompanied by a large increase in protein synthetic rate. Bell and Reeder (1967) have also demonstrated clearly that fertilization does not initiate protein synthesis in the surf clam, Spisula; in preloading experiments with these eggs, a 3- to 4-fold increase in amino acid incorporation occurred after fertilization. Postfertilization
RN,4 Synthesis
The information about RNA synthesis after fertilization in Urechis is too limited for a detailed comparison with the situation in sea urchins and frogs, although such a comparison could be of considerable interest in terms of possible differences between RNA metabolism in regulative and mosaic eggs. (Mosaic development has not been demonstrated directly in I:rechis, but is likely in view of the spiral cleavage pattern.) However, RNA metabolism in Cv-echis embryos does resemble in at least two respects that described in other organsims. The first is the low apparent rate of RNA synthesis during early cleavage. This observation has also been made on embryos of the sea urchin (e.g., Xemer, 1963; Glisin and Glisin, 1964), Xenopus (Brown and
494
GOULD
Littna, 1964), the marine snail Ilyanassa (Collier, 1961), and the ascidian Phallusia (D’Anna, 196“). The second is the apparent cessation of ribosomal RNA synthesis during early development. Ribosomal RNA synthesis is resumed in the gastrulae (Brown and Littna, 1964) of starfish (Barros et al., 1966) and Xenopus and in postgastrulation stages of sea urchins (Nemer, 1963; Comb et al., 1965) and loaches (Ajtkhoxin et al., 1964). In Urechis, ribosomal RNA synthesis also appears to resume during gastrulation and is therefore not markedly precocious. The reappearance of definitive nucleoli and resumption of ribosomal RNA synthesis are concomitant in Xenopus embryos (Gurdon and Brown, 1965); however, RBA-containing “nucleolar bodies” whose function is currently unknown do occur in earlier stages, and even in anucleolate mutants (Hay and Gurdon, 1967). Whether in Urechis the early (40-cell stage) “nucleoli” are synthesizin small amounts of ribosomal RNA, or whether they are analogous to “nucleolar bodies” and their radioactive RNA is nonribosomal, remains to be learned. A third observation, of uncertain significance at present, relates to the nontransfer RNA synthesized during early development through blastula stages. In Xenopus (Brown and Littna, 1964; Brown and Gurdon, 1966), the sea urchin (e.g., Nemer, 1963; Infante and Nemer, 1968; Spirin and Nemer, 1965), and CJrechis, this RXA sediments very heterogeneously in sucrose gradients. It would be very interesting to know whether any of this RNA is transported into the cytoplasm and functions there as messenger RNA. In Urechis embryos, most of the RSA labeled from fertilization through blastula formation is nuclear (Figs. 8 and 9). The occasional metaphase cells (not shown) encountered in these preparations have substantial numbers of grains dispersed throughout the cytoplasm. Either this R?JA returns to the nuclei during telophase or it is degraded prior to the next interphase. Evidence on nuclear us cytoplasmic distribution of radioactive RNA during cleavage in other organisms is scanty. One autoradiographic study on sea urchin embryos (Ficq et al., 1963) does suggest a primarily nuclear accumulation of labeled RNA through blastula formation. In many other autoradiographic studies, either incorporation was too low or labeling periods too short, to permit con elusions about cytoplasmic labeling. Likewise, to my knowledge, studies based on embryo homogenates have generally not provided the clean separation of cytoplasmic from nuclear material (including nuclear material that may be transiently dispersed into the cytoplasm in meta-
RN.4
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
495
phase cells) critical for deciding whether messenger RNA is transported into the cytoplasm during early development. ,~e Fate, ajter Fertilization,
of the
RNA Synthesized by Mature Oocytes
Although it is clear that, at least some of the RSA synthesized in unfertilized Urecllis eggs persists after fertilization, its functional significance, if any, remains to be determined. Further discussion of the possible significance of synthesis in mature oocytes is found in Gould (1969). Although many of the observations reported in this paper and in Gould (1969) are not explained in developmental terms at present, they provide necessary background for continuing studies on the biochemistry of oogenesis and early development. These st’udies have also established that Ulechis, because of its developmental characteristics and suitability 1 biochemical studies, is potentially of considerable value for solving problems in oogenesis and early development. For this reason, it is to be hoped that existing populations will not be used wastefully. After fertilization in Urechis eggs, the apparent rate of protein synthesis increases approximately only twofold, when experiments are designed to avoid the complication of increased permeability to amino acids in fertilized eggs. RNA synthesis after fertilization drops to virtually undetectable levels until the completion of meiosis. The RiSA synthesized during early cleavage sediments heterogeneously in sucrose gradients, but includes no distinct peak of “45 S” RNA, and shows higher levels of relative incorporation into RNA sedimenting at 18 S and less than the RNA synthesized by unfertilized eggs. Ribosomal RNA synthesis appears to cease until gastrulation. During the first 7 hours of development (to ciliated blastula) most of the RnTA label is nuclear in autoradiograms of eggs exposed continuously to uridine-3H after fertilization. At least some of the RNA synthesized in unfertilized eggs persists after fertilization. The distribution in sucrose gradients of this RN-4 remains essentially unchanged through completion of meiosis, and 30 S ribosomal RNA labeled prior to fertilization with methionine-methylJ4C into the blastula stage. Autoradiograms show a dispersal of labeled unfertilized egg RNA into the cytoplasm following germinal vesicle breakdown, and no reentry into the interphase nuclei at the 2-cell stage. The author able criticisms
would like to thank Drs. Fred Wilt and Norman Wessells for valuof this and the preceding manuscript, (Gould, 1969), and t,o express
496
GOULD
her deep gratitnde to Dr. Wessells, who generorlsly during the conrse of these studies.
sllpplied
laboratory
facilities
REFERENCES AJTKHOBIN, M., B~I,ITSIN,~, N., and SPIRIN, A. (1964). Nucleic acids dnring earlJ development of fish embryos (AJiagurnus fossilis). (In Russian.) Biokhimiya 29, 169. BIRROS, C., HAND, G., and MONROY, A. (1906). Cotltrol of gastrulation in the starfish, Asterias jorbesii. Exptl. Cell Res. 43, 167. BELL, E., and 1., and Gul
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
497
NEW-BY, W. (1932). The early embryology of the Echiuroid, Urechis. Biol. Bull. 63, 387. NEWHY, W. (1940). “The Embryology of the Echiuroid Worm Urechis caupo,” Xlemoirs, American Philosophical Society, Philadelphia, Pennsylvania. SMITH, L., ECICER, R., and SUHTISLNY, S. (1966). The initiation of protein synthesis in eggs of Rana pipiens. Proc. Satl. Acad. Sci. U.S. 66, 1724. SPIRIN, A., and NICMICR, M. (19G5). Messenger RNA in early sea urchin embryos: cytoplnsmic particles. Science 160, 214. TYLER, -4., PIATIG~RSK~, J., and OZAKI, H. (1966). Influence of individual amino acids on uptake and incorporation of valine, gllutamic acid and arginine by Ilnfertilixed and fertilized sea urchin eggs. Biol. Bull. 131, 204.
BIOLOGY
19,
A Comparison
482497
(1969)
of RNA
Fertilized
and
and
Unfertilized
n[EREDITH of Biological
in
Eggs
caupo’
of Urechis
Department
ProteinzSynthesis
~.GOULD'
Sciences, Stanford
University,
Accepted January
Stanford,
California
$4305
22, 1969
INTRODUCTION
In the preceding paper(Gould, 1969), some features of the RNA and protein synthesis which occur in the unfertilized eggs of UTechis caupo were described. One of the first questions to arise concerning the developmental significance of this activity was whether it continued unchanged in fertilized eggs, or was unique to the fully grown oocytes. The results of a comparison of patterns of RNA and protein synthesis in unfertilized and fertilized eggs are reported here. A second reason for examining patterns of synthesis during early development in Urechis has already been discussed (Gould, 1969). Very little is known about the biochemistry of early development of organisms in which meiosis occurs after fertilization, or in which cleavage is spiral. [For a review on the biochemistry of “mosaic” eggs, see Collier (1965).] Comparative data of this sort will be necessary before generalizations can be drawn correlating biochemical and developmental events in early development. The experiments reported here include a comparison of apparent rates of RNA and protein synthesis before and after fertilization, characterization of the RNA synthesized during early development by sucrose gradient sedimentation and autoradiography, and some observations on the fate of labeled unfertilized egg RNA after fertilization. MATERIALS
AND
METHODS
Methods for obtaining and incubating Urechis eggs, and the techniques employed to characterize RNA and protein synthesis have been de1 Supported by National Institutes of Health grants GM-10060 and 5-Fl-GM21,696. 2 Present address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada. 482
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
TABLE TIMETABLE
VS UNFERTILIZED
EGGS
483
1
OF NORMAL DEVELOPMENT
IN Urechis
caupo AT 17”Ca Time
stage Sperm addition Nucleolus breaks down Germinal vesicle breakdown completed 1st polar body 2nd polar body 2 cells 4 cells 16 cells 32 cells 40 cells 64 cells, ciliated blastula Gastrulation (148 cells at start)
0 l(t12 minutes 15 minutes 35 minutes 45 minutes 90 minutes 2 hours 23g hours 3% hours 5 hours 7 hours l(i-18 hours
Feedirlg larva Metamorphosis
About 40 hours GOdays
n From Gould (1967). scribed (Gould, 1967, 1969). Sperm was obtained from the storage sacs of worms by the same method used for eggs. One hundred percent fertilization, and normal synchronous development through gastrulation, were obtained routinely with eggs, from healthy females, incubated at 17°C in 100 volumes of seawater containing sperm diluted 1: 400,000. A time table of normal development is given in Table 1. Consult Newby (1932, 1940) for a thorough description of Urechis development, including cell lineage through early development. male
RESULTS
Protein
Synthesis
When rates of amino acid incorporation are compared in the con tinuous presence of external precursor, fertilized eggs appear considerably more active than unfertilized eggs (lower curves, D’ig. 1). However, the total uptake of radioactivity into fertilized eggs is also much greater (upper curves, lcig. l), which suggests that the greater incorporation in fertilized eggs might result simply from a higher proportion of isotope in the internal pool of amino acids available for protein synthesis. To avoid this complication of differential uptake, fertilized and un fertilized eggs with the same internal isotope content were compared in the absence of external precursor. This was accomplished by preincubating unfertilized eggs in 14C-labeled amino acids, washing them thoroughly
484
GOULD
.60
? 0 -
.40 20
* .F E ' 2 0
0
20
40
60
60
100
minutes
FIG. l.rAmino acid uptake and incorporation by fertilized and unfertilized eggs incubated with W-labeled algal hydrolyzate. Eggs (0.7 cc) were incubated in 46 ml of seawater with 9pC of 1%. Sperm and isotope were added at zero time. Sample size: 0.042 cc of eggs. Lower curve and left ordinate, TCA-insoluble counts per minute; upper curve and right ordinate, tot,al count,s per minute -O-e-, fertilized eggs; -O-O-, unfertilized eggs.
in unlabeled seawater, and fertilizing half of them. Pleasured in this manner, only a small (about two fold) increase in the rate of protein synthesis occurs after fertilization (Fig. 2 is typical of several experiments). The fact that incorporation does not reach a plateau, but continues to rise as shown by the final sample at 1446 hours, indicates that rates of incorporation were not limited by the rapid exhaustion of internal label available for protein synthesis. (Although values for total isotope content at 14% hours were not obtained, data from similar experiments indicate that essentially no loss of total isotope occurs during such long “chase” periods.) RNA Synthesis RNA synthesis is very low during the first 4-5 hours after fertilization (Fig. 3a). Measurable amounts of precursor are incorporated into RNA during early cleavage, but RNA synthesis between fertilization and completion of the meiotic divisions is virtually undetectable (Fig. 3b). When incorporation is compared in fertilized and unfertilized eggs preloaded with uridine-3H, the apparent rate of RNA synthesis actually decreases after fertilization (Fig. 4).
RNA
AND
PROTEIN
SYNTHESIS:
I
FERTILIZED
: : : : : 0 20 40
VS UNFERTILIZED
: : : : I'; :
minutes
60
60
EGGS
485,
J
14 ‘I ‘2 hours
FIG. 2. Amino acid uptake and incorporation by fert.ilized and unfertilized eggs preloaded with W-labeled amino acids. Eggs (1.1 CC) were incltbated in 25 ml of seawater with 1OrC ‘%-L-amino acid mixture. The eggs were labeled for 8 minutes, then washed repeatedly during the next, 40 minutes. Two equal portions were each resuspended in 50 ml of seawater, and one portion was fertilized at zero t,ime. Sample size: 0.044 cc of eggs. Untreated and TCA-extracted egg pel1et.s were both dissolved ill 0.5 ml of concentrat,ed formic acid and colmted at similar efficiencies. The data are plotted as described for Fig. 1. There are several differences bet\veen the RXA synthesized by unfertilized eggs and that synthesized after fertilization. IGg. 5 shows the sedimentation patterns of RNA synthesized by fertilized and unfertilized eggs during 39; hours of incubation. The more heterogeneous distribution of high molecular weight RNA label from fertilized eggs, and the proportionately greater synthesis of lower molecular weight RNA (1s S and less) are characteristic of stages from early cleavage through ciliated blastula (7 hours). So distinct peaks of radioactivity were associated with ribosomal RNA in preparations from fertilized eggs labeled with uridine-3H or 3”P04 at any stage through ciliation. Hobvever, by 30 hours (trochophore larvae), radioactivity and optical density are coincident in 28 S and 18 ‘S RXA (Fig. 6). To determine Avhether ribosomal RNA synthesis occurs earlier in development, eggs were labeled Tvith methionine-methyl-14C, which is incorporated preferentially into ribosomal and transfer RNA. In these preparations (Vig. 7) essentially no rapidly sedimenting RNA is labeled throuwhb 54$ hours, although considerable slowly sedimenting in-
486
GOULD
1
2
3
4
5
6
7
8
9
10 11 12
Hours FIG. 3. Incorporation of uridine-3H into RNA after (0.8 cc) were incubat)ed in 50 ml of seawater, with sperm zero time. Sample size: 0.048 cc of eggs. (b) Eggs (1.0 cc) of seawater with 50 pC 3H and sperm added at zero time. eggs.
fertilization. (a) Eggs and 15 #.Z’ 3H added at were incubated in 75 ml Sample size: 0.053 cc of
1200. IOOO/
.z 600.. .E z
I'
6oo-400t
PU
"--L-+0
__------
j I:'
&P-
I 200 t
e
minutes
F-J 5‘13 hours
FIG. 4. Uridine-3H incorporation in preloaded fertilized and unfertilized eggs. Eggs (0.8 cc) were incubated in 35 ml of seawater with 50 PC $H for 1% hours, washed, and resuspended in two equal portions of 40 ml each. Sperm addition at zero time. Sample size: 0.03 cc of eggs. -O-O--, fertilized eggs; -O-O--, unfertilized eggs.
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
487
.-IO0 .; -00 5 " 40 --1 Fraction
no
FIG. 5. RNA synthesis in fertilized and unfertilized eggs. Fertilized or unfertilized eggs (0.75 cc) were incubated in 72 ml of seawater with 100 FC uridine-3H for 335 hours. Isotope was added 15 minut,es after fertilization. RNA was extracted with phenol-SDS, including DNase digestion (see Ciould, 1969). --, ODxo; unfertilized eggs; -a-@-, counts per minute, . . ) counts per minute, fertilized eggs.
IO 0.0 0 d2 0.6
0% x 6 .; m
d 0.4
4z
0.2
2
5
IO
15 Fraction
20
25
30
no.
FIG. 6. RNA-V accumulated in 30-hour embryos labeled since early cleavage. Eggs (0.4 cc) were incubated in 40 ml of seawater wit,h 190 PC 3LP04 added at the &cell stage. --, ODzsO; -*-a-, counts per minute.
corporation is evident. A small amount of rapidly sedimenting incorporation of uncertain nature appears by 7% hours, and is not increased significantly through 11 hours (data not shown). By 16% hours (midgastrula) a distinct peak of 28-30 S RNA label is seen, indicating resump-
488
GOULD
r
2800 0.6
1
0
5
IO
15 Fraction
20
25
no
FIG. 7. Incorporation of L-methionine-methyl-% into RNA after fertilization. The 5x- and ig-hotlr radioactivity profiles are from an experiment in which 1.0 cc of eggs (0.33 cc per sample) were incubated in 90 ml of seawater with 10 PC l*C added 1 hour after fertilization. The 16?4 hollr radioactivity profile is from an experiment in which 0.6 cc eggs (0.3 cc per sample) were incubated in 65 ml seawater with 5 pC 14C added 13 minutes after fertilization. RNA extraction included pronase digestion. --, ODB~; . counts per minute at 5?$ hours; -O-O-, at 7% hours; -O-O--, at 1634 hours.
tion of ribosomal RNA synthesis. An earlier low level synthesis is of course not excluded by the data. In autoradiograms of fertilized eggs incubated continuously with uridine-3H. most of the RNA label is nuclear in stages through ciliation (Figs. 8 and 9), although some cytoplasmic label is detected as early as 4% hours after fertilization (3240 cells; Fig. 8). “Nucleoli” reappear soon after the 40-cell stage (5 hours), frequently overlain by a cluster of grains (Fig. 9) absent in ribonuclease-digested controls (not shown). Small dense bodies resembling nucleoli are also seen by phase contrast microscopy in living cells at this time. The Fate, after Fertilization,
of RNA Synthesized in Unfertilized Eggs
Although pulse-chase labeling conditions cannot ordinarily be achieved in Urechis eggs, the near cessation of RNA synthesis after fertilization
RNA
AND
PROTEIK
SYXTHESIH:
FERTILIZED
VS UNFERTILIZED
EGGS
489
Fro. 8. Aatoradiograms of 4% hour blastrdae labeled with uridine-W. Eggs (0.8 cc) were incrlbated in 80 ml of seawater with 140 PC :‘H added at fertilization. Autoradiograms were exposed 4 weeks. Hematoxylin stained. X 425. (a) DNasedigested section. (b) RNase-digested section. FIG. 9. Autoradiogram of ti$i hour blastula labeled with uridine-3H. Eggs (1.0 cc) were incubated in 75 ml of seawater with 50 PC “H added at) fertilization. Arrow points to nucleollls. Other det,ails as for Fig. 8.
allows uridine-3H-labeled RNA synthesized in unfertilized eggs to be traced at least through first cleavage. When labeled unfertilized egg RNA is traced through the completion of meiosis (45 minutes after fertilization), no significant changes are seen in the gradient profiles of phenol-SDS extracted RNA (Fig. 10) or of radioactive RNA from the
490
GOULD
-800 -700 0.6..
-600
0.5-
-500
0.4.-
-400
0.3-e
-300
0.2 --
-200
0. I .-
-100
.’
5
IO
15 Fraction
20
25
E 2 0
30
no.
FIG. 10. The fate, after fertilization, of RNA synthesized in unfertilized eggs: sedimentation profile of ext,racted RNA. Eggs (2.0 cc) were incubated in 100 ml of seawater with 500 rC 3zPOd for 15 hours. After washing, 0.3 cc of eggs each were resuspended in two flasks with 50 ml of seawater each, and one portion was fertilized. Both portions were harvested 40 minutes later. RNA from the fertilized eggs is shown; that from the unfertilized eggs sedimented identically. --, O&60; -@-•--, counts per minute.
15,000 g supernatant of egg homogenates (Fig. 11; compare also with Fig. Sa in Gould, 1969). Followed in autoradiograms, however, the intracellular distribution of the labeled unfertilized egg RSA does change after fertilization. These changes are summarized in the series of autoradiograms in Fig. 12. As the nucleolus breaks down prior to the meiotic divisions, RNA spreads throughout the germinal vesicle area. This spread is accompanied by a marked increase in RNA basophilia (by Azure B staining, not shown) in the nucleus. Much of the label remains associated with the more basophilic spindle-forming region during meiosis (Figs. 12b and c). By first cleavage label is spread rather evenly throughout the cytoplasm (12e). The bulk of the radioactive RnTA apparently does not reenter the interphase nuclei (12f). When unfertilized egg RNA labeled with methionine-met,hyl-14C is
RNA
AND
PROTEIK
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
I
1.6. 1.6
EGGS
-320
I1.4 .4.-
-200
1.2.. 1.2 I
--240 -200
1.0.. 1.01 00: : & 0.6.. & 0.6..
.-I60
i
66
0.6.. 0.6..
I
0.4..
o.2
491
c .E ;
--I 20 .- 60
Y
i
.. 40
pv-
”
5
IO
15
20
Fraction
25
30
no
FIG. 11. The fate, after fertilization, of RNA synthesized in unfertilized eggs: distribution in egg homogenate. Eggs (1.0 cc) were incubated in 100 ml of seawater with 170 PC uridineJH for 10 holux, then washed, divided into two equal portions, and resuspended in 25 ml of seawater each. Sperm was added to one portion, and egg both were homogenized (see Gould, 1969) 30 minutes lat,er. The fertilized preparation is shown; the unfertilized egg preparation was identical. p, ODz60; -@-a--, cormts per minute; , colmts per minute after IlNase (2 rg for 2 hours on ice prior to gradient, cent rifugation).
followed through early cleavage, the 28-30 S RNA appears to persist through 645 hours (Fig. 13; no 14C-labeled Z-30 S RNA was detected during this period in eggs labeled with methionine-14C after fertilization, Fig. 7). DISCUSSION
Protein Synthesis before and afte? Fehlization The differing uptake of amino acids by fertilized and unfertilized eggs complicates attempts to compare rates of protein synthesis. Nevertheless, data from preloading experiments (which rest on the assumption that internal amino acid pools do not change markedly after fertilization) indicate that the increase in amino acid incorporation after fertilization in Urechis (‘L-fold) is not as dramatic as that in the sea urchin (5 to 16-fold, as estimated by Epel, 1967). In a slightly different approach to the prob-
492
GOULD
FIG. 12. The fate, aft,er fertilization, of RNA synthesized in unfertilized eggs: autoradiograms. Eggs (0.4 cc) were incltbated in 12 ml of seawater with 50 PC uridine-3H for 2 hours, washed, and resuspended in 42 ml of seawater with 52 pg of unlabeled uridine, then fertilized (a-e; for f, preincnbation was for 12 hours, and reincubation was in the absence of excess urllabeled uridine). Autoradiograms a-e were exposed for 4 weeks; f, for 8 weeks. Hematoxylin stained. X 540. (a) Shortly before fertilization. (b) At 1555 minutes after fertilization. (c and d) At 20 minutes after fertilization; d is RNase-digested. Arrows point to metaphase (meiosis I) chromosomes. (e) At 88 minlltes. The cleavage spindle has formed. (f) At 2 hours, interphase of the 2-cell stage. Lighter central areas are nuclei.
lem of differential uptake, Tyler et al. (1966) estimated a lo-fold increase following fertilization in sea urchin eggs by dividing the total acidsoluble uptake into the amount of incorporation. Calculated in this manner from the data in Fig. 1, a 2-fold increase in Urechis eggs is again obtained. The data of Smith et al. (1966) and Ecker and Smith (1968), who showed that unfertilized Rcma pipiens eggs microinjected with 1eucineJH were synthesizing protein at the same rate as fertilized eggs, provide
RSA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS USFERTILIZED
EGGS
493
0.2..
5
IO
15
20
Fraction
25
30
35
no
Fro. 13. The fate after fert,ilization of RNA labeled with L-methionine-methyl14C in unfertilized eggs. Eggs (0.9 cc) were incubated in 100 ml of seawater with 30 PC W for 11 hours and washed; half were fertilized. RNA extraction included -a-@--, pronase digestion. e, OD260; . . , counts per minute, unfertilized; counts per minute GjJJ hours after fertilization.
another case where fertilization is not accompanied by a large increase in protein synthetic rate. Bell and Reeder (1967) have also demonstrated clearly that fertilization does not initiate protein synthesis in the surf clam, Spisula; in preloading experiments with these eggs, a 3- to 4-fold increase in amino acid incorporation occurred after fertilization. Postfertilization
RN,4 Synthesis
The information about RNA synthesis after fertilization in Urechis is too limited for a detailed comparison with the situation in sea urchins and frogs, although such a comparison could be of considerable interest in terms of possible differences between RNA metabolism in regulative and mosaic eggs. (Mosaic development has not been demonstrated directly in I:rechis, but is likely in view of the spiral cleavage pattern.) However, RNA metabolism in Cv-echis embryos does resemble in at least two respects that described in other organsims. The first is the low apparent rate of RNA synthesis during early cleavage. This observation has also been made on embryos of the sea urchin (e.g., Xemer, 1963; Glisin and Glisin, 1964), Xenopus (Brown and
494
GOULD
Littna, 1964), the marine snail Ilyanassa (Collier, 1961), and the ascidian Phallusia (D’Anna, 196“). The second is the apparent cessation of ribosomal RNA synthesis during early development. Ribosomal RNA synthesis is resumed in the gastrulae (Brown and Littna, 1964) of starfish (Barros et al., 1966) and Xenopus and in postgastrulation stages of sea urchins (Nemer, 1963; Comb et al., 1965) and loaches (Ajtkhoxin et al., 1964). In Urechis, ribosomal RNA synthesis also appears to resume during gastrulation and is therefore not markedly precocious. The reappearance of definitive nucleoli and resumption of ribosomal RNA synthesis are concomitant in Xenopus embryos (Gurdon and Brown, 1965); however, RBA-containing “nucleolar bodies” whose function is currently unknown do occur in earlier stages, and even in anucleolate mutants (Hay and Gurdon, 1967). Whether in Urechis the early (40-cell stage) “nucleoli” are synthesizin small amounts of ribosomal RNA, or whether they are analogous to “nucleolar bodies” and their radioactive RNA is nonribosomal, remains to be learned. A third observation, of uncertain significance at present, relates to the nontransfer RNA synthesized during early development through blastula stages. In Xenopus (Brown and Littna, 1964; Brown and Gurdon, 1966), the sea urchin (e.g., Nemer, 1963; Infante and Nemer, 1968; Spirin and Nemer, 1965), and CJrechis, this RXA sediments very heterogeneously in sucrose gradients. It would be very interesting to know whether any of this RNA is transported into the cytoplasm and functions there as messenger RNA. In Urechis embryos, most of the RSA labeled from fertilization through blastula formation is nuclear (Figs. 8 and 9). The occasional metaphase cells (not shown) encountered in these preparations have substantial numbers of grains dispersed throughout the cytoplasm. Either this R?JA returns to the nuclei during telophase or it is degraded prior to the next interphase. Evidence on nuclear us cytoplasmic distribution of radioactive RNA during cleavage in other organisms is scanty. One autoradiographic study on sea urchin embryos (Ficq et al., 1963) does suggest a primarily nuclear accumulation of labeled RNA through blastula formation. In many other autoradiographic studies, either incorporation was too low or labeling periods too short, to permit con elusions about cytoplasmic labeling. Likewise, to my knowledge, studies based on embryo homogenates have generally not provided the clean separation of cytoplasmic from nuclear material (including nuclear material that may be transiently dispersed into the cytoplasm in meta-
RN.4
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
495
phase cells) critical for deciding whether messenger RNA is transported into the cytoplasm during early development. ,~e Fate, ajter Fertilization,
of the
RNA Synthesized by Mature Oocytes
Although it is clear that, at least some of the RSA synthesized in unfertilized Urecllis eggs persists after fertilization, its functional significance, if any, remains to be determined. Further discussion of the possible significance of synthesis in mature oocytes is found in Gould (1969). Although many of the observations reported in this paper and in Gould (1969) are not explained in developmental terms at present, they provide necessary background for continuing studies on the biochemistry of oogenesis and early development. These st’udies have also established that Ulechis, because of its developmental characteristics and suitability 1 biochemical studies, is potentially of considerable value for solving problems in oogenesis and early development. For this reason, it is to be hoped that existing populations will not be used wastefully. After fertilization in Urechis eggs, the apparent rate of protein synthesis increases approximately only twofold, when experiments are designed to avoid the complication of increased permeability to amino acids in fertilized eggs. RNA synthesis after fertilization drops to virtually undetectable levels until the completion of meiosis. The RiSA synthesized during early cleavage sediments heterogeneously in sucrose gradients, but includes no distinct peak of “45 S” RNA, and shows higher levels of relative incorporation into RNA sedimenting at 18 S and less than the RNA synthesized by unfertilized eggs. Ribosomal RNA synthesis appears to cease until gastrulation. During the first 7 hours of development (to ciliated blastula) most of the RnTA label is nuclear in autoradiograms of eggs exposed continuously to uridine-3H after fertilization. At least some of the RNA synthesized in unfertilized eggs persists after fertilization. The distribution in sucrose gradients of this RN-4 remains essentially unchanged through completion of meiosis, and 30 S ribosomal RNA labeled prior to fertilization with methionine-methylJ4C into the blastula stage. Autoradiograms show a dispersal of labeled unfertilized egg RNA into the cytoplasm following germinal vesicle breakdown, and no reentry into the interphase nuclei at the 2-cell stage. The author able criticisms
would like to thank Drs. Fred Wilt and Norman Wessells for valuof this and the preceding manuscript, (Gould, 1969), and t,o express
496
GOULD
her deep gratitnde to Dr. Wessells, who generorlsly during the conrse of these studies.
sllpplied
laboratory
facilities
REFERENCES AJTKHOBIN, M., B~I,ITSIN,~, N., and SPIRIN, A. (1964). Nucleic acids dnring earlJ development of fish embryos (AJiagurnus fossilis). (In Russian.) Biokhimiya 29, 169. BIRROS, C., HAND, G., and MONROY, A. (1906). Cotltrol of gastrulation in the starfish, Asterias jorbesii. Exptl. Cell Res. 43, 167. BELL, E., and 1
RNA
AND
PROTEIN
SYNTHESIS:
FERTILIZED
VS UNFERTILIZED
EGGS
497
NEW-BY, W. (1932). The early embryology of the Echiuroid, Urechis. Biol. Bull. 63, 387. NEWHY, W. (1940). “The Embryology of the Echiuroid Worm Urechis caupo,” Xlemoirs, American Philosophical Society, Philadelphia, Pennsylvania. SMITH, L., ECICER, R., and SUHTISLNY, S. (1966). The initiation of protein synthesis in eggs of Rana pipiens. Proc. Satl. Acad. Sci. U.S. 66, 1724. SPIRIN, A., and NICMICR, M. (19G5). Messenger RNA in early sea urchin embryos: cytoplnsmic particles. Science 160, 214. TYLER, -4., PIATIG~RSK~, J., and OZAKI, H. (1966). Influence of individual amino acids on uptake and incorporation of valine, gllutamic acid and arginine by Ilnfertilixed and fertilized sea urchin eggs. Biol. Bull. 131, 204.