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Messenger RNA synthesis, transport, and storage in silkmoth ovarian follicles
DEVELOPMENTAL
BIOLOGY
Messenger
51,
173-181
(19761
RNA Synthesis, Transport, and Storage Ovarian Follicles’ L. M. PAGLIA,’ of Bzo1og.v.
Department
S. J. BERRY,:’ Weslcyn
Unioersity,
AND
in Silkmoth
W. H. KASTERN
Middletou~n,
Connectwut
06457
Developing oocytes of the silkmoths Actias lurm and Antheraea polyphemus were shown to contain significant amounts of polyadcnylatcd RNA. This presumed messenger RNA comprises 0.2 to 0.3Cr of the total extractable RNA, and is found exclusively in subribosomal fractions of ooplasm. Pulse-chase and other time-course studies indicate that the nurse cells are the probable source of this as well as other fractions of ooplasmic RNA. Labeled polyaderiylated RNA extracted from both nurse cells and ooplasm appears in similar, heterodisperse, fractions with a major peak at 18 S in sucrose gradients, No evidence of the presence of polysomes or of active protein or RNA synthesis was detected in the oocytes during vitellogenesis. INTRODUCTION
Developing oocytes of giant silkmoths provide very favorable material for the investigation of the “maternal” contribution to the early embryo. The mosaic development of many insect embryos in which regions of the egg seem to be determined before fertilization suggests that the contribution of the maternal genome may be of critical importance in directing the events of later embryogenesis. The burst of actinomycin-insensitive protein synthesis observed in the eggs of many animals (Brachet and Denis, 1963; Gross and Cousineau, 1963; Balthus, 1965; etc.) including insects (Lockshin, 1966) shortly after fertilization may represent utilization of maternally derived information. Insensitivity to actinomycin inhibition suggests that both general machinery (ribosomes, transfer RNA, etc.) and the specific programming molecules (mRNA) for protein synthesis are stored in the oocyte and activated at fertilization. There is considerable direct evidence for ’ Supported by Career Development Award (to SJB) No. 5.K4-Hd50195-02 and National Science Foundation Grant No. GB34155. ’ Current address: NIDR, NIH, Rethesda, Maryland 20014. ” Address reprint requests to S. J. Berry,
stored messenger RNA in amphibian (Brachet and Denis, 1963; Ecker et al., 1968) and sea urchin eggs (Gross et al., 1964; Tyler, 1966), although the mechanism by which protein synthesis is inhibited until fertilization is unknown. Various authors (Bell and Reeder, 1967; Epel, 1967, and others) have shown that, a low rate of prefertilization translation is detectable in sea urchin oocytes. In this communication we describe the synthesis and transport of RNA containing polyadenylated regions to the ooplasm during the early events of oogenesis. The ability to bind this RNA on poly(U)-sepharose and the similarity of the sedimentation behavior in sucrose gradients to messenger RNA derived from other systems (Greenberg, 1975) leads us to suggest that this fraction represents mRNA. We chose the ovarian follicles of silkmoths for this investigation because they can be easily dissected to isolate specific cell types, RNA is readily labeled in extirpated follicles and because no RNA synthesis can be demonstrated in the oocyte nucleus during vitellogenesis (Pollack and Telfer, 1969). The follicles consist of a palisade of small columnar follicle cells of mesodermal origin surrounding the developing oocyte and its seven sister trophocytes
173 Copyright G 19% by Acadeinic Press, Inc. All rights of reproduction in any form reserved.
174
DEVELOPMENTAL
BIOLOGY
all of which are derived from the primordial germ cells. The trophocytes or nurse cells are attached to each other and to the oocyte by cytoplasmic connectives (see King and Aggarwal, 1965, for details in Cecropia). We had previously demonstrated that ribosomes are synthesized in the nurse cells and transferred to the oocytes (Hughes and Berry, 1970) and we show here that a similar mechanism is involved in mRNA and transfer RNA synthesis. Since the major fractions of RNA and their associated proteins are not synthesized in the oocyte, but in the nurse cells, we suggest the possibility that newly synthesized protein found in oocytes before fertilization represents ribosomal and “informasomal” proteins rather than yolk or other cellular components. MATERIALS
Follicle
Preparation
AND
METHODS
and Labeling
Pupae of A. polyphemus and A. luna were obtained from either commercial sources or were reared in the field, and stored at 4°C until use. Adult development was initiated by return to 25°C at long day (17-hr light, 7-hr dark) photoperiod. Ovaries were removed from females at Day 13 of pupal-adult development (Neusch, 1965) and strings of follicles were dissected free of the surrounding ovariole sheath in plasma-free Grace’s medium (Grand Island Biological). Nurse cell caps were separated from oocytes using watchmaker’s forceps and ooplasm was isolated by gently tearing open the tunica propria. In uiuo labeling of cytoplasmic elements was accomplished by up to three injections of 100 &i of [3H]uridine (New England Nuclear) into developing adults at 3-day intervals. Upon emergence of the adult, whole chorionated eggs were removed from the ovary, rinsed in insect Ringer (0.13 M NaCl, 2 mM KCl, 1.5 mM CaCl,) and opened individually. In vitro labeling of RNA was carried out by immersing either whole follicles or isolated nurse cells in Grace’s medium containing 200 &i of
VOLUME
51. 1976
VHluridine for varying periods up to 20 hr. Pulse-chase labeling was accomplished by preincubating follicles in 200 PCi of 13Hladenosine for 1 to 2 hr, followed by rinsing in Grace’s medium containing 25 pglml of actinomycin D (Calbiochem). In vitro labeling with amino acids involved incubation of follicles in medium containing 50 PCi of a mixture of “H-labeled amino acid mix (New England Nuclear). To ensure maximum synthetic activity, only follicles in which the nurse cell cap comprised l/z to 2/3 the volume of the follicles were selected. Fractionation
Procedure
Following incubation, nurse cells were suspended in 2 ml of low magnesium (LM) buffer (0.01 M MgS04, 0.005 M KCl, 0.01 M Tris, pH 7.4) in a prechilled Dounce homogenizer. Cells were allowed to stand for 5-10 min in buffer and then were gently homogenized by several strokes of a loose-fitting “A” pestle. Nurse cell cytoplasm or ooplasm washed free of follicular cells was centrifuged for 3 min at 1200 g to pellet nuclei and other large cell debris, and the resulting supernatant was decanted and recentrifuged for 20 min at 18,000g. The final supernatant was either extracted with phenol:choloroform or applied directly to sucrose gradients. RNA Extraction lation
and Messenger RNA Iso-
Samples to be extracted were adjusted to pH 9 in 0.1 M Tris, and an equal volume of redistilled pheno1:chlorofor-m (1:l) and SDS to a final concentration of 0.5% were added. The aqueous phase was repeatedly extracted since large amounts of yolk proteins were present in ooplasm samples. After extraction, RNA was precipitated with 2 volumes of cold 95% ethanol containing 2 mM sodium acetate at -20°C. The precipitate was washed with 95% ethanol prior to drying and resuspension. Messenger RNA was separated from other RNA fractions by column chroma-
PAGLIA,
BERRY
AND KASTERN
tography on poly(U)-sepharose according to techniques of Wagner et al. (1971). Poly(U)-sepharose was stored in buffered (0.01 M Tris, 0.01 M EDTA, 0.2% SDS, pH 7.4) 90% ether-extracted formamide at 4°C. The binding efficiency of the matrix was checked periodically by binding [“HIpoly(A) (Schwarz/Mann) to the column. Normal binding efficiency varied between 97-lOO%, and columns binding less than 95% were rejected. Columns consisted of 3-5 ml of packed poly(U)-sepharose, washed with buffered 90% formamide, and equilibrated with NETS buffer (0.1 M NaCl, 0.001 M EDTA, 0.1 M Tris, 0.2% SDS, pH 7.4). RNA samples were dissolved in 0.5-l ml of NETS and eluted from the column at a rate of 1 ml/30 min. Poly(A) + RNA was eluted by addition of 900/o buffered formamide, and ethanol precipitated with 50 pg of yeast tRNA (Sigma) added as carrier. Sucrose Gradient Analysis
Sedimentation
and
RNA samples were run on 15-30% sucrose gradients in NETS buffer at 40,000 r-pm for 6 hr on a SW 41 rotor (Beckman, Fullerton, Calif.) in a Beckman L2 65B preparative ultracentrifuge. Under these conditions, 18 S RNA sedimented approximately half way down the tube. Ribonucleoprotein samples were separated on 2035% sucrose gradients made up in low salt buffer (0.01 M Tris, pH 7.4) and run for 5 hr. Under these conditions the monoribosomal peak migrated nearly to the bottom of the tube. Each 13-ml gradient was scanned at 260 nm on a Gilford recording spectrophotometer and collected in 0.5-ml aliquots. Polysome Analysis After incubation for 1 hr with labeled amino acids, follicles were dissected and the ooplasm was collected in LM buffer. The sample was then treated with 0.5% Triton X-100 and 0.2% deoxycholic acid prior to fractionation. The supernatant
Messenger
RNA
175
Synthesis
after centrifugation at 1500 g for 10 min was divided, and one-half treated with 30 n&f EDTA. Both fractions were separated on 20-35% sucrose (SchwarzlMann ultrapure) gradients at 190,OOOg for 1 hr. Other tissues were prepared in a similar manner except that cells were disrupted in a Dounce homogenizer. Radioactivity
Analysis
All samples were counted on a Packard Tricarb scintillation counter. Gradient aliquots were precipitated with 10% trichloroacetic acid at 4°C with 0.1 ml added BSA carrier protein (1 mg/ml) and incorporated label was trapped by filtration through glass fiber filters (Reeve Angel, Clifton, N.J.). RESULTS
Presence of Polyadenylated Oocyte Cytoplasm
RNA in
Poly(A) + RNA extracted from chorionated eggs after in vivo labeling amounted to 0.2-0.3% of the total radioactivity in RNA. Both poly(U) bound and nonbound fractions were centrifuged on sucrose gradients and the resulting profiles are shown in Fig. 1. Poly(A) - RNA exhibited peaks at 28, 18, and 4-5 S while the polyadenylated fraction migrated over a wide range from approximately 7-28 S, with the major peak at 18 S. This type of analysis was repeated with in vitro labeled follicles as well. Ooplasm was removed and treated as described above. Essentially identical results were obtained, with Poly(A) + RNA amounting to 0.2-0.3% of total radioactivity. The labeled fraction in both experiments was base-sensitive and DNAse-resistant. These treatments were particularly important considering the long labeling periods. In vitro labeling produced little if any radioactive DNA. Activity
of Oocyte Messenger RNA
The relative absence of protein sis in ooplasm prior to fertilization
synthein in-
176
DEVELOPMENTAL
BIOLOGY
VOLUME
51, 1976
Developing adults were labeled by a series of three injections of [“Hluridine totaling 400 &I. Three days after the final injection, RNA was extracted from 200 eggs. Gradients of 15.30% sucrose were centrifuged for 6 hr at 40,000 rpm. The top profile shows nonmessenger RNA with peaks at 28, 18, and 4-5 S. Below is the profile of polyadenylated RNA displaying a broad range on either side of the 18 S peak.
this region. The experiment was repeated using [3H]uridine and the follicles were incubated for 12 hr to ensure a high specific activity of the RNA present. Wing tissue was pulsed for 2 hr and this proved sufficient to label polysomes as shown in Fig. 3. The nurse cells again exhibited EDTA-sensitive polysomal radioactivity, while the egg did not. This data conf%-ms electron micrographs (Berry, unpublished) showing an absence of oocyte polyribosomes and, thus, protein synthesis. Since the presence of newly synthesized messenger RNA and its apparent lack of biosynthetic activity were established, an attempt was made to characterize the cytoplasmic state of mRNA. Follicles were labeled with i3H]uridine for lo-20 hr in vitro and an S-15 from ooplasm was centrifuged for 5 hr on sucrose gradients. Following collection of O.&ml fractions, aliquots were pooled from various regions of the gradients as indicated in Fig. 4. RNA was extracted from each gradient fraction and passed through poly(U)-sepharose. Only
sects has been noted by a number of workers (Bier, 1963, 1967; King and Aggarwal, 1965; Pollack and Telfer, 1969); however, this presumption has been based almost entirely upon cytological and autoradiographic evidence. We have obtained corroborative data from attempts to isolate synthetically active, labeled polyribosomes from oocyte cytoplasm after incubation with “H amino acids. The resulting profiles and comparable profiles from wing and nurse cells are shown in Fig. 2. The EDTA-treated samples show a shift in o.d. from the monosome to subunits; however, there is no evidence of polysomally derived radioactivity in either oocyte sample. No precursor transport problem was apparent in these eggs since there is much non-TCA precipitable radioactivity in the cytoplasm. Gradients obtained from wing cells have significant amounts of polysomally derived RNA, while the nurse cells exhibit small, but detectable amounts of label in
FIG. 2. Polysome profiles after labeling with :‘H amino acid. Wing tissue and 100 follicles from developing pupa were labeled with 50 &i of “H amino acid mix for 1 hr. Nurse cells and ooplasm were isolated, samples were divided, and 30 mM EDTA was added to one aliquot prior to centrifugation.
FRACTION FIG.
NUMBER
1. Oocyte RNA.
PAGLIA, Wl”g
BERRY
AND KASTERN
1 Iwing EDTA
I
FIG. 3. Polysome profiles after labeling with [~‘Hluridine. Profiles similar to those shown in Fig. 2. Tissue was incubated with 100 FCi i”H]uridine, wing for 2 hr and follicles for 12 hr.
the hatched region of approximately lo-35 S contained significant amounts of poly(A) + RNA, with some 510% appearing in the region of low molecular weight material at the top of the gradient. No labeled poly(A) + RNA was found associated with either the monosome region or in the heavier range below it. This mRNA fraction was recentrifuged on sucrose gradients and exhibited the same profile as that extracted and isolated from previous ooplasm samples illustrated in Fig. 1.
Messenger
RNA
177
Synthesis
cell ribosomes are present as monosomes in the process of maturation or transport and that only a very small percentage is engaged in protein synthesis. The cytoplasmic profiles obtained from nurse cells (Figs. 2 and 3) with their considerable monosome peaks illustrate this phenomenon. To determine the fate of newly synthesized nurse cell mRNA, both intact follicles and isolated nurse cells were labeled in vitro. Labeling periods of 4-8 hr were employed and the ratio of poly(A)+ to poly(A) - RNA was much the same as that found in the oocyte (0.24-0.3%). With shorter pulses, (i.e., l-2 hr) a greater percentage of newly synthesized RNA messenger was found. However, when nurse cells were labeled with nucleotides and the cytoplasm separated on sucrose gradients, less than 10% of the poly(A)+ RNA was found in the polysomal region. Lengthening the centrifugation time to 5 hr allowed analysis of cytoplasmic elements smaller than the monosome. Labeled nurse cell cytoplasm analyzed in this manner is illustrated in Fig. 5, with cuts made in the
Source of Oocyte Messenger RNA The nurse cells associated with the egg are responsible for the synthesis and transport of ribosomal and transfer RNA to the oocyte and newly synthesized RNA does not accumulate in oocytes deprived of nurse cells (Hughes and Berry, 1970). Since the germinal vesicle has been shown to be inactive in RNA synthesis, and the follicle cells make no direct contribution to the egg cytoplasm, the nurse cells are likely candidates as the source of this messenger RNA as well. Hughes and Berry (1970) have also shown that most nurse
FRACTION 4. Oocyte
NUMeER
ribonucleoprotein. Two-hundredfifty follicles were labeled for 10 hr with 200 PC1 I:‘Hluridine and the ooplasm centrifuged for 5 hr. Broken lines represent pooled fractions of the gradient. The hatched area represents those fractions containing poly(A)-associated RNA. FIG.
178
DEVELOPMENTAL
FRACTION
BIOLOGY
51, 1976
NUMBER
cell ribonucleoprotein. Onehundred-fifty nurse cell caps (approx 1000 cells) were incubated for 6 hr with 100 &i LJH]uridine. Cytoplasm was isolated and separated on 30-35% sucrose gradients. Hatched areas show mRNA in both polysomal and subribosomal regions with 9095% found in the lower molecular weight fraction. PIG.
VOLUME
5. Nurse
gradients as shown. Appropriate aliquots from these gradients were pooled and the RNA extracted prior to binding on poly(U)-sepharose columns. The majority of cytoplasmic poly(A)+ mRNA (90-95%), was found in a region of 7-28 S, which is similar to the pattern of distribution of these elements in ooplasm. This mRNA fraction was recentrifuged on 1530% sucrose gradients and exhibited a size profile much like egg messenger as is shown in Fig. 6. To demonstrate the transport of mRNA from nurse cell to oocyte, whole follicles were incubated with [3Hluridine for various lengths of time, after which the egg cytoplasm was isolated and centrifuged on sucrose gradients. Figure 7 shows ooplasm profiles from 2, 4, 8, and 20 hr of incubation. At 2 hr, only labeled transfer and some poly(A)+ RNA have been transported as well as presumably mature, but unlabeled, ribosomes. At 4 hr, increased amounts of both mRNA and tRNA were observed. Occasionally, small, but insignificant, amounts of labeled monosomes
FIG. 6. Nurse cell poly(A) + RNA. Two-hundred nurse cell caps were incubated with 200 &i PH]uridine for 6 hr. Following homogenization and fractionation RNA was extracted from cytoplasm and separated on poly(U)-sepharose. The poly(A) + RNA fraction was then centrifuged for 6 hr on 1530% sucrose gradients.
4-
2hr
BOS
3-
” I 0
’
* 4 I
3
:2 I
40 30 20 IO
FRACTION
20 NUMBER
FIG. 7. Ribonucleoprotein transport. For each profile, 100 follicles were labeled with 100 &i [3H]uridine. Ooplasm was centrifuged on 20-35% sucrose gradients.
PAGLIA,
BERRY
Messenger
AND KASTERN
were evident at this time. By 8 hr, however, the ribosome peak predominated and poly(A)+ RNA represented a smaller percentage of total radioactivity, a situation which persisted through the 20-hr incubation. To illustrate the concommitent depletion of RNA from nurse cells with the increase of ooplasmic elements, pulse-chase experiments were employed. Follicles were incubated in vitro with [“Hluridine (400 &i/ml) for 1 hr and chased in fresh medium with added, unlabeled uridine (5 x lop4 M) for various periods of time. Total radioactivity in both eggs and nurse cells increased during the chase, apparently indicating that the unlabeled uridine was not sufficient to purge the cells of their large radioactive precursor pool. This experiment was repeated using an actinomytin D mediated shutdown of RNA synthesis in place of the chase. Follicles were incubated for l-2 hr with [“Hladenosine (200 $.X/ml) and, following several rinses in unlabeled medium, transferred to unlabeled Grace’s medium containing 25 pglml of actinomycin D for up to 6 hr. Both nurse cells and ooplasm were isolated after each chase and RNA extracted prior to fractionation on poly(U)-sepharose. Table I illustrates a rise in both poly(A)+ and poly(A)RNA in the egg over time with a
Cell type
Nurse cell
Oocyte
OF RNA
179
Synthesis
corresponding drop in labeled nurse cell RNA. As a rough control, follicles deprived of nurse cells were incubated for 5 hr with 100 PC1 of [sH]adenosine and uridine. In both cases insignificant amounts of incorporation were detected in ooplasmic RNA. CONCLUSIONS
At present, the most reliable criterion for nonpolysome-associated messenger RNA is the presence of a polyadenylic acid moiety, and based on the results reported here, mRNA is indeed present in unfertilized silkmoth oocytes. This fact is not surprising in view of the abundant evidence for its existence in other systems, most notably amphibians and echinoderms (Smith, Ecker, and Subtelny, 1966; Bell and Reeder, 1967; Tyler, 1967; Gould, 1969; Rosbach and Ford, 1974). Poly(U)-sepharose affinity chromatography of total ooplasmic RNA indicates a messenger complement of 0.2-0.3% of the total RNA, a range consistent in both in vivo and in vitro labeling experiments. This figure is much lower than that found in other insects cells and other systems, but is to be expected in view of the relatively vast store of ribosomes and transfer RNA in unfertilized eggs and early embryos. The presence of this poly(A)RNA in the unfer-
TABLE TRANSFER
RNA
1
FROM NURSE
CELW
TO OOCYTES”
Pulse (hr)
Act D chase (hr)
Poly(A) - RNA (cpm)
Poly (A) RNA (8)
Poly (A) + RNA
(cpm)
Poly (A) + RNA (So)
1 2 2 2
None None 2 6
6,767 2,850,480 749,591 115,121
97.1 99.7 99.8 99.7
199 8,542 1,876 340
2.85 0.34 0.25 0.29
1
None None 2 6
5,687 68,976 3,700,322 5,304,121
97.4 97.1 99.7 99.7
151 2,092 11,684 15,912
2.59 2.94 0.31 0.30
2 2 2
” For each time point, 200 follicles were labeled with 200 PCi [“Hladenosine in Grace’s medium. Actinomytin D 25 pg/ml was employed as a “chase.” Nurse cells were dissected from follicles at the termination of the “pulse” or “chase” period. Total extractable RNA was applied to poly(U)-sepharose columns to separate poly (A)+ and poly(A)fractions. Total acid-precipitable counts for each fraction were determined and counts per minute (cpm) represent the average of three experiments.
180
DEVELOPMENTAL
BIOLOGY
tilized egg is of interest in view of the apparent postfertilization polyadenylation of sea urchin oocyte mRNA (Slater et al., 1972, 1973; Wilt, 1973). The absence of messenger translation in the oocyte is of particular significance, especially in regard to the storage of maternal mRNA in eggs prior to fertilization for use in early embryogenesis. Considerable evidence for the presence of messengers stored in oocytes has come to light; however, it is still not certain whether or not some or all of these molecules are truly inactive in translation. The rapid increase in protein synthesis at fertilization has been studied in sea urchins (Slater et al., 1973) and amphibians (Rosbach and Ford, 1974). Particular attention is drawn in the Slater paper to the shift of mRNA from subribosomal to polysome regions of sucrose gradients. Although the subribosoma1 fraction was not well characterized, the apparent flow of this poly(A)+ RNA to the polysomal region was sufficient to propose a translational activation upon fertilization. The shift from subribosomal to the polysomal fraction is not unique to eggs and embryos, but may be a general characteristic of cells undergoing a transition from a quiescent to an active metabolic state (e.g., Rudland, 1975). Since active polyribosomes were not found in silkmoth eggs, using techniques that demonstrate activity in other cells, it seems reasonable to assume that this oocyte mRNA is biosynthetically inactive at least during vitellogenesis. Incubation with tritiated amino acids demonstrated active polysomes in wing tissues, with low amounts in nurse cells, and none in ooplasm. Repetition of this experiment using tritiated uridine was biased in favor of finding protein-synthetic activity in nurse cells and eggs because of the long incubation which ensured a high specific activity of the RNA. Under these conditions, polysomes were evident in wing cells and nurse cells but again absent in oocytes. The flow of newly synthesized ribosomes
VOLUME
51, 1976
from nurse cells to oocyte and the lack of appreciable accumulation of labeled RNA in oocytes deprived of nurse cells has already been documented (Hughes and Berry, 1970) and the experiments described above indicate an accelerated transport of mRNA. The pulse-chase experiment demonstrates a decrease in labeled nurse cell RNA coordinated with the increase in ooplasmic label. The near identity of the percentage of poly(A)+ RNA as compared with poly(A)RNA in nurse-cell cytoplasm and ooplasm after the 6-hr chase in particular suggests that addition of poly(A) in the ooplasm cannot account for the appearance of poly(A)+ RNA. These observations draw further attention to the unusual situation in which mRNA is synthesized by one cell type and used by another. While the profiles for subribosomal poly(A)+ RNA from both sources are quite similar, one cannot be certain that they are identical without demonstrating sequence homologies between them, and thus alternative hypotheses cannot be absolutely ruled out. The role of maternal messenger RNA is central to investigations of the mosaic development of insect eggs. In many insects, blastula formation represents the first embryonic cellularization. Prior to cortical migration nuclei appear to be totipotent (Illmensee, 1972) and cellular determination may well be the result of cytoplasmic elements, at least in the case of the presumptive germ plasm (Mahowald and Illmensee, 1974). Mahowald (1968) also suggests that localized mRNA may be involved in this determination although cytochemical analysis reveals only the presence of unspecified species of RNA in the polar granules associated with this phenomenon. Whether or not some of the poly(A) + RNA stored in silkmoth eggs has such a function is still to be determined. REFERENCES BALTHIJS,
E., QUERTIER,
J., FICQ, A., and BRACHET,
PAGLIA,
BERRY
AND KASTERN
J. (1965). Biochemical studies on nucleate and anucleate fragments isolated from sea urchin eggs. A comparison between fertilization and parthenogenetic activations. B&hem. Biophys. Acta 95, 408-417. BELL, E., and REEDER, R. (1967). The effect of fertiiization on protein synthesis in the egg of the surf clam Spisula solidissima. Biochem. Biophys. Acta 142, 500-511. BIER, K. (1963). Synthese,
interzellularer Transport, und Abbua von Ribokleinsaure im Ovar Den Tubenfliege Musca domestica. J. Cell. Biol. 16, 426-440. BIER, K. (1967). Oogenese, das Wachstum von Riesenzellen. Naturwissenschaften 54, 189-195. BRACHET, J., and DENIS, H. (1963). Effects of actinomycin D on morphogenesis. Nature ilondonl 198, 205-206. ECKER, R. E., SMITH, L. D., and SUBTELNY, S. (1968). Kinetics of protein synthesis in enucleate frog oocytes. Science 160, 1115-1117. EPEL, D. (1967). Protein synthesis in sea urchin eggs: A “late” response to fertilization. Proc. Nut. Acad. Sci. USA 57, 899-906. GOULD, M. C. (1969). A comparison of RNA and protein synthesis in fertilized and unfertilized eggs of Urechis caupo. Develop. Biol. 19, 482-497. GREENBERG, J. R. (1975). Messenger RNA metabolism of animal cells: Possible involvement of untranslated sequences and mRNA-associated proteins. J. Cell Biol. 64, 269-288. GROSS, P. R., and COUSINEAU, G. H. (1963). Effects of actinomycin D on macromolecule synthesis and early de.velopment in sea urchin eggs. Bioph,ys. Biochem. Res. Commun. 10, 321-367. GROSS, P. R., MALKIN, L. I., and MOYER, W. A. (1964). Template for the first proteins of embryonic development. Proc. Nat. Acad. Sci. USA 51, 407-414. HUGHES, M., and BERRY, S. J. (1970). The synthesis and secretion of ribosomes by nurse cells of Antherea polyphemus. Develop. Biol. 23, 651-664. ILLMENSEE, K. (1972). Developmental potencies of nuclei from cleavage, preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster. Wil. Roux Arch. 170, 267-298. KING, R. C., and AGGARWAL, S. K. (1965). Oogenesis in Hyalophora cecropia. Growth 29, 17-83. LOCKSHIN, R. A. (1966). Insect embryogenesis: Mac-
Messenger romolecular
RNA
Synthesis
synthesis
during
Science 154, 775-776. MAHOWALD, A. P. (1968). Polar
181 early development.
granules in Drosophila. II. Ultrastructural changes during early embryogenesis. J. Ezp. Zool. 167, 237-262. MAHOWALD, A. P., and ILLMENSEE, K. (19741. Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Nut. Acad. Sci. USA 71, 1016-1020. NUESCH, H. (1965). Die Imaginal-Entwicklung VOII Antheraea polyphemus Cr (lepidoptera). Zool. Jb. Anat. 82, 393-446. POLLACK, S. B., and TELFER, W. H. (19691. RNA in cecropia moth ovaries: Sites of synthesis, transport, and storage. J. Exp. Zool. 170, l-24. ROSBACH, M., and FORD, P. J. (1974). Polyadenylic acid containing RNA inxenopus laevis oocytes. J. Mol. Biol. 85, 87-101. RUDLAND, P. S., WEIL, S., and HINTON, A. R. (1975). Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts. J. Mol. Biol. 96, 745-766. SLATER, D. W., SLATER, I., and GILLESPIE, D. (1972). Post fertilization synthesis of polyadenylic acid in sea urchin embryos. Nature (London) 240, 333337. SLATER, I., GILLESPIE, D., and SLATER, D. W. (1973). Cytoplasmic adenylation and processing of maternal RNA. Proc. Nat. Acad. Sci. USA 70, 406-411. SMITH, L. D., ECKER, R. E., and SUBTELNY, S. (19661. The initiation of protein synthesis in eggs of Rana pipiens. Proc. Nat. Acad. Sci. USA 36, 1124-1128. TYLER, A. (1966). Incorporation of amino acids into protein by artificially activated non-nucleate fragments of sea urchin eggs. Biol. Bull. 130,450461. TYLER, A. (1967). Masked messenger RNA and cytoplasmic DNA in relation to protein synthesis and processes of fertilization and determination in embryonic development. S.ymp. Sot. Dev. Biol. 1, 170-226. WAGNER, A. F., BUGIANESI, R. L., and SHEN, T. Y. (1971). Preparation of sepharose-bound Poly(rI:rC). Biophys. Biochem. Res. Commun. 45, 184-189. WILT, F. H. (1973). Polyadenylation of maternal RNA of sea urchin eggs after fertilization. Proc. Nat. Acad. Sci. USA 70, 2345-2349.
BIOLOGY
Messenger
51,
173-181
(19761
RNA Synthesis, Transport, and Storage Ovarian Follicles’ L. M. PAGLIA,’ of Bzo1og.v.
Department
S. J. BERRY,:’ Weslcyn
Unioersity,
AND
in Silkmoth
W. H. KASTERN
Middletou~n,
Connectwut
06457
Developing oocytes of the silkmoths Actias lurm and Antheraea polyphemus were shown to contain significant amounts of polyadcnylatcd RNA. This presumed messenger RNA comprises 0.2 to 0.3Cr of the total extractable RNA, and is found exclusively in subribosomal fractions of ooplasm. Pulse-chase and other time-course studies indicate that the nurse cells are the probable source of this as well as other fractions of ooplasmic RNA. Labeled polyaderiylated RNA extracted from both nurse cells and ooplasm appears in similar, heterodisperse, fractions with a major peak at 18 S in sucrose gradients, No evidence of the presence of polysomes or of active protein or RNA synthesis was detected in the oocytes during vitellogenesis. INTRODUCTION
Developing oocytes of giant silkmoths provide very favorable material for the investigation of the “maternal” contribution to the early embryo. The mosaic development of many insect embryos in which regions of the egg seem to be determined before fertilization suggests that the contribution of the maternal genome may be of critical importance in directing the events of later embryogenesis. The burst of actinomycin-insensitive protein synthesis observed in the eggs of many animals (Brachet and Denis, 1963; Gross and Cousineau, 1963; Balthus, 1965; etc.) including insects (Lockshin, 1966) shortly after fertilization may represent utilization of maternally derived information. Insensitivity to actinomycin inhibition suggests that both general machinery (ribosomes, transfer RNA, etc.) and the specific programming molecules (mRNA) for protein synthesis are stored in the oocyte and activated at fertilization. There is considerable direct evidence for ’ Supported by Career Development Award (to SJB) No. 5.K4-Hd50195-02 and National Science Foundation Grant No. GB34155. ’ Current address: NIDR, NIH, Rethesda, Maryland 20014. ” Address reprint requests to S. J. Berry,
stored messenger RNA in amphibian (Brachet and Denis, 1963; Ecker et al., 1968) and sea urchin eggs (Gross et al., 1964; Tyler, 1966), although the mechanism by which protein synthesis is inhibited until fertilization is unknown. Various authors (Bell and Reeder, 1967; Epel, 1967, and others) have shown that, a low rate of prefertilization translation is detectable in sea urchin oocytes. In this communication we describe the synthesis and transport of RNA containing polyadenylated regions to the ooplasm during the early events of oogenesis. The ability to bind this RNA on poly(U)-sepharose and the similarity of the sedimentation behavior in sucrose gradients to messenger RNA derived from other systems (Greenberg, 1975) leads us to suggest that this fraction represents mRNA. We chose the ovarian follicles of silkmoths for this investigation because they can be easily dissected to isolate specific cell types, RNA is readily labeled in extirpated follicles and because no RNA synthesis can be demonstrated in the oocyte nucleus during vitellogenesis (Pollack and Telfer, 1969). The follicles consist of a palisade of small columnar follicle cells of mesodermal origin surrounding the developing oocyte and its seven sister trophocytes
173 Copyright G 19% by Acadeinic Press, Inc. All rights of reproduction in any form reserved.
174
DEVELOPMENTAL
BIOLOGY
all of which are derived from the primordial germ cells. The trophocytes or nurse cells are attached to each other and to the oocyte by cytoplasmic connectives (see King and Aggarwal, 1965, for details in Cecropia). We had previously demonstrated that ribosomes are synthesized in the nurse cells and transferred to the oocytes (Hughes and Berry, 1970) and we show here that a similar mechanism is involved in mRNA and transfer RNA synthesis. Since the major fractions of RNA and their associated proteins are not synthesized in the oocyte, but in the nurse cells, we suggest the possibility that newly synthesized protein found in oocytes before fertilization represents ribosomal and “informasomal” proteins rather than yolk or other cellular components. MATERIALS
Follicle
Preparation
AND
METHODS
and Labeling
Pupae of A. polyphemus and A. luna were obtained from either commercial sources or were reared in the field, and stored at 4°C until use. Adult development was initiated by return to 25°C at long day (17-hr light, 7-hr dark) photoperiod. Ovaries were removed from females at Day 13 of pupal-adult development (Neusch, 1965) and strings of follicles were dissected free of the surrounding ovariole sheath in plasma-free Grace’s medium (Grand Island Biological). Nurse cell caps were separated from oocytes using watchmaker’s forceps and ooplasm was isolated by gently tearing open the tunica propria. In uiuo labeling of cytoplasmic elements was accomplished by up to three injections of 100 &i of [3H]uridine (New England Nuclear) into developing adults at 3-day intervals. Upon emergence of the adult, whole chorionated eggs were removed from the ovary, rinsed in insect Ringer (0.13 M NaCl, 2 mM KCl, 1.5 mM CaCl,) and opened individually. In vitro labeling of RNA was carried out by immersing either whole follicles or isolated nurse cells in Grace’s medium containing 200 &i of
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51. 1976
VHluridine for varying periods up to 20 hr. Pulse-chase labeling was accomplished by preincubating follicles in 200 PCi of 13Hladenosine for 1 to 2 hr, followed by rinsing in Grace’s medium containing 25 pglml of actinomycin D (Calbiochem). In vitro labeling with amino acids involved incubation of follicles in medium containing 50 PCi of a mixture of “H-labeled amino acid mix (New England Nuclear). To ensure maximum synthetic activity, only follicles in which the nurse cell cap comprised l/z to 2/3 the volume of the follicles were selected. Fractionation
Procedure
Following incubation, nurse cells were suspended in 2 ml of low magnesium (LM) buffer (0.01 M MgS04, 0.005 M KCl, 0.01 M Tris, pH 7.4) in a prechilled Dounce homogenizer. Cells were allowed to stand for 5-10 min in buffer and then were gently homogenized by several strokes of a loose-fitting “A” pestle. Nurse cell cytoplasm or ooplasm washed free of follicular cells was centrifuged for 3 min at 1200 g to pellet nuclei and other large cell debris, and the resulting supernatant was decanted and recentrifuged for 20 min at 18,000g. The final supernatant was either extracted with phenol:choloroform or applied directly to sucrose gradients. RNA Extraction lation
and Messenger RNA Iso-
Samples to be extracted were adjusted to pH 9 in 0.1 M Tris, and an equal volume of redistilled pheno1:chlorofor-m (1:l) and SDS to a final concentration of 0.5% were added. The aqueous phase was repeatedly extracted since large amounts of yolk proteins were present in ooplasm samples. After extraction, RNA was precipitated with 2 volumes of cold 95% ethanol containing 2 mM sodium acetate at -20°C. The precipitate was washed with 95% ethanol prior to drying and resuspension. Messenger RNA was separated from other RNA fractions by column chroma-
PAGLIA,
BERRY
AND KASTERN
tography on poly(U)-sepharose according to techniques of Wagner et al. (1971). Poly(U)-sepharose was stored in buffered (0.01 M Tris, 0.01 M EDTA, 0.2% SDS, pH 7.4) 90% ether-extracted formamide at 4°C. The binding efficiency of the matrix was checked periodically by binding [“HIpoly(A) (Schwarz/Mann) to the column. Normal binding efficiency varied between 97-lOO%, and columns binding less than 95% were rejected. Columns consisted of 3-5 ml of packed poly(U)-sepharose, washed with buffered 90% formamide, and equilibrated with NETS buffer (0.1 M NaCl, 0.001 M EDTA, 0.1 M Tris, 0.2% SDS, pH 7.4). RNA samples were dissolved in 0.5-l ml of NETS and eluted from the column at a rate of 1 ml/30 min. Poly(A) + RNA was eluted by addition of 900/o buffered formamide, and ethanol precipitated with 50 pg of yeast tRNA (Sigma) added as carrier. Sucrose Gradient Analysis
Sedimentation
and
RNA samples were run on 15-30% sucrose gradients in NETS buffer at 40,000 r-pm for 6 hr on a SW 41 rotor (Beckman, Fullerton, Calif.) in a Beckman L2 65B preparative ultracentrifuge. Under these conditions, 18 S RNA sedimented approximately half way down the tube. Ribonucleoprotein samples were separated on 2035% sucrose gradients made up in low salt buffer (0.01 M Tris, pH 7.4) and run for 5 hr. Under these conditions the monoribosomal peak migrated nearly to the bottom of the tube. Each 13-ml gradient was scanned at 260 nm on a Gilford recording spectrophotometer and collected in 0.5-ml aliquots. Polysome Analysis After incubation for 1 hr with labeled amino acids, follicles were dissected and the ooplasm was collected in LM buffer. The sample was then treated with 0.5% Triton X-100 and 0.2% deoxycholic acid prior to fractionation. The supernatant
Messenger
RNA
175
Synthesis
after centrifugation at 1500 g for 10 min was divided, and one-half treated with 30 n&f EDTA. Both fractions were separated on 20-35% sucrose (SchwarzlMann ultrapure) gradients at 190,OOOg for 1 hr. Other tissues were prepared in a similar manner except that cells were disrupted in a Dounce homogenizer. Radioactivity
Analysis
All samples were counted on a Packard Tricarb scintillation counter. Gradient aliquots were precipitated with 10% trichloroacetic acid at 4°C with 0.1 ml added BSA carrier protein (1 mg/ml) and incorporated label was trapped by filtration through glass fiber filters (Reeve Angel, Clifton, N.J.). RESULTS
Presence of Polyadenylated Oocyte Cytoplasm
RNA in
Poly(A) + RNA extracted from chorionated eggs after in vivo labeling amounted to 0.2-0.3% of the total radioactivity in RNA. Both poly(U) bound and nonbound fractions were centrifuged on sucrose gradients and the resulting profiles are shown in Fig. 1. Poly(A) - RNA exhibited peaks at 28, 18, and 4-5 S while the polyadenylated fraction migrated over a wide range from approximately 7-28 S, with the major peak at 18 S. This type of analysis was repeated with in vitro labeled follicles as well. Ooplasm was removed and treated as described above. Essentially identical results were obtained, with Poly(A) + RNA amounting to 0.2-0.3% of total radioactivity. The labeled fraction in both experiments was base-sensitive and DNAse-resistant. These treatments were particularly important considering the long labeling periods. In vitro labeling produced little if any radioactive DNA. Activity
of Oocyte Messenger RNA
The relative absence of protein sis in ooplasm prior to fertilization
synthein in-
176
DEVELOPMENTAL
BIOLOGY
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51, 1976
Developing adults were labeled by a series of three injections of [“Hluridine totaling 400 &I. Three days after the final injection, RNA was extracted from 200 eggs. Gradients of 15.30% sucrose were centrifuged for 6 hr at 40,000 rpm. The top profile shows nonmessenger RNA with peaks at 28, 18, and 4-5 S. Below is the profile of polyadenylated RNA displaying a broad range on either side of the 18 S peak.
this region. The experiment was repeated using [3H]uridine and the follicles were incubated for 12 hr to ensure a high specific activity of the RNA present. Wing tissue was pulsed for 2 hr and this proved sufficient to label polysomes as shown in Fig. 3. The nurse cells again exhibited EDTA-sensitive polysomal radioactivity, while the egg did not. This data conf%-ms electron micrographs (Berry, unpublished) showing an absence of oocyte polyribosomes and, thus, protein synthesis. Since the presence of newly synthesized messenger RNA and its apparent lack of biosynthetic activity were established, an attempt was made to characterize the cytoplasmic state of mRNA. Follicles were labeled with i3H]uridine for lo-20 hr in vitro and an S-15 from ooplasm was centrifuged for 5 hr on sucrose gradients. Following collection of O.&ml fractions, aliquots were pooled from various regions of the gradients as indicated in Fig. 4. RNA was extracted from each gradient fraction and passed through poly(U)-sepharose. Only
sects has been noted by a number of workers (Bier, 1963, 1967; King and Aggarwal, 1965; Pollack and Telfer, 1969); however, this presumption has been based almost entirely upon cytological and autoradiographic evidence. We have obtained corroborative data from attempts to isolate synthetically active, labeled polyribosomes from oocyte cytoplasm after incubation with “H amino acids. The resulting profiles and comparable profiles from wing and nurse cells are shown in Fig. 2. The EDTA-treated samples show a shift in o.d. from the monosome to subunits; however, there is no evidence of polysomally derived radioactivity in either oocyte sample. No precursor transport problem was apparent in these eggs since there is much non-TCA precipitable radioactivity in the cytoplasm. Gradients obtained from wing cells have significant amounts of polysomally derived RNA, while the nurse cells exhibit small, but detectable amounts of label in
FIG. 2. Polysome profiles after labeling with :‘H amino acid. Wing tissue and 100 follicles from developing pupa were labeled with 50 &i of “H amino acid mix for 1 hr. Nurse cells and ooplasm were isolated, samples were divided, and 30 mM EDTA was added to one aliquot prior to centrifugation.
FRACTION FIG.
NUMBER
1. Oocyte RNA.
PAGLIA, Wl”g
BERRY
AND KASTERN
1 Iwing EDTA
I
FIG. 3. Polysome profiles after labeling with [~‘Hluridine. Profiles similar to those shown in Fig. 2. Tissue was incubated with 100 FCi i”H]uridine, wing for 2 hr and follicles for 12 hr.
the hatched region of approximately lo-35 S contained significant amounts of poly(A) + RNA, with some 510% appearing in the region of low molecular weight material at the top of the gradient. No labeled poly(A) + RNA was found associated with either the monosome region or in the heavier range below it. This mRNA fraction was recentrifuged on sucrose gradients and exhibited the same profile as that extracted and isolated from previous ooplasm samples illustrated in Fig. 1.
Messenger
RNA
177
Synthesis
cell ribosomes are present as monosomes in the process of maturation or transport and that only a very small percentage is engaged in protein synthesis. The cytoplasmic profiles obtained from nurse cells (Figs. 2 and 3) with their considerable monosome peaks illustrate this phenomenon. To determine the fate of newly synthesized nurse cell mRNA, both intact follicles and isolated nurse cells were labeled in vitro. Labeling periods of 4-8 hr were employed and the ratio of poly(A)+ to poly(A) - RNA was much the same as that found in the oocyte (0.24-0.3%). With shorter pulses, (i.e., l-2 hr) a greater percentage of newly synthesized RNA messenger was found. However, when nurse cells were labeled with nucleotides and the cytoplasm separated on sucrose gradients, less than 10% of the poly(A)+ RNA was found in the polysomal region. Lengthening the centrifugation time to 5 hr allowed analysis of cytoplasmic elements smaller than the monosome. Labeled nurse cell cytoplasm analyzed in this manner is illustrated in Fig. 5, with cuts made in the
Source of Oocyte Messenger RNA The nurse cells associated with the egg are responsible for the synthesis and transport of ribosomal and transfer RNA to the oocyte and newly synthesized RNA does not accumulate in oocytes deprived of nurse cells (Hughes and Berry, 1970). Since the germinal vesicle has been shown to be inactive in RNA synthesis, and the follicle cells make no direct contribution to the egg cytoplasm, the nurse cells are likely candidates as the source of this messenger RNA as well. Hughes and Berry (1970) have also shown that most nurse
FRACTION 4. Oocyte
NUMeER
ribonucleoprotein. Two-hundredfifty follicles were labeled for 10 hr with 200 PC1 I:‘Hluridine and the ooplasm centrifuged for 5 hr. Broken lines represent pooled fractions of the gradient. The hatched area represents those fractions containing poly(A)-associated RNA. FIG.
178
DEVELOPMENTAL
FRACTION
BIOLOGY
51, 1976
NUMBER
cell ribonucleoprotein. Onehundred-fifty nurse cell caps (approx 1000 cells) were incubated for 6 hr with 100 &i LJH]uridine. Cytoplasm was isolated and separated on 30-35% sucrose gradients. Hatched areas show mRNA in both polysomal and subribosomal regions with 9095% found in the lower molecular weight fraction. PIG.
VOLUME
5. Nurse
gradients as shown. Appropriate aliquots from these gradients were pooled and the RNA extracted prior to binding on poly(U)-sepharose columns. The majority of cytoplasmic poly(A)+ mRNA (90-95%), was found in a region of 7-28 S, which is similar to the pattern of distribution of these elements in ooplasm. This mRNA fraction was recentrifuged on 1530% sucrose gradients and exhibited a size profile much like egg messenger as is shown in Fig. 6. To demonstrate the transport of mRNA from nurse cell to oocyte, whole follicles were incubated with [3Hluridine for various lengths of time, after which the egg cytoplasm was isolated and centrifuged on sucrose gradients. Figure 7 shows ooplasm profiles from 2, 4, 8, and 20 hr of incubation. At 2 hr, only labeled transfer and some poly(A)+ RNA have been transported as well as presumably mature, but unlabeled, ribosomes. At 4 hr, increased amounts of both mRNA and tRNA were observed. Occasionally, small, but insignificant, amounts of labeled monosomes
FIG. 6. Nurse cell poly(A) + RNA. Two-hundred nurse cell caps were incubated with 200 &i PH]uridine for 6 hr. Following homogenization and fractionation RNA was extracted from cytoplasm and separated on poly(U)-sepharose. The poly(A) + RNA fraction was then centrifuged for 6 hr on 1530% sucrose gradients.
4-
2hr
BOS
3-
” I 0
’
* 4 I
3
:2 I
40 30 20 IO
FRACTION
20 NUMBER
FIG. 7. Ribonucleoprotein transport. For each profile, 100 follicles were labeled with 100 &i [3H]uridine. Ooplasm was centrifuged on 20-35% sucrose gradients.
PAGLIA,
BERRY
Messenger
AND KASTERN
were evident at this time. By 8 hr, however, the ribosome peak predominated and poly(A)+ RNA represented a smaller percentage of total radioactivity, a situation which persisted through the 20-hr incubation. To illustrate the concommitent depletion of RNA from nurse cells with the increase of ooplasmic elements, pulse-chase experiments were employed. Follicles were incubated in vitro with [“Hluridine (400 &i/ml) for 1 hr and chased in fresh medium with added, unlabeled uridine (5 x lop4 M) for various periods of time. Total radioactivity in both eggs and nurse cells increased during the chase, apparently indicating that the unlabeled uridine was not sufficient to purge the cells of their large radioactive precursor pool. This experiment was repeated using an actinomytin D mediated shutdown of RNA synthesis in place of the chase. Follicles were incubated for l-2 hr with [“Hladenosine (200 $.X/ml) and, following several rinses in unlabeled medium, transferred to unlabeled Grace’s medium containing 25 pglml of actinomycin D for up to 6 hr. Both nurse cells and ooplasm were isolated after each chase and RNA extracted prior to fractionation on poly(U)-sepharose. Table I illustrates a rise in both poly(A)+ and poly(A)RNA in the egg over time with a
Cell type
Nurse cell
Oocyte
OF RNA
179
Synthesis
corresponding drop in labeled nurse cell RNA. As a rough control, follicles deprived of nurse cells were incubated for 5 hr with 100 PC1 of [sH]adenosine and uridine. In both cases insignificant amounts of incorporation were detected in ooplasmic RNA. CONCLUSIONS
At present, the most reliable criterion for nonpolysome-associated messenger RNA is the presence of a polyadenylic acid moiety, and based on the results reported here, mRNA is indeed present in unfertilized silkmoth oocytes. This fact is not surprising in view of the abundant evidence for its existence in other systems, most notably amphibians and echinoderms (Smith, Ecker, and Subtelny, 1966; Bell and Reeder, 1967; Tyler, 1967; Gould, 1969; Rosbach and Ford, 1974). Poly(U)-sepharose affinity chromatography of total ooplasmic RNA indicates a messenger complement of 0.2-0.3% of the total RNA, a range consistent in both in vivo and in vitro labeling experiments. This figure is much lower than that found in other insects cells and other systems, but is to be expected in view of the relatively vast store of ribosomes and transfer RNA in unfertilized eggs and early embryos. The presence of this poly(A)RNA in the unfer-
TABLE TRANSFER
RNA
1
FROM NURSE
CELW
TO OOCYTES”
Pulse (hr)
Act D chase (hr)
Poly(A) - RNA (cpm)
Poly (A) RNA (8)
Poly (A) + RNA
(cpm)
Poly (A) + RNA (So)
1 2 2 2
None None 2 6
6,767 2,850,480 749,591 115,121
97.1 99.7 99.8 99.7
199 8,542 1,876 340
2.85 0.34 0.25 0.29
1
None None 2 6
5,687 68,976 3,700,322 5,304,121
97.4 97.1 99.7 99.7
151 2,092 11,684 15,912
2.59 2.94 0.31 0.30
2 2 2
” For each time point, 200 follicles were labeled with 200 PCi [“Hladenosine in Grace’s medium. Actinomytin D 25 pg/ml was employed as a “chase.” Nurse cells were dissected from follicles at the termination of the “pulse” or “chase” period. Total extractable RNA was applied to poly(U)-sepharose columns to separate poly (A)+ and poly(A)fractions. Total acid-precipitable counts for each fraction were determined and counts per minute (cpm) represent the average of three experiments.
180
DEVELOPMENTAL
BIOLOGY
tilized egg is of interest in view of the apparent postfertilization polyadenylation of sea urchin oocyte mRNA (Slater et al., 1972, 1973; Wilt, 1973). The absence of messenger translation in the oocyte is of particular significance, especially in regard to the storage of maternal mRNA in eggs prior to fertilization for use in early embryogenesis. Considerable evidence for the presence of messengers stored in oocytes has come to light; however, it is still not certain whether or not some or all of these molecules are truly inactive in translation. The rapid increase in protein synthesis at fertilization has been studied in sea urchins (Slater et al., 1973) and amphibians (Rosbach and Ford, 1974). Particular attention is drawn in the Slater paper to the shift of mRNA from subribosomal to polysome regions of sucrose gradients. Although the subribosoma1 fraction was not well characterized, the apparent flow of this poly(A)+ RNA to the polysomal region was sufficient to propose a translational activation upon fertilization. The shift from subribosomal to the polysomal fraction is not unique to eggs and embryos, but may be a general characteristic of cells undergoing a transition from a quiescent to an active metabolic state (e.g., Rudland, 1975). Since active polyribosomes were not found in silkmoth eggs, using techniques that demonstrate activity in other cells, it seems reasonable to assume that this oocyte mRNA is biosynthetically inactive at least during vitellogenesis. Incubation with tritiated amino acids demonstrated active polysomes in wing tissues, with low amounts in nurse cells, and none in ooplasm. Repetition of this experiment using tritiated uridine was biased in favor of finding protein-synthetic activity in nurse cells and eggs because of the long incubation which ensured a high specific activity of the RNA. Under these conditions, polysomes were evident in wing cells and nurse cells but again absent in oocytes. The flow of newly synthesized ribosomes
VOLUME
51, 1976
from nurse cells to oocyte and the lack of appreciable accumulation of labeled RNA in oocytes deprived of nurse cells has already been documented (Hughes and Berry, 1970) and the experiments described above indicate an accelerated transport of mRNA. The pulse-chase experiment demonstrates a decrease in labeled nurse cell RNA coordinated with the increase in ooplasmic label. The near identity of the percentage of poly(A)+ RNA as compared with poly(A)RNA in nurse-cell cytoplasm and ooplasm after the 6-hr chase in particular suggests that addition of poly(A) in the ooplasm cannot account for the appearance of poly(A)+ RNA. These observations draw further attention to the unusual situation in which mRNA is synthesized by one cell type and used by another. While the profiles for subribosomal poly(A)+ RNA from both sources are quite similar, one cannot be certain that they are identical without demonstrating sequence homologies between them, and thus alternative hypotheses cannot be absolutely ruled out. The role of maternal messenger RNA is central to investigations of the mosaic development of insect eggs. In many insects, blastula formation represents the first embryonic cellularization. Prior to cortical migration nuclei appear to be totipotent (Illmensee, 1972) and cellular determination may well be the result of cytoplasmic elements, at least in the case of the presumptive germ plasm (Mahowald and Illmensee, 1974). Mahowald (1968) also suggests that localized mRNA may be involved in this determination although cytochemical analysis reveals only the presence of unspecified species of RNA in the polar granules associated with this phenomenon. Whether or not some of the poly(A) + RNA stored in silkmoth eggs has such a function is still to be determined. REFERENCES BALTHIJS,
E., QUERTIER,
J., FICQ, A., and BRACHET,
PAGLIA,
BERRY
AND KASTERN
J. (1965). Biochemical studies on nucleate and anucleate fragments isolated from sea urchin eggs. A comparison between fertilization and parthenogenetic activations. B&hem. Biophys. Acta 95, 408-417. BELL, E., and REEDER, R. (1967). The effect of fertiiization on protein synthesis in the egg of the surf clam Spisula solidissima. Biochem. Biophys. Acta 142, 500-511. BIER, K. (1963). Synthese,
interzellularer Transport, und Abbua von Ribokleinsaure im Ovar Den Tubenfliege Musca domestica. J. Cell. Biol. 16, 426-440. BIER, K. (1967). Oogenese, das Wachstum von Riesenzellen. Naturwissenschaften 54, 189-195. BRACHET, J., and DENIS, H. (1963). Effects of actinomycin D on morphogenesis. Nature ilondonl 198, 205-206. ECKER, R. E., SMITH, L. D., and SUBTELNY, S. (1968). Kinetics of protein synthesis in enucleate frog oocytes. Science 160, 1115-1117. EPEL, D. (1967). Protein synthesis in sea urchin eggs: A “late” response to fertilization. Proc. Nut. Acad. Sci. USA 57, 899-906. GOULD, M. C. (1969). A comparison of RNA and protein synthesis in fertilized and unfertilized eggs of Urechis caupo. Develop. Biol. 19, 482-497. GREENBERG, J. R. (1975). Messenger RNA metabolism of animal cells: Possible involvement of untranslated sequences and mRNA-associated proteins. J. Cell Biol. 64, 269-288. GROSS, P. R., and COUSINEAU, G. H. (1963). Effects of actinomycin D on macromolecule synthesis and early de.velopment in sea urchin eggs. Bioph,ys. Biochem. Res. Commun. 10, 321-367. GROSS, P. R., MALKIN, L. I., and MOYER, W. A. (1964). Template for the first proteins of embryonic development. Proc. Nat. Acad. Sci. USA 51, 407-414. HUGHES, M., and BERRY, S. J. (1970). The synthesis and secretion of ribosomes by nurse cells of Antherea polyphemus. Develop. Biol. 23, 651-664. ILLMENSEE, K. (1972). Developmental potencies of nuclei from cleavage, preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs ofDrosophila melanogaster. Wil. Roux Arch. 170, 267-298. KING, R. C., and AGGARWAL, S. K. (1965). Oogenesis in Hyalophora cecropia. Growth 29, 17-83. LOCKSHIN, R. A. (1966). Insect embryogenesis: Mac-
Messenger romolecular
RNA
Synthesis
synthesis
during
Science 154, 775-776. MAHOWALD, A. P. (1968). Polar
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granules in Drosophila. II. Ultrastructural changes during early embryogenesis. J. Ezp. Zool. 167, 237-262. MAHOWALD, A. P., and ILLMENSEE, K. (19741. Transplantation of posterior polar plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. Proc. Nut. Acad. Sci. USA 71, 1016-1020. NUESCH, H. (1965). Die Imaginal-Entwicklung VOII Antheraea polyphemus Cr (lepidoptera). Zool. Jb. Anat. 82, 393-446. POLLACK, S. B., and TELFER, W. H. (19691. RNA in cecropia moth ovaries: Sites of synthesis, transport, and storage. J. Exp. Zool. 170, l-24. ROSBACH, M., and FORD, P. J. (1974). Polyadenylic acid containing RNA inxenopus laevis oocytes. J. Mol. Biol. 85, 87-101. RUDLAND, P. S., WEIL, S., and HINTON, A. R. (1975). Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts. J. Mol. Biol. 96, 745-766. SLATER, D. W., SLATER, I., and GILLESPIE, D. (1972). Post fertilization synthesis of polyadenylic acid in sea urchin embryos. Nature (London) 240, 333337. SLATER, I., GILLESPIE, D., and SLATER, D. W. (1973). Cytoplasmic adenylation and processing of maternal RNA. Proc. Nat. Acad. Sci. USA 70, 406-411. SMITH, L. D., ECKER, R. E., and SUBTELNY, S. (19661. The initiation of protein synthesis in eggs of Rana pipiens. Proc. Nat. Acad. Sci. USA 36, 1124-1128. TYLER, A. (1966). Incorporation of amino acids into protein by artificially activated non-nucleate fragments of sea urchin eggs. Biol. Bull. 130,450461. TYLER, A. (1967). Masked messenger RNA and cytoplasmic DNA in relation to protein synthesis and processes of fertilization and determination in embryonic development. S.ymp. Sot. Dev. Biol. 1, 170-226. WAGNER, A. F., BUGIANESI, R. L., and SHEN, T. Y. (1971). Preparation of sepharose-bound Poly(rI:rC). Biophys. Biochem. Res. Commun. 45, 184-189. WILT, F. H. (1973). Polyadenylation of maternal RNA of sea urchin eggs after fertilization. Proc. Nat. Acad. Sci. USA 70, 2345-2349.