Two-dimensional electrophoresis of labeled proteins and hybridization of mRNAs with specific gene probes was used to demonstrate changes in protein synthesis, and the cytoplasmic control of actin maternal mRNAs during the early development of llyanassa.

The isolated polar lobe was used as a nucleus-free egg fragment to study the regulation of translation. It was shown that actin mRNAs are present in the unfertilized egg and are therefore maternal in origin, are inactive during early cleavage, and are translated in normal, lobeless, actinomycin D-treated blastulae and in isolated polar lobes that have been aged for 24 h. Thus, the activation of actin mRNAs is controlled by cytoplasmic factors that function independently of cleavage and nuclear activity. I suggest that the running of a cytoplasmic clock determines when maternal mRNAs are activated, and that this clock is made and set running during oogenesis.

Changes in protein synthesis that occurred during early cleavage were shown to also involve the diminution of some early cleavage proteins, and it was suggested that this diminution is controlled by cytoplasmic factors localized in the blastomeres of the lobeless egg but absent from the polar lobe.

During the embryogenesis of the marine mud snail llyanassa obsoleta, a transition in the pattern of protein synthesis occurs after one day of development when the embryo consists of 28 to 30 cells (Collier & McCarthy, 1981; Brandhorst & Newrock, 1981). This transition is exemplified by deletions, additions and changes in the rate of accumulation of proteins synthesized at this stage.

In this paper, I have used the llyanassa polar lobe, an isolatable and anucleate protrusion of vegetal ooplasm, as a means to study the cytoplasmic regulation of protein synthesis during early development. I report a detailed study of earlier observations (Collier & McCarthy, 1981; Brandhorst & Newrock, 1981) on the synthesis of the actins, which are an integral part of the early changes in protein synthesis, and show that this part of the transition is controlled by cytoplasmic factors that operate independently of cleavage and nuclear activity.

Rearing of embryos

Snails collected from Plumb Beach in Brooklyn, New York were brought into the laboratory and maintained in a tank of recirculating sea water where, when fed a clam on alternate days, they laid eggs daily. Embryos were reared at 20°C in Woods Hole sea water, which had been filtered through a 0·45 μm pore Millipore membrane and contained 50 μg ml-1 each of penicillin and streptomycin. Polar lobes were isolated by agitation in Ca2+- and Mg2+-low sea water, as described previously (Collier, 1981).

In vivo labeling of proteins and sample preparation

Embryos or polar lobes were reared to the desired stage, incubated for 4h in 120 μCiml-1 of L-[35S]methionine (New England Nuclear, 800–1100 Cimmole-1), washed in Ca2+- and Mg2+-free sea water, homogenized in 0·02m-Tris (pH 8·8) containing 0·002 M-CaCl2 and 0·05 mg ml-1 of micrococcal nuclease (Sigma Chemical Co.), quick-frozen and lyophilized. The dried homogenate, usually prepared from 200 to 400 embryos or polar lobes, was dissolved in 0·1 ml of a sample buffer containing 9·2 M-urea (Schwarz/Mann Ultra Pure), 2·0 % NP-40 (Tergito, type NP-40 from Sigma Chemical Co.), 0·1 M-dithiothreitol, 0·4% pH3–10 ampholyte and 1·6% pH 5–7 ampholyte (all ampholytes were from BioRad Laboratories), and stored at –70°C.

Electrophoresis

Isoelectric focusing of the thawed protein samples was carried out as described by O’Farrell (1975) except for a final ampholyte concentration of 2·0% (1·6% pH 5–7; 0·4% pH3–10) in the focusing gels. The focusing gels were polymerized in 20x 0·15 cm glass tubes, and electrophoresis was at 25 °C for 19000 volt-hours. Second dimension electrophoresis was on 10% polyacrylamide separating gels and 5·4 % stacking gels as described by Laemmli (1970).

Actins were identified by coelectrophoresis of known actins with radioactive proteins extracted from Ilyanassa embryos. After the initial identification of actins on two-dimensional gels, all subsequent identifications and comparisons were done by aligning the numbered reference proteins (numbers 1 through 17 in Fig. 2) of autoradiographs superimposed and viewed over a light-box.

Isolation and hybridization of mRNAs

Messenger RNA was isolated from 1200 mature eggs or 400 24 h embryos by homogenization in a Dounce homogenizer in lOmm-vanadyl nucleoside complex, 0·5 % Brij-35, and 0·5 % sodium deoxycholate. The homogenate was digested for 30 min at 37°C with Proteinase K (200 μg ml-1) added directly to the homogenate. One volume of supersaturated Nal was added to the proteinase-digested homogenate, which was then diluted with one volume of saturated Nal and filtered through a nitrocellulose filter mounted in a Schleicher and Shuell ‘dot-blot’ apparatus. This is the Nal ‘dot-blot’ assay developed by Gillespie (Bresser et al. 1983) in which it has been shown that mRNA, treated as described above, binds selectively to nitrocellulose in 12·2M-NaI.

An actin gene probe was prepared from a chicken alpha actin gene (from Cleveland et al. 1980), inserted into Wzndlll digested pBR322 and cloned in E. coli HB101. The use of a chicken gene as a probe was based on the demonstrated similarity between tryptic peptide fingerprints of Ilyanassa and chicken actin (Schmidt et al. 1980). The plasmid was isolated and purified by centrifugation on a CsCl-ethidium bromide gradient and made radioactive by nick-translation (Rigby et al. 1977) with [32P]dCTP.

RNase sensitivity of the hybridization reaction, and therefore its RNA dependence, is shown in Fig. 3A. The RNA-nitrocellulose filters were air-dried and prehybridized in 6×SSC, 1% SDS, 0·2 % polyvinylpyrrolidone, 0·2 % Ficoll, 0·2 % BSA, 10 mm-vanadyl nucleoside complex, 50 μg ml-1each of Poly(A) and salmon sperm DNA for 24 h at 42 °C. Hybridization was in 50 % formamide, 6×SSC, 1 % SDS and a radioactive actin probe (prepared as described above) at 42 °C for 24 h. Hybridized filters were washed extensively and autoradiographed with Kodak XAR X-ray film.

Changes in protein synthesis

A major transition in the proteins synthesized by the Ilyanassa embryo occurs after 24 h of development when the embryo has an average of 29 cells. This is the mesentoblast stage of development as designated previously (Collier & McCarthy, 1981).

This transition involves, as shown in Fig. 1A and 1B, the disappearance of 23 peptides that were present during early cleavage (2- to 8-cell) and the appearance of 26 peptides that were not detectable earlier. Changes unique to either stage are marked by curved arrows in Fig. 1A and 1B, i.e. the marked proteins in Fig. 1A disappear in the mesentoblast embryo and those marked in Fig. IB are new proteins synthesized by the mesentoblast embryo. All other proteins shown in these figures are common to both cleavage- and mesento-blast-stage embryos; however, significant changes in the rate of accumulation of many proteins occur between these two stages of development. Some of these quantitative changes (Fig. 1A and 1B) are marked as examples by ‘up’ and ‘down’ arrowheads to show increases and decreases, respectively, in the rates of accumulation.

Fig. 1.

Changes in protein synthesis. (A) Early-cleavage-stage embryos pulsed for 4h from the second polar body to the 8-cell stage; exposed for 2×109 disintegrations. (B) Mesentoblast-stage embryos pulsed from 20 to 24h of development; exposed for 2·3 ×109 disintegrations. Curved arrows in (A) mark those proteins that disappear in the mesentoblast embryo and in (B) proteins that are first synthesized by the mesentoblast embryo. The ‘up’ and ‘down’ arrows indicate increases and decreases, respectively, in the intensity of spots that occur between cleavage- and mesentoblast-stages of development. In (A) the open arrow indicates the position occupied by actins when present, in (B) actins are marked with a curved arrow labeled Ac.

Fig. 1.

Changes in protein synthesis. (A) Early-cleavage-stage embryos pulsed for 4h from the second polar body to the 8-cell stage; exposed for 2×109 disintegrations. (B) Mesentoblast-stage embryos pulsed from 20 to 24h of development; exposed for 2·3 ×109 disintegrations. Curved arrows in (A) mark those proteins that disappear in the mesentoblast embryo and in (B) proteins that are first synthesized by the mesentoblast embryo. The ‘up’ and ‘down’ arrows indicate increases and decreases, respectively, in the intensity of spots that occur between cleavage- and mesentoblast-stages of development. In (A) the open arrow indicates the position occupied by actins when present, in (B) actins are marked with a curved arrow labeled Ac.

Fig. 2.

Synthesis of Actins. All embryos and isolates were pulsed for 4h with [35S]methionine; cleavage-stage embryos were pulsed from the second polar body to the 8-cell stage. (A) 2- to 8-cell normal embryos exposed for 6·5 × 1010 disintegrations, (B) 24 h normal embryos exposed for 4·6 × 1010 disintegrations (C) 2- to 8-cell lobeless embryos exposed for 2·3 × 109 disintegrations, (D) 24h lobeless embryos exposed for 2·3 × 109 disintegrations, (E) polar lobes isolated and pulsed at the trefoil stage and exposed for IT × lO10 disintegrations, (F) embryos reared continuously for the first 24 h of development in actinomycin D and exposed for 6×109 disintegrations, and (G) isolated polar lobes ‘aged’ for 24 h, pulsed for 4h with [35S]methionine and exposed for 5·6×1010 disintegrations. Actins are marked with unnumbered arrowheads, expected position of actins in Figs 3A, 3C, and 3E are marked by open circles, numbered arrowheads indicate the position of reference proteins in relation to actins, and arrowheads lettered (a), (b) and (c) mark proteins that are normally diminished in intensity at the mesentoblast stage of development.

Fig. 2.

Synthesis of Actins. All embryos and isolates were pulsed for 4h with [35S]methionine; cleavage-stage embryos were pulsed from the second polar body to the 8-cell stage. (A) 2- to 8-cell normal embryos exposed for 6·5 × 1010 disintegrations, (B) 24 h normal embryos exposed for 4·6 × 1010 disintegrations (C) 2- to 8-cell lobeless embryos exposed for 2·3 × 109 disintegrations, (D) 24h lobeless embryos exposed for 2·3 × 109 disintegrations, (E) polar lobes isolated and pulsed at the trefoil stage and exposed for IT × lO10 disintegrations, (F) embryos reared continuously for the first 24 h of development in actinomycin D and exposed for 6×109 disintegrations, and (G) isolated polar lobes ‘aged’ for 24 h, pulsed for 4h with [35S]methionine and exposed for 5·6×1010 disintegrations. Actins are marked with unnumbered arrowheads, expected position of actins in Figs 3A, 3C, and 3E are marked by open circles, numbered arrowheads indicate the position of reference proteins in relation to actins, and arrowheads lettered (a), (b) and (c) mark proteins that are normally diminished in intensity at the mesentoblast stage of development.

Fig. 3.

Hybridization of mRNAs with an actin gene probe. (A) Mesentoblast RNA; RNA extracted from mesentoblast-stage embryos and hybridized with an actin gene probe. pAl, pBR322 plasmid containing an actin gene insert; pBR, pBR322 plasmid; E. coli E. coli DNA; RNase, RNA blot from mesentoblast-stage embryos digested with RNase before hybridization. (B) Egg RNA; blot of RNA extracted from mature unfertilized eggs hybridized with an actin gene probe.

Fig. 3.

Hybridization of mRNAs with an actin gene probe. (A) Mesentoblast RNA; RNA extracted from mesentoblast-stage embryos and hybridized with an actin gene probe. pAl, pBR322 plasmid containing an actin gene insert; pBR, pBR322 plasmid; E. coli E. coli DNA; RNase, RNA blot from mesentoblast-stage embryos digested with RNase before hybridization. (B) Egg RNA; blot of RNA extracted from mature unfertilized eggs hybridized with an actin gene probe.

The electrophoresis of the gels shown in Fig. 1 were under slightly different conditions to the gels in Fig. 2 (see below) and the position of specific proteins are not identical. The actins in Fig. IB are marked by a curved arrow labeled Ac, their absence in Fig. 1A is indicated by an open arrow.

In previous observations, Brandhorst & Newrock (1981) identified 4 polypeptides that disappeared by the mesentoblast stage while Collier & McCarthy (1981) recorded the disappearance of 12 polypeptides at this stage. In Fig. 1A and IB, the 23 peptides that have disappeared and the 26 previously undetected peptides are visible because the gels in Fig. 1A and IB are clearer than those previously published. Further, gels published in this paper are not readily comparable to those published earlier by Brandhorst & Newrock (1981) and by Collier & McCarthy (1981). The major difference in these sets of gels comes from the digestion of the embryo proteins with micrococcal nuclease before electrophoresis, and the greater resolution obtained by the use of long narrow focusing gels (20×0·15 cm) in the present study.

Actin mRNAs

That the actin mRNAs are transcribed during oogenesis was indicated by their synthesis in embryos incubated continuously from the trefoil stage to the 25-cell stage (24 h) in actinomycin D (Fig. 2F) at a concentration of 50 μg ml-1 which has been shown to be sufficient to repress 95 % of the incorporation of uridine into RNA (Collier, 1966). All of the proteins made during early cleavage and 98 % of those made by the mesentoblast-stage embryo, including the actins, are insensitive to actinomycin D and are therefore probably translated from oogenetic transcripts (Collier & McCarthy, 1981). Although the translation of the actins is unaffected by actinomycin D, many proteins surrounding the actins are atypically expressed (compare Fig. 2F with Fig. 2G and 2D). It is of special interest that the proteins labeled a, b and c in Fig. 2A, which are normally absent or greatly reduced in amount in mesentoblast-stage embryos (Fig. 2B and 2D), are also absent in mesentoblast embryos treated with actinomycin D.

In order to demonstrate directly that the actin mRNAs are maternal in origin, RNA extracted from mature unfertilized eggs was hybridized with a radioactive beta-actin gene probe (Fig. 3B). The gene probe not only hybridized with RNA from mature eggs (Fig. 3B), but also with RNAs extracted from mesentoblast embryos (pAl in Fig. 3A). Thus actin mRNAs are present in the mature unfertilized egg and in mesentoblast-stage embryos.

Stage-specific synthesis of actins

Even though actin mRNAs are present in the mature egg they are not translated during early cleavage (Fig. 2A). Only after 24 h of development at the mesentoblast stage does actin synthesis occur (Fig. 2B). The absence of actin synthesis during early cleavage is not related to repressive interactions between the polar lobe and the lobeless egg because neither of these parts of the embryo synthesize actins in isolation (Fig. 2C and 2E).

The actins are synthesized by both normal and lobeless mesentoblast-stage embryos (Fig. 2B and 2E), therefore their synthesis is not dependent on the polar lobe.

Stage-specific changes in protein synthesis by isolated polar lobes

Actin maternal mRNAs are available in mature eggs (Fig. 3B), but inactive during early cleavage in normal and lobeless embryos (Fig. 2A and 2C) and in polar lobes isolated and pulsed at the trefoil stage (Fig. 2E). The actin mRNAs are translated by normal mesentoblast-stage embryos (Fig. 2B) and 24 h lobeless embryos (Fig. 2D), and by isolated polar lobes that have been aged for 24h in sea water at 20°C (Fig. 2G). After 24 h, normal eggs from the same lot used for polar lobe isolation had developed into mesentoblast embryos with an average of 29 cells. In the ‘aged’ isolated polar lobe, three polypeptides labeled a, b, and c (Fig. 2G; compare with Fig. 2A) were not markedly reduced in intensity as they were in normal and lobeless embryos (Fig. 2B and 2D).

Actin mRNAs are present in the unfertilized egg and are therefore maternal in origin, are inactive during early cleavage, and are translated in normal, lobeless, actinomycin D-treated blastulae and in isolated polar lobes that have been aged for 24 h. That the actin mRNAs are maternal in origin was shown by the hybridization of mRNAs extracted from mature unfertilized eggs with an actin gene probe. This observation confirms a similar conclusion suggested by the synthesis of actins by mesentoblast-stage embryos reared continuously in actinomycin D. The synthesis of actins by aged polar lobes demonstrates both the presence and stability (for at least 24 h) of actin mRNAs in the polar lobe. The production of actins in the polar lobe must be from the translation of maternal mRNAs because the polar lobe was isolated at the trefoil stage, and, therefore, had not been exposed to a transcriptionally active zygote nucleus.

It is likely, but not demonstrated by my data, that zygotic transcripts are being translated into actins by the blastula. That maternal actin mRNAs are translated in the blastula is also suggested by the synthesis of the actins by blastulae that have been continuously reared in actinomycin D. A dual origin from both the maternal and the zygotic genome for actin mRNAs has also been demonstrated in ascidian embryos by Tomlinson et al. (1987).

Because the anucleate polar lobe, when isolated from the egg, synthesizes actins after aging, it is clear that the activation of actin maternal mRNAs is under cytoplasmic control and that the controllers are products of the maternal genome. Similarly, any requirements for cleavage, DNA synthesis and the transcription of the nuclear genome as a controlling mechanism are also eliminated.

Swenson et al. (1987) have demonstrated that three maternal mRNAs are stored in the cytoplasm of Spisula oocytes. Because the llyanassa germinal vesicle breaks down before the formation of the polar lobe, I cannot rule out the possibility that the actin mRNAs were not originally localized in the egg germinal vesicle; however, it is clear that these mRNAs are not exclusively localized in either the animal or vegetal hemisphere of the llyanassa egg.

Rosenthal et al. (1983) and Rosenthal & Ruderman (1987) have found a correlation between translation and the extensive increases in the adenylation of maternal mRNAs in Spisula oocytes, however, no causal relationship was established. A decrease in the poly (A) titre between early cleavage and the mesentoblast-stage of llyanassa embryos was observed by Collier (1975) and Clark & Kidder (1977). Because these assays for poly(A) were not sensitive enough to detect polyadenylation of individual mRNAs, it is uncertain whether polyadenylation is associated with translational regulation of actin mRNAs at this stage of llyanassa development. The possibilities that adenylation and capping are responsible for the translational regulation of the actins, as well as the possibility that the actin mRNAs are bound to a ‘masking factor’ (Spirin, 1966; Young & Raff, 1979; Rosenthal et al. 1980; Grainger & Winkler, 1987) remain to be investigated in the llyanassa embryo. There is no information on terminal capping of mRNAs or methylation of mRNAs caps in llyanassa, though there is a correlation between cap methylation and translational activation of mRNAs in sea urchin eggs (Caldwell & Emerson, 1985).

The cleavage-stage proteins a, b, and c marked in Fig. 2A, which either disappear or are greatly diminished in their intensity after 24 h of development, are also diminished in 24 h embryos reared continuously in actinomycin D; thus, the factor(s) responsible for the diminution of these proteins is probably not dependent on transcription of the zygotic genome.

It is of special interest that these three cleavage-stage proteins are diminished in 24 h lobeless embryos (Fig. 2D) but not in the 24 h ‘aged’ isolated polar lobes (Fig. 2G). It would thus appear that the translation of the mRNAs for these proteins is controlled by nuclear factors other than RNA synthesis, which would be blocked by actinomycin D, and/or by cytoplasmic factors localized in lobeless blastomeres but absent from the vegetal cytoplasm contained in the third polar lobe. Because the translation of these three proteins occurs undiminished in ‘aged’ polar lobes, it is unlikely that their diminution in the normal and lobeless embryo is caused by mRNA degradation. Deadenylation of the mRNAs for these proteins is a possible controlling mechanism.

Although I have not established the precise time when the ‘aged’ polar lobes begin to make actin, there appears to be a timing factor involved because freshly isolated polar lobes do not translate actin mRNAs while ‘aged’ ones do. Thus, I suggest that there is a biological clock running to determine when stored maternal messenger RNAs are to be activated, that this clock resides in the cytoplasm and that it was made and set during oogenesis.

Morgan’s (1935) experiments with llyanassa eggs in which he observed rhythmic changes in the form of isolated polar lobes that were in time with the cleavage cycle suggest an autonomy in the polar lobe similar to its autonomous regulation of translation. Similarly, Conklin’s (1938) observations on the cleavage of Crepidula eggs suggests the continued running of a cytoplasmic clock after nuclear events were suppressed by low temperatures. Gather et al. (1986) have shown by cleavage inhibition experiments with llyanassa eggs that there is a ‘clock’ mechanism specifying cleavage time in this egg. These authors have reviewed the evidence that this ‘clock’ resides in the cytoplasm. In amphibian eggs, cyclic contractions correlated with the division cycle in anucleate eggs also suggest that there are biological clocks localized in the cytoplasm (Sawai, 1975; Sakai & Kubota, 1981).

While the presence of maternal mRNAs in oocytes has been known for a long time, this paper demonstrates that a mechanism for activating maternal mRNAs is also maternally encoded and stored in the egg cytoplasm. This mechanism of mRNA activation performs like a clock that was made, stored in the cytoplasm and set running during oogenesis. What sets this clock running and how it tells time is unknown; however, the observations made in this paper point to a way for exploring this problem.

This work was supported by NSF Grant PCM78-02773 and grants 665132 and 668144 from the City University of New York PSC-CUNY Award Program.

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