Sea urchin thymidylate synthetase

Experimental Cell Research 101 (1976) 59-62

SEA URCHIN

THYMIDYLATE

Changes of Activity

during Embryonic

E. PARIS1 and BENITA Laboratorio

di Embriologia

SYNTHETASE

Molecolare,

Development

De PETROCELLIS 80072 Arco Felice, Napoli,

Italy

SUMMARY Thymidylate synthetase activity has been determined during Sphaerechinus granularis development. After fertilization and during cleavage the enzyme activity does not change; at the blastula stage the enzyme activity undergoes a transient marked decrease in concomitance with the drop of the mitotic activity; at the gastrula stage with the resumption of cell proliferation, the enzyme level increases again. These results suggest that the enzyme might be involved in the regulation of DNA replication during embryogenesis.

In the sea urchin most of the enzymes for cleotide reductase during development of DNA synthesis are already present in the the sea urchin embryos [5]. In this context also thymidylate syntheunfertilized egg, having been synthesized during oogenesis [l-5]. Nevertheless the tase occupies a very important position, egg reacquires the ability to synthesize since the dTMP production is known to be DNA only after fertilization or partheno- a rate-limiting factor for DNA synthesis, as genetic activation. During cleavage, the demonstrated by the rapid inhibition of rate of DNA synthesis increases exponen- DNA synthesis in animal cells brought tially until the blastula stage; at this time about by inhibitors of this enzyme [8]. the rate of DNA synthesis drops and reThe present investigation was undertaken mains at a low level for several hours. At to determine whether or not there is a corthe onset of gastrulation, cell division and relation between thymidylate synthetase acDNA synthesis are resumed [2]. For sev- tivity and DNA synthesis during sea urchin eral years we have been interested in the embryonic development. molecular mechanisms controlling DNA The observed changes in the activity of synthesis during embryonic development thymidylate synthetase suggest that this en[6,7]. A key factor in the regulation of DNA zyme may play a critical role in DNA synsynthesis is the supply of deoxyribonucleothesis and cell division during embryonic tides and hence the activity of the enzymes development. on which their synthesis depends. This has been clearly illustrated by the correlation MATERIALS AND METHODS between the deoxyribonucleotide pool, Sea urchins of the species Sphaerechinus granularis DNA synthesis, and activity of the ribonu- were obtained from the Zoological Station of Naples. Exp

Cell

RPS 101 (1976)

60

Parisi and De Petrocelli.\ taining toluene-liquiflor and ,oluene (9. I \ Iv 1.and a~sayed for tritium in a Beckman scintilla[lon counter. A correction was made for a control in which the enzyme was inactivated by perchloric acid soon after the addition of the substrate. As a further control another 0.2 ml aliquot was dried by lyophilization and counted in the same conditions. Determinations done in triplicate on the same homogenate showed a difference con-

tained within 15%. Protein concentration was determined according to the method of Lowry et al. [IO].

RESULTS Thymidylate synthetase activity present in cell-free extracts prepared from eggs and embryos increased linearly with the incuba5 ,o ‘15 Fig. 1. Abscissa: time (mitt); ordinate: nAtoms of tion time (fig. 1) and was proportional to the tritium released. amount of homogenate protein present in Time course of thymidylate synthetase activity. Assays were carried out using I mg of homogenate the reaction mixture (table 1). Omission of protein from unfertilized eggs. All values have been tetrahydrofolate or of the enzymatic extract corrected for a zero time control (0.027 nAtoms). resulted in a reduction of the tritium released to a background level represented by the amount remaining after lyophilization Deoxyuridine-5’-monophosphate[5-3H] was from SchwarzlMann; unlabelled deoxyuridine monophos- (table 1). The synchrony of the first cell diphate and dl-L-tetrahydrofolic acid (grade III) were vision among eggs from the same female from Sigma Chemical Co. Active charcoal was from Barnberg-Cheney Co., Columbus, Ohio. All other has enabled us to ask the question as to reagents were of analytical grade. whether or not the activity of the enzyme Deoxyuridine-5’-monophosphate[5-3H] was repuriundergoes any change during the cleavage fied by lyophilization, and redissolved in the original volume of distilled water. Stock solutions of dl-L- cycle. The activity was referred to protein tetrahydrofolate were prepared by dissolving 25 mg of concentration, since the protein content/ dl+tetrahydrofolic acid in 3 ml of I M t-mercaptoethanol containing 0.05 M NaHCO,. The solutions embryo is constant throughout developwere stored under vacuum for no longer than one ment [ll]. The results reported in table 2 week. The active charcoal was washed with 0.2 N HCI and then with water to neutral pH. Sea urchin embryos show that during the first cell cycle the were cultured as previously described [I]. For the ensynthetase activity did not zymatic assay the embryos were collected at the de- thymidylate sired stage by low speed centrifugation, washed twice with 0.63 M NaCl and disrupted by intermittent sonication at 0°C in about 5 vol of 0.025 M Tris-HCI, pH 7.6. Thymidylate synthetase activity was assayed according to Lomax & Greenberg [9] by measuring the amount of 3H released from deoxymidine-5’-monophosphate[5-3H] into water. The reaction mixture contained, in a volume of 0.2 ml, 100 FM [3H]dUMP (183 000 cpm/nmole); 0.042 M Tris-HCI pH 7.6, 0.026 M MgCIZ; 1.06 mM EDTA; 0.0158 M formaldehyde; 0.1 M 2-mercaptoethanol; 37 PM tetrahydrofolate; and 0.5-1.0 mg homogenate protein. The reaction was initiated by the addition of [sH]dUMP. The incubation was carried out under argon for 5 min, or longer, at 37°C. The reaction was stopped by addition of 0.5 ml of 5 % perchloric acid; after 10 min at 0°C the samples were centrifuged for 15 min at 15000 g. The supernatant was absorbed on a short (0.5X 1.0 cm) charcoal column and recycled 3 times. A 0.2 ml aliquot of the eluate was added to 10 ml of scintillation fluid conExp Cell Res 101 (1976)

Table 1. Tritium release from [3H]diJMP Tritium released (nAtoms/lOmin) Reaction mixture

A

B

Complete+25 ~1 homogenate Complete+50 ~1 homogenate -Tetrahydrofolate+ZS ~1 homogenate -Homogenate

0.110 0.230

0.028 0.027

0.038 0.020

0.033 -

Assays were carried out as under Methods, using a homogenate prepared from embryos at early blastula stage. (A) total tritium released: (B) amount of tritium not removed by lyophilization.

dTMP synthetase in sea urchin embryo Table 2. Thymidylate synthetase activity in sea urchin embryos during the first division cycle nAtoms of tritium released in 5 minlmg protein

Stage Unfertilized 10 min after 40 min after 80 min after 130min after (2 cells)

fertilization fertilization fertilization fertilization

0.282 0.284 0.277 0.303 0.264

dropped to a level which was from 8 to 20 times lower than that of the unfertilized egg. After this period, at the gastrula stage, the enzyme activity raised again to a level comparable to that found during cleavage. The data of table 3 also show that the extracts from eggs and embryos at the mesenchyme blastula stage gave, when mixed together, an activity that was the average of the snecific activities of the single extracts (0.132 against the expected valie of 0.138).

Assays were carried out in triplicate, as described under Methods, using eggs collected from a single sea urchin. All values have been corrected for a zero time control.

change significantly. The level of enzyme activity present in extracts from embryos at different stages of development is reported in table 3. The thymidylate synthetase activity did not change during early cleavage up to the hatching blastula stage. From this stage on, during the period coinciding with the formation of the primary mesenchyme and the decline in mitotic activity, the enzyme activity underwent a transient but marked decrease; indeed it

61

DISCUSSION The results reported in this paper represent a further step in our studies on the enzymes of DNA biosynthesis in sea urchin embryos. Sea urchin eggs contain a high level of thymidylate synthetase activity comparable to that of other systems characterized by a high rate of cell proliferation, such as regenerating rat liver [12] and fast growing tumors [13, 141. This is not surprising in view of the high demand of DNA synthesis in developing embryos. The level of the en-

Table 3. Thymidylate synthetase activity in sea urchin embryos at different stages of development nAtoms of tritium released in 5 min/mg protein Time after fertilization 0 10 min 15 h 174h 19 h 23 h 27 h 40 h 43 h

Stage

Expt 1

Expt 2

Unfertilized Fertilized Hatching blastula Swimming blastula Early mesench. blastula Mesenchyme blastula Mesenchyme blastula Early gastrula Gastrula Unfertilized+ early mesench. blastula

0.245 0.256 0.208

0.309 0.252

0.032 0.062 0.266

0.013 0.010 0.011 0.019 0.193

0.132 (0.138 expected value)

Enzyme activity was determined as described under Methods. Assays were carried out in triplicate. In the experiment with mixed homogenates, the extracts from eggs and early mesenchyme blastula (19 h) were combined in equal amounts at the moment of the assay. Exp Ce//Res I01 (1976)

zyme activity does neither increase in concomitance with the onset of DNA synthesis nor during early cleavage. Most likely the enzyme present in the egg is able to support the formation of dTMP in an amount sufficient for the replication of DNA during the period of active cell division. This situation is not uncommon, since some enzymes involved in DNA synthesis, either at the level of the deoxynucleotide pool [ 1, 51 or at the level of polymer [2-41, are stored in the egg during oogenesis. The only exception is the case of ribonucleotide reductase, whose activity increases several-fold after fertilization [.5, 151. How the enzymes stockpiled in the egg are brought into action after fertilization is still unknown. It is possible that, like DNA polymerase [4] and glucose 6-phosphate dehydrogenase [ 161, other enzymes become active as a result of decompartmentalization. An alternative explanation for the constant level of the enzyme activity observed during cleavage is that during this period the rate of enzyme synthesis equals the rate of degradation. The sudden drop in thymidylate synthetase activity, observed after the blastula stage, is noteworthy since it occurs at the very moment when DNA synthesis and cell division also decrease to very low levels [2]. The enzyme activity increases again at the gastrula stage in concomitance with the resumption of the mitotic activity. Since the experiments with mixed homogenates indicate the absence of specific inhibitors, the changes of thymidylate synthetase activity may be due to rapid degradation of the protein followed by de novo synthesis of the enzyme. The pattern of thymidylate synthetase strikingly resembles that of another enzyme, a DNA endonuclease, whose activity also fluctuates in the course of embryonic development [2]. For DNase we E,p Cd Rr.\ 101 (IY7rS)

have shown the existence of a rather complicated system of regulation acting at a post-transcriptional level [ 171. The existence of a similar mechanism for thymidylate synthetase remains to be proved. In conclusion the results of the present paper suggest that thymidylate synthetase may be an important factor in the regulation of dTTP pool during sea urchin embryonic development . We thank Mr A. Capasso for his excellent assistance.

technical

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,,ebeived December 22. 1975 Accepted March 3, 1976