The onset of collagen synthesis in sea urchin embryos

Biochimica et Biophysica Acta, 349 (1974) 135--141 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA Report BBA 91387

THE ONSET OF COLLAGEN SYNTHESIS IN SEA URCHIN EMBRYOS

R. G O L O B , C.J. C H E T S A N G A *

and P. D O T Y

Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Mass. 02138 (U.S.A.) ( R e c e i v e d February 27th, 1974.)

Summary In Arbacia punctulata and Strongylocentrotus purpuratus, two species of sea urchins, collagen synthesis begins during gastrulation and increases manyfold before reaching a plateau in the late pluteus stage. A collagen extraction method involving treatment with 0.1 M NaOH and hot 10% trichloroacetic acid provided the basis for a sensitive assay of collagen synthesis.

During early embryogenesis, differentiation begins when a group of cells acquires the capacity to produce specific proteins. In order to study this developmental period at the biochemical level, assays for specific characteristic proteins are essential. Collagen is representative of a specific protein which can be identified easily due to the existence of a built-in marker, hydroxyproline. With the minor exception of elastin, collagen is the only protein containing hydroxyproline [1]. Therefore, when cells are incubated with radioactive proline, the presence of radioactive hydroxyproline can be assumed to indicate the presence of collagen, and, at any developmental stage, the ratio of radioactivity of hydroxyproline in collagen to that of proline in total protein can be used to measure semiquantitatively the rate of collagen synthesis relative to that of total protein synthesis [2--4]. On this basis we have examined the course of early collagen synthesis in the embryonic development of two sea urchin species, Arbacia punctulata and Strongylocentrotus purpuratus. A. punctulata embryos were cultured in Millipore-filtered sea water at 22 °C. At selected developmental stages, 1.5 ml of packed embryos were incubated for 3 h in 10 ml of sea water and 250 ~Ci of [2,3-3H]proline (New * Present address: Department,of Natural Science, The University of Michigan, Dearborn, Mich. 48128 (U.S.A.).

136

TABLE I C O L L A G E N S Y N T H E S I S IN P U L S E L A B E L E D A R B A C I A T i m e (h)

Pro 10 15 23 28 39 51 63

blastula gastrula pluteus pluteus pluteus pluteus pluteus

PUNCTULATA

EMBRYOS

CPM

Stage

--1 h +4h +15 h +27 h +39 h

1 1 1 1 2

146 183 265 366 448 948 204

Hypro 900 200 000 000 000 000 000

1 3 5 9 10

103 650 644 415 793 765 140

H y p r o / P r o X 100 0.07 0.08 0.13 0.25 0.40 0.45 0.46

England Nuclear: 40 Ci/mmole). Following pulse labeling, the embryos were washed once in sea water, twice in a solution of 1.0 M dextrose and 0.01 M Tris--HC1, pH 8, and once more in sea water. After each washing, the embryos were collected by centrifugation for 1.5 minutes at 1500 X g. The washed embryos were homogenized in 2 ml of distilled water, and the homogenates were dialyzed exhaustively against distilled water at 2 °C, concentrated by lyophilization, and then hydrolyzed in 0.1 ml of 6 M HC1 for 16 h at 110 °C in vacuo. The hydrolyzates, along with pro and hypro standards, were chromatographed on Whatman No. 3 paper, using a butanol--acetic acid--water (63:27:10, by vol.) solvent at 18 °C for 20 h. The strips containing the amino acid standards were developed with 0.4% ninhydrin in ethanol, while the strips containing the hydrolyzates were cut into 1 cm long pieces and then counted on a Beckman Model LS250 Scintillation Counter, with Aquasol (New England Nuclear) as the scintillation fluid. On all of the strips, the pro spot migrated 5 cm farther than the hypro spot but never more than 30 cm from the origin. The cpm of the hypro and pro spots and the ratio of hypro/pro X 100 at each selected post-fertilization time are shown in Table I. The ratios of hypro/pro X 100 have not been corrected for the loss of tritium during the h y d r o x y l a t i o n of [2,3-3H]proline. In Fig. 1A the cpm of the hypro spots and the ratios of hypro/pro X 100 are plotted versus time. These results show that collagen synthesis begins during gastrulation, increases rapidly between 20 and 40 h, and then levels off at about 50 h. The synthesis follows a sigmoidal curve, with a m a x i m u m rate increase at 30 h. The incorporation of [3H]hypro increases almost linearly as a function of time. Since proline is incorporated into both collagen and non-collagen proteins, changes in the hypro/pro ratio can reflect fluctuations either in the rate of synthesis of collagen or non-collagen proteins or both. However, hypro is only incorporated into collagen, and therefore the increases in hypro incorporation shown in Table I and Fig. 1A provide direct evidence that the rate of collagen synthesis is increasing. Thus the observed increases in hypro cpm suggest t h a t the increases in the hypro/pro ratio were caused primarily by

137

"

I0000

8_

:~:

~00o

2.0 0 0 x o 1.5 ,V

x

b l o $ 1 ~

,

,

,

:z:

~: 1.0 0.,5

I0

ZO

3O

40

Time in HOurS

5O

6O

I0

20

30

40

50

60

Time in Hours

Fig. i . (A) The differential rate of collagen synthesis in pulse labeled Arbacia punctulata e m b r y o s , e x t r a c t e d f o r t o t a l p r o t e i n . D e t a i l s in the t e x t . o - - o , h y p r o / p r o X 1 0 0 ; e - - e , h y p r o cpm. (B) The d i f f e r e n t i a l r a t e o f collagen s y n t h e s i s in pulse labeled Arbacia punetulata e m b r y o s , e x t r a c t e d to enrich f o r c o l l a g e n . Details in the t e x t . o - - - o , h y p r o / p r o X 1 0 0 ; • ~, h y p r o c p m .

increases in the rate of collagen synthesis, rather than, for example, decreases in the rate of non-collagen protein synthesis. These increases in collagen synthesis have been shown to occur concurrently with the formation of the spicule matrix, and, according to PucciMinafra et al. [4] most of the synthesized collagen is concentrated in the spicule matrix. These results suggest that collagen synthesis plays an important role in spicule differentiation. The maximum ratio of hypro/pro X 100 is 0.46%, representing a 7-fold increase in collagen synthesis from the earliest observations to the maximum. When compared with the maximum ratio observed in Xenopus laevis of 2.6% (not corrected for loss of tritium during proline hydroxylation) [2], the 0.46% implies that invertebrates may produce smaller relative quantities of collagen than do vertebrates. In order to detect low hypro levels relative to high pro levels and, thereby, to obtain a more sensitive measurement of collagen synthesis in developing A. punctulata embryos, a method was developed to extract collagen from the total protein pool. The extraction consisted of dissolving the sample in 0.1 M NaOH for 2.5 h at 50 °C with occasional vortexing, adding an equal volume of hot 20% trichloroacetic acid, and heating the hot 10% trichloroacetic acid suspension for 3.5 h at 85 °C with occasional mixing. The suspension was then filtered through Whatman FG/C filters, and the filtrate was dialyzed, hydrolyzed, and chromatographed as described above. When 250-pg samples of calf skin collagen and bovine serum albumin were subjected to this extraction method, 85% of the calf skin collagen but only 12% of the bovine serum albumin, as measured by the Folin procedure, were recovered in the filtrate [ 5]. These percentages indicate that this method provided very substantial enrichment of collagenous proteins by selective solubilization.

138

To determine whether the dilute alkali and hot trichloroacetic acid were hydrolyzing the collagen into dialyzable polypeptides, some labeled e m b r y o s were lysed in cold 10% trichloroacetic acid, and the homogenate was dialyzed exhaustively against distilled water at 2 °C before treatment with 0.1 M NaOH and hot 10% trichloroacetic acid. An equal amount of labeled embryos was lysed in 2 ml of distilled water and then subjected to the normal extraction method. There was no detectable difference in radioactivity between the filtrate of the pre-extraction dialyzed sample and the filtrate of the postextraction dialyzed sample: this demonstrated negligable hydrolysis into dialyzable peptides. This extraction method was then used in a pulse label experiment, closely paralleling the first experiment in both purpose and execution. The procedures {incubation, washing, lysing, extraction, dialysis, chromatography) followed in this experiment were as described above; at each selected developmental stage, the embryos were incubated, however, with only 50--100 pCi of [2,3-3H]proline in 10 ml of sea water. The results compiled in Table II show that the extraction method resulted in a significant enrichment of collagen relative to total protein. When the maximum ratio of hypro/pro × 100 for total protein is compared with that for collagen-eniichment protein, a 5-fold increase in the collagen concentration is observed. In addition, Table II shows that [3H]proline is incorporated in significant amounts throughout early embryogenesis, whereas [3 H ] h y d r o x y p r o l i n e is present at significant levels only after blastula.

T A B L E II C O L L A G E N S Y N T H E S I S IN P U L S E L A B E L E D A R B A C I A

T i m e (h)

Stage

10 15 24 26 28 35 51 63

blastula gastrula pluteus pluteus ÷2 p l u t e u s +4 p l u t e u s +11 p l u t e u s +27 pluteus +39

PUNCTULATA

EMBRYOS*

CPM Pro

h h h h h

11 251 381 386 350 304 458 591

Hypro 270 500 500 400 800 200 900 700

1 1 2 4 8 13

5 679 541 854 315 228 994 020

Hypro/Pro X 100 0.04 0.27 0.40 0.48 0.66 1.39 1.96 2.20

*Enriched for c o l l a g e n as described in the text.

Collagen synthesis, as shown in Fig. 1B, follows a sigmoidal curve, identical to that in Fig. 1A. Once again synthesis begins during the gastrula stage and increases many-fold before reaching a plateau at a b o u t 50 h, in late pluteus. The similarity in curve shapes in Fig. 1A and Fig. 1B {both in the h y p r o / p r o × 100 versus time graphs and in the hypro cpm versus time graphs) suggests that the extraction method enriched for collagen w i t h o u t distorting the kinetics of collagen synthesis as measured in Expt 1.

139 From blastula to pluteus + 40 h, the increase in the differential rate of collagen synthesis is seen to be about 50-fold. If we multiply the corresponding increase from Expt 1 by the collagen enrichment factor, the 6-fold increase translates into a 30-fold increase, which agrees, within experimental error, with the observed 50-fold increase. Although the pulse label experiments demonstrate that the differential rate of collagen synthesis increases many-fold from gastrulation until late pluteus, they do not provide any data about collagen accumulations during the same period. A continuous label experiment was performed to measure collagen accumulations from blastula until pluteus + 40 h. A. punctulata embryos were incubated with [2,3-3H]proline from just after fertilization until the selected developmental stages and then harvested and worked up as in Expt 2. T A B L E III C O L L A G E N S Y N T H E S I S IN C O N T I N U O U S L Y T i m e (h)

Stage

10 24 27 39 51 63

blastula pluteus pluteus pluteus pinteus pluteus

L A B E L E D ARBACIA PUNCTULATA*

CPM Pro

+3 +15 +27 +39

h h h h

2 783 657 594 815 622

Hypro 850 800 800 100 500 400

5 6 7 14 11

3 402 054 327 400 900

Hypro/Pro × 100 0.01 0.69 0.92: 1.23: 1,77 ~ 1.91 ~

* E n r i c h e d f o r c o l l a g e n as d e s c r i b e d Ln t h e t e x t .

The ratios of hypro/pro × 100 in Table III were used as measurements of the differential amount of collagen that had accumulated in the embryos from fertilization until the selected stage. Once again, the increases in hypro cpm suggest that the changes in the hypro/pro ratio were caused by changes in collagen, not non-collagen, accumulations. As plotted in Fig. 2, the increase in collagen accumulations coincides with the increase in synthesis rate, as plotted in Fig. 1B, and these increases coincide with the increase in spicule formation. The simultaneity of these

2O

3O

4O

~

60

T~le m I.~rs

Fig. 2. The differential rate o f eollagen s y n t h e s i s in c o n t i n u o u s l y l a b e l e d Arbacia punctulata e m b r y o s . The e m b r y o s were i n c u b a t e d w i t h [ 2 , 3 o S H ] p r o l i n e f r o m J u s t after fertilization until the selected d e v e l o p m e n t a l stages and t h e n harvested a n d w o r k e d up as d e s c r i b e d in t h e t e x t . The h y p r o / p r o X 1 0 0 ratios have n o t b e e n c o r r e c t e d for t h e loss o f t r i t i u m d u r i n g proline h y d r o x y l a t i o n .

140

increases links the mediation of collagen synthesis, once again, to the differentiation of the spicule matrix and is consistent with the expectation of very little turnover or excretion of collagen during embryonic development. In order to examine the generality of these results collagen synthesis was then studied in S. purpuratus, a sea urchin species that develops more slowly than A. punctulata [6, 7]. The S. purpuratus embryos were treated according to the procedures of Expt 2. The results graphed in Fig. 3 again place the beginning of collagen synthesis in the gastrula stage. Although the exact time of the t a k e o f f of collagen synthesis is not well defined because no measurements were made between 20 and 40 h, the general course is seen to be the same except on a time scale less than half as fast. 6 5

0 0 D x £

O

4

pluteus ~ gostrulo ~

3

Q_ o 2 I

blastulo

~

lo/

0

/ 0 l

,

,

,

60

80

I00

,

,

,

,

I

L~ 0

20

40

120

140 160

180

Time in Hours Fig. 3. T h e d i f f e r e n t i a l r a t e of c o l l a g e n s y n t h e s i s in p u l s e labeled Strongylocentrotus purpuratus e m b r y o s . Details in t h e t e x t . T h e h y p r o / p r o X 1 0 0 r a t i o s h a v e n o t b e e n c o r r e c t e d for the loss o f t r i t i u m dtLring p r o l i n e h y d r o x y l a t i o n .

Whereas other studies have investigated collagen synthesis in different parts of the sea urchin [8--10], our studies have focused on the onset of collagen synthesis in the embryo. Based on our measurements of collagen synthesis in A. punctulata and S. purpuratus, and on those reported for P. lividus [4] it seems probable that, among sea urchins, collagen synthesis usually begins during gastrulation and undergoes a many-fold increase before reaching its m a x i m u m level in late pluteus. We thank Dr. Helga Boedtker for her valuable comments during both the planning of these experiments and the preparation of this manuscript. In addition, we t h a n k Rick Schwarz for developing the collagen extraction method. This work was supported by National Institutes of Health Grant HD-01229.

141

References I 2 3 4 5 6 7 8 9 10

Matthews, M. (1967) Biol. Rev. Cambridge Phil. Soc. 42, 499 Green, H., Goldberg, B., Schwartz, B. and Brown, D.D. (1968) Develop. Biol. 18, 391 Klose, J. and Fllckinger, R.A. (1971) Biochim. Biophys. Acta 232, 207 Pucci-Minafra, I., Casano, C. and La Rosa, C. (1972) Cell Differ. VoL 1, No. 3, 157 LowTy, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265 Harvey, E.B. (1956) In the American Arbacia and Other Sea Urchins, Princeton University Press, Princeton, N.J. Roeder, R.G. and Rutter, W.J. (1970) Biochemistry 9, 2543 Watson, M.R. and Sllverster, X.X. (1959) Biochem. J. 71, 578 Gadeyne, C. and Francois, C. C1970) Arch. Internat. Physiol. Biochim. 78, 427 Klein, L. and Currey, J.D. (1970) Science 169, 1209