Printed in Sweden Copyright @ 1978 by Academic Press, Inc.

All rights of reproduction in any form reserved 0014~827/78/1151-0089$02.00/0

Experimental Cell Research II5 (1978) 89-94

ACTIN AND TUBULIN SYNTHESIS IN MURINE

BLASTOCYST OUTGROWTHS

SERGIO L. ABREW* and RALPH L. BRINSTER

University of Pennsylvania, School of Veterinary Medicine, Laboratory of Reproductive Physiology, Philadelphia, PA 19103, USA

SUMMARY

The actin and tubulin contents of blastocysts grown in vitro for 72 h were estimated by densi- tometric analysis of Coomasie-blue stained SDS-gels. Actin represents 7% and tubulin 13% of total blastocyst outgrowth protein. The relative synthesis of these two proteins was measured by two-dimensional electrophoresis utilizing unlabeled and isotopically labeled actin and tubulin internal markers. Actin synthesis constituted 6.3 % and tubulin synthesis 1.5 % of the total protein synthesis. These values are not significantly different from those we have reported previously for mouse preimplantation blastocysts recovered from uteri. It appears then that the relative propor- tion of synthesis does not change significantly during the developmental period that encompasses the blastocyst stage and early implantation as represented by the in vitro hatching, attachment and outgrowth of the blastocyst. Data on the characteristics of growth and culture of the outgrowths is also presented.

We have recently studied the synthesis of tubulin and actin during the preimplantation development of the mouse [I]. The rates of protein synthesis in the mouse preimplanta- tion embryo remains low until the S-cell stage at which point a rapid increase is evident.

The synthesis of actin was found to re- main at a “low” level, less than 0.3 % of to- tal synthesis in unfertilized, fertilized and 2- cell embryos [I]. However, in the develop- mental interval between the 2-cell and the S-cell cleavage stages it increases rapidly. It constitutes about 6% of total protein syn- thesis in the blastocyst and the synthetic rate is 90-fold higher than in the unfertilized and fertilized ovum. This increase in actin synthesis contrasts with a 7-fold increase in total protein synthesis during the same developmental period. The rate of tubulin synthesis more closely parallels the in- crements in total protein synthetic rates and

by the blastocyst stage it increases 14-fold, representing about 2 % of total protein syn- thesis [ 11.

It is of interest to determine whether or not these high synthetic rates of actin and tubulin, observed at the end of preimplanta- tion development, are maintained during the subsequent developmental events of blastocyst hatching, attachment and im- plantation. The biochemical study of im- planting blastocysts is complicated by the difficulty of dissecting the embryos free from any maternal tissue. Biastocysts, how- ever, can attach to the surface of culture dishes, and the trophoblast cells outgrow as a monolayer [2]. These in vitro activities are considered to reflect the processes which lead to in utero implantation [3-51.

1 Present address: Sloan-Kettering Institute for Can- cer Research, Section 6144, 1275 York Avenue, New York, NY 10021, USA. 2 To whom reprint requests should be addressed.

Exp CellRev 115(1978)

90 Abreu and Brinster

Fig. 2. Blastocysts after 72 h in culture, 152 pf. Blas- phase contrast microscopy of isolated outgrowth. I’, tocysts were cultured in MEM containing 10% FCS. Trophoblast; I, inner cell mass. a, X100; b, X200; (a) Group of outgrowths; (b) isolated outgrowth; (c) c, x380.

In this communication we report on the synthesis of actin and tubulin by “synchro- nized” blastocysts cultured for three days in vitro.

MATERIALS AND METHODS

Blastocyst collection, culture and “synchronization” Swiss-albino mice (6-8 weeks old) were maintained in a 10 h dark cycle and fertilization of superovulated

Table 1. Characteristics of blastocyst out- growths

Diameter/outgrowth Estimated cell no./outgrowth

387&43 /.&La 1 105*133*

Protein/outgrowth Tubulin (% of total protein)

245f21 ngc 14.8d 11.3

Actin (% of total protein) 8.2d 6.5

a-e Values expressed* S.E.M. were obtained from three sets of determinations. The diameter of out- growths was measured with an ocular micrometer in a Wild dissecting microscope. Cell number of three ex- perimental sets of outgrowths was determined, after trypsinization, in a Neubauer hemocytometer. Pro- tein content was determined by the method of Lowry et al. [21]. d These two sets of values were obtained from den- sitometric evaluation of total blastocyst outgrowth protein, e.g. fig. 2.

Exp Cell Res 115 (1978)

mice was assumed to have taken place midway (2 a.m.) in this cycle. Blastocvsts were flushed and col- lected 80 h post-fertilization (pf) as described [6].

Groups of lm200 blastocysts were cultured in 35 mm tissue culture dishes (Falcon Plastics) containing 3.5 ml of Dulbecco’s Modified Eagle’s Medium (DME) made 10 % in fetal calf serum (FCS) (G&co).

“Synchronization” of blastocvsts (obtaining blasto- cysts-at approximately the same developmen& point) was attempted as follows. Blastocysts of similar ap- pearance, e.g. same diameter and blastocoelic cavity size, were initially chosen from a common pool of newly collected embryos. These blastocysts were placed in the culture conditions described above and monitored for attachment (tested by gently swirling the culture dish) to the plastic substratum. When 50% of the blastocysts had attached (about 120 h post- fertil. or 40 h in culture), those not attached were re- moved. It was estimated that the difference in attach- ment of this 50% did not exceed 4 h. That is, all blastocysts selected had attached within 120-+_4 h. Cul- ture of these embryos continued for a total of 72 h or until 152 h post-fertilization (pf).

Amino acid incorporation Blastocyst outgrowths, 152 h pf, were washed three times with Basal Eagle’s Medium (BME), Earle’s salts, and then pulsed for 2 h with 1 ml of BME containing 10% dialysed FCS; 200 PCi of [sFIjleucine (1 Ci/ mmole, Amersham Searle) and 2x10-’ M of each of the other 19 unlabeled amino acids. At this time the labeled medium was removed and the outgrowth were chased for 15 min with 3.5 ml of BME-10% FCS con- taining all 20 amino acids, unlabeled, at a concentra- tion of 2~ lo-’ M each. After the chase. the outarowths were washed twice with 4 ml of the. same &edium. This was removed and the cells were lysed by the ad- dition of 1 ml of a 0.5% sodium dodecyl sulphate

Blastocyst outgrowth synthesis of actin and tub&in 91

Fig. 2. Densitomettic scan of stained, 8%, SDS-gels. (Top) 75 pg of total outgrowth protein; @ortom) 10 pg of purified tubulin and 5 pg of purified actin. The Laemmli system [24] utilized here, does not always separate the tubulin subunits. Consistent separation is obtained with SDS-urea systems. cr, /3, tubulin sub- units.

(SDS), 1.0% @mercaptoethanol solution. The lysate was precipitated overnight with 10 vol of acetone at 4°C.

Assays of protein synthesis Total protein synthesis assays, two-dimensional elec- trophoresis, preparation of marker proteins and double label-analyses were performed as previously described RI.

RESULTS

Blastocyst culture Culture of blastocysts for 72 h in DME- 10% FCS resulted in their efficient hatch- ing, attachment and outgrowth. Compar- able results with this medium have been re- ported [7]. We obtained virtually 100% hatching of the culture blastocysts and 95 % attachment and growth. In all instances af- ter 72 h of culture an inner cell mass was apparent on the outgrowth of trophoblast (fig. 1). The time scale of hatching and at- tachment was very similar to that reported by Sherman & Salomon [8], with 50% of our blastocysts attaching by 40 h of incuba- tion.

“Synchronization” of blastocyst growth Because we intended to study the synthesis of actin and tubulin at one point during blas- tocyst outgrowth, it appeared to us neces- sary to obtain a population of outgrowths which did not differ significantly in develop- ment. If the entire pool of collected blasto- cysts was used (instead of a selected homo- geneous population), a great deal of vari- ance in outgrowth diameter resulted at the end of 72 h of incubation (data not shown). This was due to the fact that blastocyst at- tachment was highly asynchronous.

As shown in table 1 our “synchroniza-

Table 2. Total protein synthesis and synthesis of actin and tubulin

[3H]Leu incorporated % of total protein mlhl

(pm/h/outgrowth) synthesis outgrowth

Total protein synthesis 1.9+0.21” Tubulin synthesis 0.028+0.003’ i.5 2i8b Actin synthesis 0.120+0.017= 6.3 730=

(2 These values + S.E.M. represent the results of three determinations. Total protein synthesis was determined by TCA precipitation of labeled protein; and actin and tubulin synthesis by two-dimensional electrophoretic analyses as described [l]. * To obtain the picograms of tubulin synthesized per hour per outgrowth a subunit mol. wt of 55 000 D and an average leucine content of 7.4%; (z, 7.2%; p, 7.5%, was assumed [22]. c Actin was assumed to have a mol. wt of 42 000 D [23] and a leucine content of 6.95 % [23].

Exp Cell Res 115 (1978)

92 Abreu and Brinster

Fin. 3. Identification and cmantitation of actin and tubulin by two-dimensional polyacrylamide electro- uhoresis. Fluoronram 1251 of outgrowths labeled with PHjleucine, no internal tubulin and actin markers were added to this gel. Mengovirus proteins, extreme left,

tion” attempt (see Materials and Methods) was successful inasmuch as parameters such as outgrowth diameter, cell number and protein content for each mass were fairly constant.

Actin and tub&n content Densitometric analyses of Coomassie-blue stained SDS-gels (table 1, fig. 2) indicate that the tubulin peak area represents about 13 % of the total scan area and actin about 7% in these outgrowths. There is no data available on tubulin and actin content in the preimplantation embryo, but antibodies to these proteins are now available [e.g. 9, lo] which should make it possible to estimate their amount in preimplantation stages by immunoassays.

High tubulin and actin contents have

Exp Cell Res 115 (1978)

were used as mol. wt (X 10e3) markers in the second dimension. o, p, Tubulin subunits; A, actin. X-ray film was exposed for a small period of time, 5 days, since we are here primarily interested in demonstrat- ing the tubulin and actin pattern.

been reported for a number of non-muscle cells and developing systems. For instance, actin accounts for 8.5% of total cellular protein in cultures of chick skin fibroblasts [ill, for 8% in 3T3 cells [12] and for 9% of the soluble protein fraction in developing chick embryo brain [ 131. Tubulin is perhaps one of the most abundant eukaryotic pro- teins. Its contents in cells and developing systems range from 0.4 to 30% of total pro- tein [26], with most reported amounts com- prising at least several per cent of the total protein.

Actin and tubulin synthesis The synthesis of these two proteins was determined by two-dimensional gel electro- phoresis [14] as previously described [l]. Actin was found to represent 6.3% and

Blastocyst outgrowth synthesis of actin and tub&n 93

7r

6-

s-

4-

3; r

i.-Gzcl U F 2 8 B 0

Fig. 4. Abscissa: developmental stage (U, unfertilized; F, fertilized ovum; 2,2-cell embryo; &&cell embryo; B, blastocyst; 0, blastocyst outgrowth at 72 h); ordi- nate; % of total [SH]leucine incorporated into O-O, actin; A-A, tubulin.

The values for 73. F. 2.8 and B were taken from ref. [l]. No significant difference was found between the amount of synthesis of tubulin and actin in the blasto- cyst and the synthesis of these two proteins in the out- growths.

tubulin 1.5 % of total protein synthesis in the blastocysts outgrowths studied here (table 2, figs 3, 4). The relative amount of synthesis of these proteins in other systems is of interest. For instance, it ranges from 6 to 22% for tubulin and from 3 to 17% for actin in developing rat brain [ 151. In growing 3T3 cells actin is 7.4% of the total synthesis and tubulin 7.2 %; interestingly, in growing SV40-transformed 3T3 cells, tubulin synthesis is reduced to 3.9% of the total [ 121. Rabbit uterine tubulin levels have been shown to increase during preimplanta- tion and following implantation, presum- ably in preparation for embryo-uterine in- teractions [ 163. The relative amount of syn- thesis which we report here does not differ significantly from those found at the blasto- cyst stage: 5.7 % of total synthesis for actin and 1.9 % for tubulin [ 11. It would seem then that the rates of actin and tubulin syn- thesis peaking at the blastocyst stage are maintained at least during early implanta- tion (fig. 4).

DISCUSSION

We have previously discussed the signifi- cance of the increase in actin and tubulin synthesis which peaks at the blastocyst stage during preimplantation development [I]. The morphogenetic considerations which we related to this increase in synthe- sis are equally applicable here. Microfila- ment and microtubule changes may play a role in the morphological and biochemical differentiation of cells [17-191. Granholm & Brenner [20] have reported that the contrac- tion and expansion of outgrowths, accom- plished by loss and regeneration of hyalo- plasmic fans, is inhibited by cytochalasin B (CB), an anti-microfilament agent. Similar- ly, CB inhibited the membrane ruffling ac- tivity associated with outward expansion of the trophoblast in outgrowths [20]. The syn- thesis of these two proteins could also be necessary for the reorganization and seg- regation of the inner cell mass. Actin and tubulin in general may influence the proper- ties of the cell surface [27]. Thus, the high rates of synthesis of these two proteins ob- served towards the end of preimplantation development and maintained at least during early implantation may reflect the morpho- genetic activity of the outgrowth.

Blastocysts and blastocyst outgrowths consist of two main cell populations: troph- oblast and inner cell mass cells. Their func- tional and morphological roles at these times and later in development are fairly well delineated in the literature [e.g. 281. The trophoblast acts during preimplanta- tion as a pump in the transfer of fluid into the blastocoelic cavity and at implantation it is actively involved in the invasion by the embryo of the uterine tissues. The inner cell mass on the other hand shows little spe- cialization until after implantation when it will give rise to the embryonic tissues. It

Exp Cell Res II5 (1978)

94 Abreu and Brinster

would be of interest then to determine the 10. individual contribution of isolated tropho- 11. blast and inner cell mass to the total out- 12.

growth synthesis of tubulin and actin. This 13. could help to further define the relationship 14. between morphogenetic activity and the 15. synthesis of these two proteins. 16.

We thank Dr Narayan Avhadani for the use of his den- 17.

sitometer. This work was supported by PHS Research Grant HD 08452, NICHHD Training Grant HD 00239 and a grant from the Population Council.

l8

19.

20. REFERENCES

21. 1. Abreu, S L & Brinster, R L, Exp cell res 114

(1978) 135. 22. 2. Cole, R J & Paul, J, Preimplantation, stages of

pregnancy (ed G E W Wolstenholme & M O’Con- 23. nor) p. 82. J & H Churchill, London (l%S).

3. Brvson. D L. Science 144 (1964) 135. 34 4. Gwatkih, R B L, J cell physiol68 (1966) 335. 55: 5. Menke, T M & McLaren, A J, J reprod fertil 23

(1970) 117. 26. 6. Brinster, R L, Growth, nutrition and metabolism

of cells in culture (ed G Rothblat & V Cristofalo) 27. p. 251. Academic Press, New York (1972).

7. Sherman, M I, Differentiation 3 (1975) 51. 28. 8. Sherman, M I & Salomon, S, The developmen-

tal biology of reproduction (ed C L Market? 8~ J Papaconstantinou) p, 277. Academic Press, New

Kowit, J D & F&on, C, J biol them 249 (1974) 3638. Bray, D & Thomas, C, Biochem j 147 (1975) 221. Fine, R E & Taylor, L, Exp cell res 102 (1976) 162. Pardee, J D & Bamburg, J R, J neurochem 26 (1976) 1093. O’Farrell, P H, J biol them 250 (1975) 4007. Schmitt, H, Gozes, I & Littauer, U Z, Brain res 121 (1977) 327. Fujimoto, G I, Saldana, L R & Gaskin, F, En- docrin res commun 3 (1976) 219. Wessells, N K, Spooner, B S, Ash, J F, Bradley, M 0. Luduena. M A. Tavlor. E L. Wrenn. J T & Yamada, K M,‘Science 171 (l971) l35. Borman, L S, Dumont, J N & Hsie, A W, Exp cell res 91 (1975) 422. Mousa, G Y & Trevithick, J R, Dev biol60 (1977) 14. Granholm, N H &z Brenner, G M, Exp cell res 101 (1976) 143. Lowrv. 0 H. Rosebrounh. N J. Farr. A L & Rand%, R J, J biol chemi93 (1951) 265.’ Bryan, J & Wilson, L, Proc natl acad sci US 68 (1971) 1762. Elzinga, M, Collin, J H, Kuehl, W M & Adelstein. R S, Proc natl acad sci US 70 (1973) 2687. Laemmli, U K, Nature 227 (1970) 680. Bonner, W M & Laskey, R A, Eur j biochem 46 (1974) 83. Soifer, D (ed), The biology of cytoplasmic microtubules. Ann NY acad xi 253 (1975). Taylor, R Bi’Duffus, P H, Raff, M C & de Petris, S, Nature new bio1233 (1971) 225. Balls, M &Wild, A E (ed), The early development of mammals. Cambridge University Press,. Lon- don (1975).

York (1974). 9. Weber, K, Pollack, R & Bibring, T, Proc natl acad

Received December 2, 1977

sci Revised version received February 6, 1978

US 72 (1975) 459. Accepted February 14, 1978

Exp Cell Res I I5 (1978)