Protein synthesis in mature frog oocytes

Experimenlal Cell Research 41, 61i-621 (1966)

614

P R O T E I N S Y N T H E S I S IN M A T U R E F R O G OOCYTES 1 R. W . M E R R I A M

2

Department of Biological Sciences, State University of New York at Stony Brook, N.Y., U.S.A.

Received August 30, 1965 THE accumulation of yolk protein in oocytes is accomplished in two general ways. Either pre-synthesized protein is transported through the ooeyte surface [21] from extraoocyte sources [13, 14, 24] or the oocyte itself synthesizes yolk protein [2, 6, 26]. The relative importance ofthesetwo mechanisms seems to vary with the particular species involved. Aside from synthesis or accumulation of yolk protein, there is probably protein synthesis which is associated with the growth of the non-yolk cytoplasm and certainly with the growth and functioning of the nucleus and chromosomes [7, 9, 12 I. Yet when oogenesis reaches completion protein synthesis, RNA synthesis, and respiration are reduced to a m u c h lower level by control mechanisms as yet not well understood. This lowered metabolism is then further reduced after maturation or ovulation until fertilization initiates the metabolic patterns of embryogenesis. For a review see [16!. In R a n a p i p i e n s , oocytes come to full size during the summer of their third year of growth. During this final rapid maturational period protein synthesis occurs at a relatively rapid rate in both the growing oocytes and their investing epithelium when incubated in vitro [15]. During the subsequent winter hibernation of almost eight months there is no growth in size and the oocytes are capable of forced ovulation, fertilization, and development [22]. Incorporation of amino acids into proteins can be demonstrated in these large cells although it is at a slow rate. Because little is known about the nature of the slow protein synthesis in full-sized winter oocytes, this paper reports experiments to characterize it. Data are presented on the role of the follicular epithelium, the relative contributions of nucleus and cytoplasm, the effects of chloramphenicol, and the immediacy of linkage between oxidative phosphorylation and the observed amino acid incorporation. 1 Supported by grants from Brown-Hagen Fund, Research Corporation and United States Public Health Service, GM 10142. 2 With the assistance of Mathilda Haefele. Experimental Cell Research 41

Protein sgnthesis irz oocgtes

MATERIALS

AND

615

METHODS

Raua pipiens females from Wisconsin were kept a t 5~ in h i b e r n a t i o n during the winter months. For use, they were removed, i m m e d i a t e l y decapitated a n d sections of whole ovary removed a n d washed in B a r t h ' s m e d i u m [1]. A p p r o x i m a t e l y equal n u m b e r s of large oocytes were placed in B a r t h ' s m e d i u m c o n t a i n i n g L-l~C-phenylalanine (5-7 m C / m M ) or L-~4C-lysine (1-2 m C / m M ) at 0.2-1.0 /~c/ml. Results with both amino acids were q u a l i t a t i v e l y similar. I n c u b a t i o n was performed at 25~ with occasional shaking. For sampling, pieces of ovary c o n t a i n i n g m a n y large oocytes were removed b y forceps a n d rinsed quickly in three changes of water. Each sample was t h e n extracted with cold and hot 10 per cent trichloroacetic acid (TCA), followed b y lipid e x t r a c t a n t s [23]. U n d e r a dissecting microscope the large oocytes of each sample were freed of smaller oocytes with forceps in a dish of acetone. The resulting clean oocytes were t h e n either homogenized directly or were broken with forceps to allow separation of nuclear a n d cytoplasmic fractions b y hand. E x t r a c t e d oocytes break easily in acetone, their white nuclei separating cleanly from the darker, pigmented cytoplasm. A c c u m u l a t e d whole cells or fractions were homogenized in acetone a n d the residue collected on pre-weighed W h a t m a n no. 42 filter p a p e r discs. After drying, a final weighing gave the weight of sample residue, referred to in this paper as protein. Discs were c o u n t e d in a gas flow proportional counter with a b a c k g r o u n d of less t h a n three counts per m i n u t e . Corrections for self-absorption were calculated from an empirically d e t e r m i n e d curve. Results are expressed as counts per m i n u t e per mg of residue or protein. For estimation of total u p t a k e of amino acid, the same i n c u b a t i o n a n d sampling procedure was used. The samples, however, were n o t extracted b u t dried at 110~ a n d homogenized in toto in cold acetone. The residue was collected on filter paper discs, weighed, a n d counted as described above. Using W h a t m a n no. 42 filter paper it was found t h a t less t h a n 0.5 per cent of the t o t a l r a d i o a c t i v i t y w e n t through the filter. RESULTS

S i n c e all of t h e m a t u r e o o c y t e s u s e d i n t h e s e s t u d i e s w e r e s u r r o u n d e d b y a s e r o s a a n d a s i m p l e e p i t h e l i u m o f follicle cells, t h e first q u e s t i o n a p p r o a c h e d w a s t h e i r role i n t h e u p t a k e a n d i n c o r p o r a t i o n o f a m i n o a c i d s .

Amino Acid Uptake and Incorporation I n t h e first e x p e r i m e n t o n e g r o u p of o o c y t e s w a s f r e e d o n l y of a d h e r e n t s m a l l o o c y t e s w h i l e a n o t h e r g r o u p f r o m the s a m e o v a r y w a s also d i s s e c t e d free o f t h e i n v e s t i n g e p i t h e l i u m . U p o n r e m o v a l o f t h e f o l l i c u l a r e p i t h e l i u m with forceps, the oocytes b e c o m e characteristically velvety in a p p e a r a n c e w i t h a f l a c i d c o n s i s t e n c y . No cells w i t h r u p t u r e d m e m b r a n e s w e r e u s e d .

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R. W. Merriam

At time zero both groups were placed into flasks containing equal aliquots of L-14C-phenylalanine solution at 0.5 /~e/ml. Two experiments were done with almost identical results, one of which is shown in Fig. 1. The results show a characteristic linear uptake and incorporation through time into both groups. The loss of epithelium has neither prevented amino /o /

~500

/

/

/ / tt

.j400

2 o o

t

0/ / J

300

X

/

~14C

~

0

~, X

/

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0

w

010(

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:E

I

"

f

0 I00

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j

X

Of

~x ~ x ~ 2o ~o Bb do MINUTES OF INCUBATION

Fig. 1.

,oo

20

40 60 MINUTES OF

80

I00

INCUBATION

Fig. 2.

Fig. 1.--Effect of epithelium on uptake and incorporation of phenylalanine. Q - - O , total uptake; x - - x , incorporated phenylalanine; - - , cells with follicular epithelium intact; . . . . , cells with epithclium removed. Data from incorporation calculated for total dry weight from the fact that extracted residue represents about 58 per cent of total dry weight. Fig. 2.--Incorporation of lysine into nuclear and cytoplasmic fractions. into cytoplasm; Q - - O , incorporation into nuclei.

•215

incorporation

acid uptake nor stopped incorporation into protein. The epithelium has, however, slowed the rate of uptake, and hence the rate of incorporation. These experiments are interpreted as evidence that the investing epithelium acts mainly as a barrier, probably slowing down the rate at which the amino acid is presented to the cell surface. When nuclear and cytoplasmic fractions were prepared, it was found that both contributed to the incorporation of amino acid as shown in Fig. 2. This type of experiment has been done several times using lysine and phenylalanine with about the same results. The apparently higher specific rate of incorporation into the nuclear fraction is due mainly to the fact that there is more inert storage protein in the cytoplasm than in the nucleus. As indicated in Fig. 1, the rate of amino acid uptake is faster than the rate of incorporation. An early experiment was performed to see if "pool" amino acid could be utilized normally in protein synthesis. An unexpected and still unique result is shown in Fig. 3. Although the radioactivity used Experimental Cell Research 41

Protein sgnthesis in oocgtes

617

in the e x p e r i m e n t was s u b - o p t i m a l , there is little d o u b t that m a t u r e oocytes o f that o v a r y were synthesizing proteins n o r m a l l y in their nuclei f r o m prev i o u s l y a c c u m u l a t e d a m i n o acid. T h e i r c y t o p l a s m i c synthesis w a s not operating. O.15 eM LABELED PHENYLALANINE

P 0.15 U M UNLABELED PHENYLALANINE

O

i

18C

~4D La

of

14C

=E IOC

20

/~

z :E G(

T

I0

? 40

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80

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Fig. 3.

280

320

o/

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o 2C

/

~

60

- - g - - a - -

120 MINUTES

9 --g

tEo 240 300 OF iNCUBATION

360

Fig. 4.

Fig. 3.--Incorporation of phenylalanine into nuclear and cytoplasmic proteins. Labeled phenylalanine was administered for 120 rain, the cells were then washed and re-suspended in the same concentration of unlabeled amino acid. Fig. 4.--Effect of cyanide and fluoride on incorporation of phenylalanine. Q--O, incorporation into whole cells in normal medium; 9169 incorporation into cells in 2 x 10-2 M cyanide; • 2 1 5 incorporation into cells in 8 • 10.2 21I fluoride. This result is i n c l u d e d b e c a u s e it suggests that the o b s e r v e d n u c l e a r a n d c y t o p l a s m i c synthesis m a y be i n f l u e n c e d or controlled differently at some p o i n t in the i n c o r p o r a t i o n process. A possible e x p l a n a t i o n is offered in the Discussion. A n u n u s u a l result s u c h as s h o w n in Fig. 3 p r o m p t e d some t h o u g h t a b o u t possible p r e p a r a t i v e artefact. T h e possibility was c o n s i d e r e d that a b r u p t i m m e r s i o n in an acid, s u c h as TCA, c o u l d cause diffusion of labeled protein w i t h i n the cells. To c h e c k on the m e t h o d , the n u c l e a r - c y t o p l a s m i c i n c o r p o r a tions were c o m p a r e d in cells f r o m the s a m e o v a r y w h i c h were treated in the u s u a l w a y with cells s a m p l e d b y freezing at - 1 9 0 ~ followed b y slow t h a w i n g in cold 10 per cent TCA a n d slow w a r m i n g to r o o m t e m p e r a t u r e . Both s a m p l e s were then extracted a n d fractionated as usual. Isolation of nuclei was c o m p l i c a t e d b y the fact that cells p r e v i o u s l y frozen did not b r e a k well a n d c y t o p l a s m t e n d e d to c o n t a m i n a t e the n u c l e a r fraction. Results are p r e s e n t e d in T a b l e I. Differences are not statistically significant a n d c a n be a c c o u n t e d for b y the greater c o n t a m i n a t i o n of the n u c l e a r fraction in cold-treated cells b y

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618

c y t o p l a s m of lower specific activity. It was c o n c l u d e d that no significant diffusion of labeled material o c c u r s d u r i n g precipitation of cellular solids in TCA.

The Source of Energy for Incorporation Mature o v a r i a n oocytes of Rana pipiens respire at a low rate [3, 15J so that it is possible that oxidative p h o s p h o r y l a t i o n c o u l d be the energy s u p p l y . TABLE I. The effect of different precipitation methods on the nucleo-cgto-

plasmic distribution of incorporated phenglalanine. C/min/mg residue, m +-sin. Treatment a TCA at room temp. TCA after freezing

Nucleus

No.b

Cytoplasm

No.

29_+11 21+ 5

3 3

11 +7 16--+6

4 6

a For explanation see text. Number of replicates.

I n a cell l o a d e d with nutritional reserves, h o w e v e r , this does not necessarily follow. To investigate the n a t u r e of the e n e r g y s u p p l y 2 • 10 -2 M s o d i u m c y a n i d e a n d 8 • 10 -2 34 s o d i u m fluoride were a d d e d to m e d i a c o n t a i n i n g labeled p h e n y l a l a n i n e . T h e rates of i n c o r p o r a t i o n were c o m p a r e d with control cells f r o m the s a m e o v a r y . In Fig. 4 the results of one of two experiments are given. I n both, fluoride a n d c y a n i d e c a u s e d an i m m e d i a t e a n d c o m p l e t e stoppage of i n c o r p o r a t i o n . These effects c o u l d be due to inhibition of electron t r a n s p o r t b y c y a n i d e a n d b l o c k a g e of p y r u v a t e p r o d u c t i o n f r o m glycolysis b y fluoride acting on enolase. Both effects c o u l d act b y stopping oxidative p h o s p h o r y l a t i o n . A n e x p e r i m e n t w a s d o n e w i t h 2 , 4 - d i n i t r o p h e n o l ( D N P ) to see if a direct u n c o u p l e r of oxidative p h o s p h o r y l a t i o n w o u l d have the s a m e effect. Using the antimetabolite at a c o n c e n t r a t i o n of 8 x 10-* 3/, there w a s no detectable i n c o r p o r a t i o n in either n u c l e a r or c y t o p l a s m i c fractions a l t h o u g h u p t a k e of the a m i n o acid into the cells w a s c o n s i d e r a b l y faster t h a n into the control cells. It was c o n c l u d e d that p r o t e i n synthesis is closely linked to oxidative p h o s p h o r y l a t i o n with c a r b o h y d r a t e reserve as the p r o b a b l e energy source.

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The Effects of Chloramphenicol C h l o r a m p h e n i c o l ( C A P ) h a s b e e n s h o w n to i n h i b i t a m i n o acid i n c o r p o r a tion in m a n y k i n d s of cells, the b l o c k a g e o c c u r r i n g at the p o i n t of t r a n s f e r of a m i n o acids f r o m t - R N A to r i b o s o m e s in b a c t e r i a [18, 20]. An e x p e r i m e n t TABLE II. The effect of chloramphenicol on 1-phenglalanine incorporation. Chloramphenicol/ml #g

C/min/mg residue, m _+S.D.

NO.

% inhibition

0

185 • 15

3

0

75 150

104 • 12 121 ! 12

3 3

44 35

a Number of replicates.

w a s p e r f o r m e d to d e t e r m i n e the effect of CAP on p r o t e i n synthesis in m a t u r e frog oocytes. T a b l e II is a t a b u l a t i o n of the results w h e n cells w e r e i n c u b a t e d in the p r e s e n c e of l a b e l e d p h e n y l a l a n i n e for 6 h r w i t h or w i t h o u t a d d e d c h l o r a m p h e n i c o l . B o t h c o n c e n t r a t i o n s p r o b a b l y g a v e m a x i m a l effect a n d it w a s c o n c l u d e d t h a t a b o u t 40 p e r cent of the o b s e r v e d a m i n o acid i n c o r p o r a tion is i n h i b i t a b l e b y the antibiotic.

DISCUSSION T h e large oocytes studied in these e x p e r i m e n t s are 1.6 to 1.7 m m in d i a m e t e r . This is the full size of the m a t u r e egg a n d i n d e e d if o v u l a t i o n is f o r c e d b y p i t u i t a r y extract, the cells are c a p a b l e of meiotic m a t u r a t i o n , fertilization, a n d d e v e l o p m e n t [22]. A l t h o u g h slow m a t u r a t i o n a l c h a n g e s d u r i n g the winter h i b e r n a t i o n c a n n o t b e r u l e d out, it is likely t h a t the obs e r v e d p r o t e i n synthesis r e p r e s e n t s t u r n o v e r r a t h e r t h a n net synthesis. T h e fact t h a t s u c h i n c o r p o r a t i o n c a n p r o c e e d in vitro a n d w i t h the investing follicle cells r e m o v e d argues strongly t h a t it is a n intrinsic p r o p e r t y of the oocyte itself. I n these short t e r m e x p e r i m e n t s p r o t e i n synthesis occurs in b o t h n u c l e u s a n d c y t o p l a s m in m o s t cases. T h e one case in w h i c h o n l y nuclei s h o w e d inc o r p o r a t i o n c o u l d b e e x p l a i n e d b y the finding of N a o r a et al. [17] t h a t frog oocytes t e n d to a c c u m u l a t e u n i n c o r p o r a t e d a m i n o acids in their nuclei. I f

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this accumulation process were active enough in the one unusual frog, especially after removal of label from the medium, it could have reduced the free amino acid in the cytoplasm to a level so low that incorporation could not be observed. Despite the presence of detectable quantities of ATP in frog ovarian ooeytes [8], the supply cannot sustain even slow protein synthesis in the absence of active oxidative phosphorylation. The effects of cyanide and DNP are immediate and complete in stopping incorporation. If fluoride is acting on glyeolysis in producing its inhibition of synthesis, then it would follow that production of pyruvate from glyeolysis is also tightly linked to respiration. Since frog embryos have been shown to have all of the glycolytie enzymes [5] and since their lactate production is inhibited by fluoride [5], it is quite possible that the fluoride effect noted in this paper is also operating on enolase of the glyeolytie pathway. The concept of carbohydrate reserves acting as the energy source for protein synthesis is made more plausible by the fact that frog ooeytes have considerable glycogen reserves [101. Inhibition of amino acid incorporation by chloramphenicol implies that at least part of the observed synthesis operates through the usual t-RNA and ribosome components. In calf thymus nuclei, for example, CAP is a strong inhibitor of amino acid incorporation [4, 11] and the point of action is after the binding of activated amino acid to t-RNA but before polymerization on the ribosome [11]. The residual CAP-insensitive incorporation could be explained in different ways. For example, the antibiotic might not penetrate to the site of some of the synthesis. On the other hand, protein synthesis even in cellfree systems of some m a m m a l s has been found (e.g. [25]) to be insensitive to high levels of CAP. A study on the effect of CAP on amino acid incorporation in rat liver fractions has shown [19] that while nuclear and mitoehondrial fl'actions are sensitive to it, incorporation into a cytoplasmic particulate fraction is quite insensitive at the same concentration. Reasoning by analogy to the rat liver system, one can speculate that it is the non-mitoehondrial cytoplasm of frog ooeytes which is resistant to CAP, accounting for about 60 per cent of the observable incorporation. The nucleus and mitoehondria would then account for that 40 per cent which is sensitive. REFERENCES

1. BAI/TH, L. C. and BARTH, L. J., J. Embrgol. Exptl Morphol. 7, 210 (1959). 2. BEAMS, H. W. and KESSEL, R. G., J. Cell Biol. 18, 621 (1963). 3. BRACrlET, J., Arch. biol. 45, 611 (1934). Experimenlal Cell Research 41

Protein synthesis in oocgtes 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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