RAPID COMMUNICATION ZIPGRAM

THE EFFECT OF INJECTED CYCLOHEXIMIDE ON

EARLY AMPHIBIAN DEVELOPMENT

HAROLD W . MANNER AND PEGGY 0. LAU Department o f Biology, Loyola Un ive rs i t y , Chicago, I l l i n o i s 60626

ABSTRACT Cycloheximide i s a known i n h i b i t o r o f cytoribosomal t r a n s l a t i o n . Prev ious ly publ ished r e s u l t s i nd i ca ted t h a t embryos c u l t u r e d i n cyc lo- heximide developed o n l y t o the midblastu la stage. invo lved i n j e c t i n g one ug o f cycloheximide i n t o each developing embryo o f Rana p ip iens. The three stages se lected f o r t he i n j e c t i o n were the two-cel l , l a t e fou r -ce l l , and morula. a t t he f o u r - c e l l , s ix teen -ce l l and midblastu la stages, respec t i ve l y . Control embryos i n j e c t e d a t the same stages w i t h H o l t f r e t e r ' s s o l u t i o n developed normal ly. The r e s u l t s would i n d i c a t e t h a t de novo p r o t e i n synthesis i s necessary, no t j u s t a t the midblastu la stage, b u t throughout the e n t i r e cleavage per iod. previous i nves t i ga t i ons .

The cu r ren t study

The embryos stopped developing

The r e s u l t s are discussed i n l i g h t o f

Cycloheximide i n h i b i t s p r o t e i n synthesis a t t he t r a n s l a t i o n a l l e v e l

(Duprat, '69).

and m i c r o i n j e c t i o n . The pe rmeab i l i t y o f embryos t o a n t i b i o t i c s has been d i s -

cussed by both Bachvarova e t a l . , ( ' 6 6 ) and F l i c k i n g e r e t a l . , ( ' 66 ) . Manner

and Lau ( ' 7 1 ) demonstrated an i n h i b i t o r y e f f e c t o f cycloheximide on e a r l y

p regas t ru la r development.

(10 ug/ml t o 50 ug/ml) a t any stage p r i o r t o b l a s t u l a t i o n , developed t o the

midblastu la b u t were prevented from gas t ru la t i ng . Our purpose i s t o demonstrate

the e f f e c t o f cycfoheximide, when in jec ted , on e a r l y amphibian development.

A n t i b i o t i c s have been app l i ed t o embryos by both incubat ion

Rana p ip iens eggs incubated i n cycloheximide

MATERIALS AND METHODS Rana p ip iens eggs were f e r t i l i z e d and subsequently

One microgkam o f cycloheximide was i n j e c t e d i n t o the animal degel led manually.

hemisphere o f each experimental embryo. This dosage proved, in previous work

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(Manner and Lau, '71), t o be p h y s i o l o g i c a l l y a c t i v e w i thou t e x h i b i t i n g t o x i c

o r l e t h a l e f f e c t s . A second group o f embryos were i n j e c t e d w i t h an equal

volume o f 100% modi f ied H o l t f r e t e r ' s so lu t i on . A f t e r i n j e c t i o n , the needle

was l e f t i n each embryo f o r approximately one minute and then removed slowly.

This needle r e t e n t i o n and delayed withdrawal prevented l o s s o f the i n j e c t e d

ma te r ia l through the puncture s i t e . Cycloheximide-injected and H o l t f r e t e r -

i n j e c t e d embryos were t rans fe r red t o 100% modi f ied Hol t f r e t e r ' s s o l u t i o n and

t h e i r development watched under a d i ssec t i ng microscope.

three d i f f e r e n t stages o f development were i n jec ted .

Embryos represent ing

Twelve cycloheximide-

i n j e c t e d and ten H o l t f r e t e r - i n j e c t e d embryos were t rea ted a t the 2 -ce l l stage;

18 cycloheximide- in jected and f o u r H o l t f r e t e r - i n j e c t e d embryos were t rea ted

a t the 4 -ce l l stage; and 15 cycloheximide- in jected and ten Hol t f r e t e r - i n j e c t e d

embryos were t rea ted a t t he morula stage. I n the 2 -ce l l stage needle penetra-

t i o n was i n t o one o f the two blastomeres. I n the 4 -ce l l stage, t he needle t i p

s t i l l entered on ly one blastomere.

undoubtedly penetrated many blastomeres.

membranes through which the cycloheximide had t o d i f f u s e i n order t o f u l l y

penetrate the egg was recognized i n the evaluat ion o f the data.

embryos were maintained a t a l abo ra to ry temperature o f 22 5 1OC.

I n the morula stage, t he needle t i p

This d i f f e r e n c e i n the number o f

A l l i n j e c t e d

RESULTS The embryos i n j e c t e d w i t h cycloheximide a t t h e 2 - c e l l stage

cleaved o n l y one more t ime w i t h development terminat ing a t the 4 -ce l l stage.

A l l t en Hol t f r e t e r - i n j e c t e d c o n t r o l s underwent normal cleavage and gast ru la ted.

Development o f t he embryos i n j e c t e d a t t h e l a t e 4 -ce l l stage proceeded a t a

re tarded r a t e and terminated a t t he 16 -ce l l stage. A l l o f t he H o l t f r e t e r -

i n j e c t e d embryos cleaved and gas t ru la ted normally.

cycloheximide a t t h e morula stage continued t o develop, b u t a t a re tarded r a t e .

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The embryos i n j e c t e d w i t h

Development terminated a t the midblastu la stage. The embryos i n j e c t e d w i t h

Hol t f r e t e r ' s s o l u t i o n underwent normal cleavage and gast ru la ted.

DISCUSSION The data repor ted i n t h i s paper show t h a t e a r l y development

o f Rana p ip iens eggs can be slowed down o r terminated by i n j e c t i o n o f cyc lo-

heximide. When embryos a re i n j e c t e d a t the e a r l y 2 -ce l l stage, development

proceeds through on ly one a d d i t i o n a l cleavage. This inmediate cessat ion

of development might be a t t r i bu ted . t o the i n h i b i t i o n o f p r o t e i n synthesis so

t h a t no t enough p ro te ins a re a v a i l a b l e f o r f u r t h e r development a f t e r the

p ro te ins already present have been u t i l i z e d . When i n j e c t e d a t t he l a t e 4 -ce l l

stage, development proceeds through two more cleavages.

accounted fo r by the f a c t t h a t c e l l membranes a c t as a b a r r i e r t o d i f f u s i o n

o f the a n t i b i o t i c i n s i d e the embryo.

between the f i r s t two blastomeres i s s t i l l n o t complete.

w i t h a l a r g e r number o f c e l l s , development proceeds t o the m idb las tu la stage.

A development o f a t l e a s t three d i v i s ons takes place.

therefore, t h a t t he e a r l i e r an embryo i s t rea ted w i t h cycloheximide, t he more

r a p i d i s the cessat ion o f development

This could be

A t t he e a r l y 2 -ce l l stage, the p a r t i t i o n

I n the morula stage,

The data i nd i ca te ,

Preblastu la p r o t e i n synthesis i s independent o f the nucleus. The templates

are provided by an unmasking o f l ong - l i ved maternal messengers. We have made

the assumption t h a t cycloheximide i n h i b i t s t h e t r a n s l a t i o n o f these messengers

and i s , therefore, responsib le f o r both the morphological abnormal i t ies t h a t

ensue and the u l t i m a t e te rm ina t ion o f development.

LITERATURE CITED

Bachvarova, R., E. H. Davidson, V. G. A l l f r e y and A. E. Mirsky 1966 A c t i v a t i o n o f RNA synthesis associated w i t h gas t ru la t i on . Nat. Acad. Sci., 55: 358-365.

Duprat, A. M. 1969 Act ion de l a cycloheximide sur des c e l l u l e s , embryonnaires d'amphibian en course de d i f f e r e n c i a t i o n - etude comparative avec l e s e f f e c t s de l a puromycine. Embry. Morph., 2: 179-190.

Proc.

Ex t r . Annal.

469

Flickinger, R. A., R. Green, D. M. Koh and M. Miyagi 1966 Patterns o f

Manner, H. W . , and P. 0. Lau 1971 Nucleic acid control o f early amphibian

synthesis o f DNA-like RNA in parts o f developing frog embryos. Proc. Nal. Acad. Sci., 56: 1712-1718.

development. Trans. Missouri Acad. Sci., 5: 129.

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