the injection of embryonic microsomes into early amphibian embryos

204 Br6ves communications - Brevi comunicazioni [EXPERIENTIA VOL, V/5]

A ~-Desoxycorticosterone Acetate

T h e r e c e n t i n t e r e s t ~ in a n a l o g u e s of d e s o x y c o r t i - co s t e rone a c e t a t e p r o m p t s us to r e c o r d t h e s y n t h e s i s a n d b io log ica l a c t i v i t y of A1-allopregnen-21-ol-3,20- d ione 2 1 - a c e t a t e (II) , for w h i c h t h e t r i v i a l n a m e " A ~- d e s o x y c o r t i c o s t e r o n e a c e t a t e " is sugges t ed .

( o n e - s i x t e e n t h t h e a c t i v i t y of p r o g e s t e r o n e ) and a n d r o g e n s ~ ( o n e - t h i r t e e n t h t h e a c t i v i t y of t e s to s t e rone ) .

C. DJER~SSL C. R. SCHOLZ, a n d J . H. LEA~HE~t

R e s e a r c h D e p a r t m e n t , D i v i s i o n of C h e m i s t r y , Ciba P h a r m a c e u t i c a l P r o d u c t s , Inc . , S u m m i t , N e w Jersey, a n d B u r e a u of Bio log ica l R e s e a r c h , R u t g e r s Un ive r s i t y , N e w B r u n s w i c k , N e w Je r s ey , J a n u a r y 20, 1949.

COOH COCH.OAc

/ \ t / l \ / \ Y \

. i o/~/ i~/ o/"x/iN/

H I H I I

COCHoOAc

I I I

A~-3-Ketoetioallocholenic ac id (I), o b t a i n e d b y s a p o n i f i c a t i o n of t h e c o r r e s p o n d i n g m e t h y l e s t e r ~, was c o n v e r t e d t o t h e ac id ch lo r ide b y t h e oxa ly l ch lo r ide m e t h o d ~ a n d t h e n c e w i t h d i a z o m e t h a n e to t h e c rys t a l - l ine A~-d i azop roges t e rone ( m . p . 183-185 ° w i t h gas evo lu t ion ) . D e c o m p o s i t i o n of t h e l a t t e r in bo i l ing ace t ic ac id s o l u t i o n g a v e a 5 0 % ove r a l l y ie ld (based on t h e ac id I) of A l - d e s o x y c o r t i c o s t e r o n e a c e t a t e (II) , m . p .

r n 26 215--217°' ~%D = + 139° (ch loroform) , (Anal . Calcd. for C~3Ha:O,: C, 74-16; H, 8 .65 ; ace ty l , 11.55. F o u n d : C, 74 .17 ; H, 8 .64 ; ace ty l , 11.10). T h e p r o d u c t e x h i b i t e d a n u l t r a - v i o l e t a b s o r p t i o n m a x i m u m a t 229 raft, log e 4.08, t y p i c a l of A ~ u n s a t u r a t e d 3 -ke to s t e ro id s ~, a n d t h e A[IVI]D v a l u e * of + 69 was in exce l l en t a g r e e m e n t w i t h t h a t o b s e r v e d ~ ( + 67) for k e t o n e s of t h i s class. T h e d i k e t o n e I I cou ld also b e p r e p a r e d b y co l l id ine d e h y d r o - b r o m i n a t i o n of 2-bromo-21-acetoxy-allopregnane-3,20- d i o n e ( I I I ) (m. p. 188 -190 ° dec.) , w h i c h was o b t a i n e d b y b r o m i n a t i n g 21-acetoxyallopregnane-3, 20-d ione in gla- c ia l ace t i c ac id w i t h p y r i d i n e h y d r o b r o m i d e p e r b r o m i d e %

A ~ - D e s o x y c o r t i c o s t e r o n e a c e t a t e ( I I ) was t e s t e d in oil s o l u t i o n for l ife m a i n t e n a n c e in a d r e n M e c t o m i z e d r a t s a n d p r o v e d t o possess a p p r o x i m a t e l y o n e - f i f t h t h e a c t i v i t y of A ~ - d e s o x y c o r t i c o s t e r o n e a c e t a t e . I t is i n t e r e s t i n g to n o t e t h a t t h e c o r r e s p o n d i n g s a t u r a t e d d e r i v a t i v e was f o u n d b y W ~ T S T ~ I N a n d I-Iu~z~KER ~ to be i n a c t i v e w h e n t e s t e d in t w e n t y t i m e s t h e e f fec t ive dose of A * - d e s o x y c o r t i c o s t e r o n e ace t a t e . A c o m p a r i s o n b e t w e e n t h e A ~-isomers a n d t h e n a t u r a l h o r m o n e s in t h e cor t ica l , p r o g e s t a t i o n a l a n d a n d r o g e n i c s t e r o i d series d e m o n s t r a t e s t h a t a sh i f t of t h e d o u b l e b o n d f r o m t h e 4,5 to t h e 1,2 p o s i t i o n s s eems to h a v e t h e l eas t e f fec t in t h e cor t i ca l h o r m o n e series as c o m p a r e d to t h e g e s t a g e n s ~

I Ctl. MEYSI'RE and A. W~rTsTgfN, Helv. chim. acta 31, 1890 (1948). - v. J. Euw and T. RgIC~STEIN, lb. 31, 2076 (1948).

2 C. DJERASSl and C. R. SCHOLZ, J. Amer. Chem. Soc. 69, 2404 (1047).

3 A.L.\VtLDS and C. H, SHANK, J. Amcr.Chem. Soc. 70,24~7 (1948). a For the calctflation of this value, it is necessary to use the

rotation of ~l-acetoxyallopregnane-3,20-dione. This known com- pound [see A. ~VETTSTEI~ ~ and F. HVNZIKER, Hclv. chim. acta 23, 764 (1940) who reported [~]D = :a~ 115° + 2 ° (chloroform)] was synthesized in this laboratory by the Wilds-Shunk procedure (reI. 3) from 3-ketoetioa~lochoIanic acid and was found to have [o:]2) 6 :is 120° + 1° (chloroform).

5 C. DJERASSL J. Org. Chem. 1G 823 (1947). 6 C. DJERASSI and C. R. Sc~m~.z, J. Amer. Chem. Soc. 70, 4t7

(1948). "1 See K. MIESCHEI% Recent Progress in Hormone Research

(Proceedings of the Laurentian Hormone Conference) 3, 47 (19i8) for a review on the relation between structure and activity of the sex hornlones.

Zusammen/assung

Die H e r s t e l l u n g y o n Al=Desoxy-corticosteronacetat wird b e s c h r i e b e n . B e i m l ~ b e r g a n g v o m g e w 6 h n l i c h e n D e s o x y - c o r t i c o s t e r o n a c e t a t (A 4) zu d ieser h i n s i e h t l i c h de r Lage d e r D o p p e l b i n d u n g i s o m e r e n V e r b i n d u n g t r i t t e in Abfa l l de r W i r k u n g i m O b e r l e b e n s t e s t an a d r e n a l e k t o m i e r t e n R a t t e n au f 1/5 ein.

1 See K. l~hzscn~g, Recent Progress in Hormone Research (Proceedings of the Laurentian Hormone Conference) 3, 47 (1948) (or a review on the relation between structure and activity of the sex hormones.

The Injection of Embryonic Microsomes into early Amphibian Embryos ~

A m o n g t h e m a n y h y p o t h e s e s w h i c h h a v e b e e n ad - v a n c e d t o e x p l a i n t h e m o d e of a c t i o n of t h e e m b r y o n i c o r g a n i z e r is t h e idea , i n d e p e n d e n t I y p u t f o r w a r d b y DhLCQ ~ a n d b y NEEDHA~ 3, t h a t t h e r e m i g h t b e a s i m i l a r i t y b e t w e e n t h e i n d u c t i o n of K n e r v o u s s y s t e m a n d t h e s p r e a d i n g of a v i ru s in fec t ion . As ha s b e e n n o t e d b y one of us (BRAcHET*), if t h e r e is a n y t h i n g c o m p a r a b l e to a v i rus in t h e e m b r y o , we s h o u l d e x p e c t to f ind i t in t h e fo rm of pa r t i c les c o m p a r a b l e in size a n d chemica l c o m p o s i t i o n to v i ruses . These c r i t e r i a desc r ibe t he so-cal led m i c r o s o m e f r a c t i o n of CLAUDE S, t h e c o m p o s i t i o n of w h i c h h a s b e e n s t u d i e d b y CLAUDE, BRAGHET a n d JEENER B, CHANTRENNE 7, a n d b y m a n y o thers , all of w h o m f o u n d t h a t t h e s e g r anu l e s a re c o m p o s e d of r i bonuc l e i c ac id a s soc ia t ed w i t h p r o t e i n s ( inc lud ing enzymes ) a n d l ipids,

I n o rde r to t e s t t h e poss ib le m o r p h o g e n e t i c a c t i v i t y of t h e s e par t i c les , t h e y were i so l a t ed f r o m bre i s of frog g a s t r u l m or n e u r u l m in t h e fo l lowing way . E m b r y o s h o m o g e n i z e d in ice-cold d i lu t e p h o s p h a t e b u f f e r were c e n t r i f u g e d for t e n m i n u t e s a t 6,000 × g in o r d e r t o ge t r id of t h e yolk , nuc le i a n d p i g m e n t g ranu le s . T h e s u p e r n a t a n t f lu id was t h e n c e n t r i f u g e d for t w e n t y m i n u t e s a t I8 ,000 X g, a n d t h e r e s u l t a n t pe l l e t s of g r a n u l e s were s u s p e n d e d , in a s m a l l v o l u m e of d i lu t e

1 Aided by a grant from the American Philosophical Society. 2 A. DALCQ, L'wU] et so~ dynamisme organisateur (Albin Michel,

Paris 1941), p. 492~ 5"26. a j . NEEDHAM, Biochemistry and Morphogenesis (Cambridge,

1942), p. 268. 4 j . BRACHEG Embryologie chimiq~e (Desoer, Li6ge 1945). s A. CLAUDE, Biol. Syrup. 10, 111 (1943). 6 j . BRACHET and R. JEE~EI¢, Enzymologia, 11, 196 (1944). 7 H. CuANrRENN~, Biochim. et biophys, acta, 1, 437 (1947) : En-

zymologia 11, 213 (1944).

[15. V. 1949] Kurze Mitteilungen - Brief Reports t)05

phosphate buffer and injected by means of fine capil- lary pipettes into the ventral halves of cleaving eggs, of Rana pipiens, _Rana [usca and of axolotl. The opera- tions were usually done on cleavage stages of from four to sixteen cells, and not too great a mor ta l i ty ensued, Many operated eggs, however, showed abnormalit ies at gastrulation and had to be discarded. Several hundred eggs were operated in this way, the location and depth of penetrat ion of the injection varying greatly. An equal number of eggs injected with phosphate buffer alone served as controls.

Only a small par t of tile material collected has thus far been sectioned, and our results still have a prel iminary interest only. However, some of the findings have been very constant. For example, in none of the operated embryos did we find double gastrulation, as would have been the case if the ventral half of the egg had been transformed into an organizer. Fur thermore, of 139 sectioned embryos, only 5 showed a secondary neural tube, and, since the neural structures were fused with the pr imary nervous system, it is likely tha t they re- suited from a mechanical disturbance of gastrulat ion movements. Thus, as regards induction, our results have thus far been negative, but i t must be remembered tha t negative findings in this connection do not rule out the virus hypothesis, since inact ivat ion during isolation of the granules is by no means excluded.

However, the embryos injected with granules showed one dist inct and interesting difference as compared with the controls. The cells surrounding the point of injection, which can usually be easily recognized, in most cases show a dist inct increase in basophilia, as demonstrated by staining with toluidine blue. The baso- philic material can be digested away with solutions of crystalline ribonuclease, and thus is presumably of ribonucleoprotein nature. These basophilic cells can be found in the ectoderm, the mesoderm or the endoderm, according to the localization and depth of injection, and are present in well localized areas, very often showing no signs of cytolysis (the latter, as shown by one of us, BRACHET 1, is also accompanied by an increase in baso- philia). In severM cases, when embryos which had been injected with granules were fixed in the late yolk-plug stage, they showed a very definite increase in baso- philia in par t of the ectoderm. There were, however, no signs of induction in these cases. Such results are of some interest, since they show tha t a localized synthesis of ribonucleic acid in the ectoderm is not of itself suf- ficient to evoke a neural induction.

On the whole, increased basophilia' near the place of injection occurred in 101 cases out of 139 (72.6%), when granules from gastrulm or young neurulm had been introduced. The same phenomenon was seen only five t imes in 62 cases (8 %) when plain phosphate buffer was injected. In these five posit ive cases, the basophitic cells were few in number and showed dist inct signs of incipient cytolysis.

These results suggest t ha t the injected particles are capable of inducing a local synthesis of ribonucleic acid, but whether they are self-reproducing units or whether they contain enzymes synthesizing ribonucleic acid cannot be decided on the basis of the present evidence.

J. BRACHET and J. R. SHAVER 2

Labora tory of Animal Morphology, Univers i ty of Brussels, and Depar tment of Zoology, Univers i ty of Pennsylvania, Philadelphia (Pa.) February 12, 1949.

1 j. BRACHET, C. R. Soc. biol., 140, 1123 (1946). 2 Fellow of the Belgian-American Educational Foundation,

1948-9.

Rdsumd

L' in jec t ion de granules provenant d 'un broyage de gastrulas ou de blastulas de grenouilles dons des oeufs segment ,s de diff~rentes esp~ces d 'Amphibiens donne, dans les stades ultdrieurs, un accroissement de la baso- phitie dons les cellules voisines de la piqfire. Les rares embryons tdmoins, injectds avec un tampon au phos- phate, qui donnent une rdaction analogue sont sur le point de se cytolyser. On n ' a pas not6 de gastrulations doubles et les quelques tubes neuraux secondaires ob- servds 6talent sans doute dus A un t raumat i sme mfca- nique.

B e o b a c h t u n g e n an v i r u s k r a n k e n Larven v o n Chironomus (Camptochlronomus) tenfans Fabr .

WEISER 1 hat an Larven von Camptochironomus tentans aus dem Drecksee bei P16n eine Infekt ion des Fet tkSrpers durch Viren beschrieben, die yon kokken- artigen Organismen begleitet waren. Im Herbst 1948 machte ich an Camptochironomuslarven yon dem glei- chen Fundplatz bei atmungsphysiologischen Studien, fiber die spAter berichtet werden wird, einige Beobach- tungen, die im Zusammenhang mit WEISERS Unter- suchungen yon Interesse sind, und daher kurz wieder- gegeben seien.

Camptochironomuslarven zeigen schon aus ihrem natiirlichen Medium (fauliger Schlamm mit deutl icher Sauerstoffzehrung) eingebracht, erheblich gesteigerte Erholungsatmung, die langsam beim Schiittetn mi t Luft im Vtarburg-Manometer geringere ~Verte anstrebt. Dos am 29. November 1948 eingebrachte Tiermater ial wurde in einer Schale mit Wasser und nur Spuren yon Schlamm im Laborator ium aufbewahrt . Nament l ich iiltere Larven zeigten verschiedentlich den kennzeichnenden weiBen, undurchsichtigen Fet tk6rper kranker Tiere. Sic starben und zerfielen racist bald; das Wasser hat te bald , fau- ligen,> Charakter und dementspreehend sicher nur ge- ringen Sauerstoffgehalt. Dos war mir erwiinscht, da ich ja Beobachtungen an Larven in Erholungsatmung an- stellen wollte. Die Folge dieser wenig pfleglichen Auf- bewahrung des Materials war jedoch auch, dab bald die Mehrzahl der Larven, ouch jiingere, nur etwa halb- erwachsene Tiere, deutliehe Krankheitserscheinungen zeigten.

"W~hre'nd friiher (z. T. auch spitter) die Erholungs- a tmung der Larven durchaus normalen Verlauf zeigte, ergaben einige Messungen am 8, 9. und 10. Dezember 1948 ein sehr merkwtirdiges Bild der Gestal tung der Er- holungsatmung: sic setzte mit normal hohem Wert ein, sank aber dann (bei halbsti indigen Messungsetappen) sehr rasch zu auBerordentlich niedrigen, kaum noch yon lebenden Tieren zu erwartenden Wer ten ab. 3 Proto- kolle ( I - I I I ) m6gen dies belegen, wiihrend die Messung IV dasVerhal ten nicht infizierter Tiere vom 10. Dezem-

Etappe I II III IV

279,9 249,5 235,4 276 176,7 65,6 157 258,5 107,7 78,7 55 233,3 81,5 25,21 43,2 233,3 45,68 - - 241,5

x j . WEXSER, Exper. 4, 317 (1948).