15. 11. 1975

Specialia

of g e n e a m p l i f i c a t i o n L T h e r e is a c l e a r i n d i c a t i o n t h a t L-triiodothyronine, though responsible for the amplificat i o n of r i b o s o m a l g e n e s i n c u l t u r e d h u m a n l i v e r cells, r e n d e r s n o i n c r e a s e d n e t s y n t h e s i s of r i b o s o m a l R N A ~ . F r o m t h e p r e s e n t r e s u l t s , it c a n f u r t h e r b e s e e n t h a t t h e a m o u n t of r i b o s o m a l D N A in l a t e r d e v e l o p m e n t a l s t a g e s of l i v e r r e m a i n s c o n s t a n t . T h i s is c o n s i s t e n t w i t h o t h e r d a t a c o n c e r n i n g g e n e d o s e in s e v e r a l t y p e s of t i s s u e o f a d u l t r a t v a r y i n g in r - R N A s y n t h e s i s ~7 F r o m all t h e d a t a , it c a n b e c o n c l u d e d t h a t a m p l i f i c a t i o n of r i b o s o m a l g e n e s s e e m s n o t t o b e a p h y s i o l o g i c a l l y r e g u l a t o r y m e c h a n i s m o p e r a t i v e in s o m a t i c cells. T h i s

1275

a l s o s e e m s t o b e t r u e of t h e a m p l i f i c a t i o n of s t r u c t u r a l g e n e s , b e c a u s e it w a s f o u n d t h a t t h e n u m b e r of g l o b i n g e n e s in h e m o g l o b i n s y n t h e s i z i n g cells w a s t h e s a m e a s i n o t h e r d i f f e r e n t i a t e d cells like l i v e r ~s. M o r e o v e r , n o d i f f e r e n c e i n t h e n u m b e r of g e n e s c o d i n g f o r t h e c o n s t a n t p a r t of i m m u n o g l o b u l i n s i n h o m o l o g o u s l i v e r o r m y e l o m a D N A c o u l d b e o b s e r v e d ~9.

Summary. T h e c o n t e n t of r i b o s o m a l D N A in m i c e l i v e r a t t h e b e g i n n i n g a s w e l l a s n e a r t h e e n d of t h e h e m a t o poietic period was measured by RNA/DNA-hybridization i n s o l u t i o n . A t b o t h s t a g e s t h e a m o u n t of r i b o s o m a l D N A w a s t h e s a m e a n d c o m p a r a b l e t o t h a t of p o s t n a t a l liver.

16 B, SCItLATTERER, in preparation.

17 j. MOifAN, A. DUNN and L. CASOLA, Nature, Lond. 223, 295 (1969). is S. PACKMAN,H. AVIV, J. Ross and PL. LEDER, Biochem. biophys. Res. Cominun. gg, 813 (1972). 19 C. H. FAUST, H. DIGGELMANN a n d B. MACH, Proc. n a t n . Acad.

Sei., USA 77, 2491 (1974).

M. ERLINGER a n d B. SCHLATTERER

Institut liar Biologische Chemic, Universitiit Hohenheim, Garbenstrasse 30, D-7 Stuttgart 70 (German Federal Republic, BRD), 24 March 1975.

O o c y t e M a t u r a t i o n i n v i t r o : C o n t r i b u t i o n of t h e O v i d u c t to T o t a l M a t u r a t i o n i n The process which transforms an amphibian oocyte into a fertilizable egg may be subdivided into several stages. The first visible external evidence that maturation h a s s t a r t e d is t h e p r e s e n c e of t h e m a t u r a t i o n s p o t . I n Rana pipiens, t h i s t a k e s 1 3 - 1 8 h a f t e r c o n t a c t w i t h p r o g e s t e r o n e , 3 6 - 4 0 h t o r e a c h t h e m e t a p h a s e of t h e s e c o n d m e i o t i c d i v i s i o n i a n d u p t o 68 h t o a t t a i n c l e a v a g e c a p a c i t y 2. Xenopus laevis o o c y t e s , p r o g e s t e r o n e - m a t u r e d in v i t r o , r e q u i r e a b o u t 24 h a f t e r c o n t a c t w i t h p r o g e s t e r o n e to r e a c h c l e a v a g e c a p a c i t y 3. Y e t , X. laevis f e m a l e s freq u e n t l y a r e a b l e t o s h e d f e r t i l i z a b l e e g g s as s o o n a s 7 h a f t e r i n j e c t i o n of g o n a d o t r o p i c h o r m o n e s . T h e p r e s e n t i n v e s t i g a t i o n c a l l s s p e c i a l a t t e n t i o n t o t h e c o n t r i b u t i o n of the oviduct during maturation. The criterion to judge t o t a l m a t u r a t i o n w a s f e r t i l i z a b i l i t y of t h e e g g a n d n o r m a l development. Material and methods. P a r t of t h e o v a r y of Xenopus laevis f e m a l e s ( w h i c h c o n t a i n e d o o c y t e s w i t h o u t a n y p i g m e n t a t i o n o n t h e i r v e g e t a t i v e pole) w a s d i s s e c t e d a s d e s c r i b e d b y SCHORDERET-SLATKINE 4. T h e o o c y t e s w e r e d e f o l l i c u l a t e d w i t h w a t c h m a k e r ' s f o r c e p s in D e B o e r ' s s o l u t i o n 5 s t o r e d in t h e s a m e s o l u t i o n s u p p l e m e n t e d with 10% fetal calf serum. Progesterone was added as d e s c r i b e d b y MERRIAM 6. A f t e r 4 h m o s t of t h e o o c y t e s

Xenopus laevis

h a d s t a r t e d m a t u r a t i o n a s j u d g e d b y t h e p r e s e n c e of t h e m a t u r a t i o n s p o t . F o r t h e e x p e r i m e n t s s u m m a r i z e d in T a b l e I, t h e o o c y t e s w e r e d i v i d e d i n t o 2 e q u a l l o t s : o n e was allowed to stay for 3 h in the maturation medium described above, the other lot was transferred into the b o d y c a v i t y of a f o s t e r f e m a l e 7 i m m o b i l i z e d w i t h M S S 222. Xenopus laevis petersi f e m a l e s w e r e u s e d a s f o s t e r females because they shed eggs heavily pigmented on the v e g e t a t i v e pole. T h e f o s t e r f e m a l e s w e r e i n j e c t e d w i t h 450 G o n a d o t r o p h i n ( O r g a n o n , H o l l a n d ) 10 h b e f o r e u s e . They were squeezed periodically during 3 h after having been injected with donor oocytes. The latter appeared, i n t e r s p e r s e d a m o n g r e g u l a r e g g s of t h e h o s t , d u r i n g t h i s period. The oocytes were then exposed to tap water w h i c h p r o v o k e s in f u l l y m a t u r e d ( u n f e r t i l i z e d or f e r t i l i z e d )

1 y. MASUI and C. L. MARKERT,J. exp. Zool. 177, 129 (1971). 2 y . MASUL j . exp. Zool. 166, 365 {1967). 3 F. HANCOCQ, A. DE SCtfUTTER, 1{. HUBERT and J. BRACHET, Differ. 2, 75 (19741. 4 S. SCHORDERET-SLATKINE,Cell Differ. 1, 179 (1972). .5 D. P. YVOLFand J. L. HEDRICK, Devl Biol. 25, 348 {1971). R. W. MERRIAM, Expl Cell Res. 68, 7.5 {1971). 7 g. H. gAVIN, J. Embryol. exp. Morph. 12, 457 (1964).

Table 1. Ooeytes matured with progesterone {criterion: presence of the maturation spot): Comparison between oocytes which were and were not in contact with an oviduct of a toster female Experiment No.

Oocytes transferred Oocytes shed Controls (ooeytes incubated in medimn a or b)

Totals

Cortex contraction (%)

Abortive cleavage (%)

268 150

531 296

(100%)

49.8

53.3

263 b

536

(100 %)

1

2

3

4

79 45

34 9

150 92

79 9

34 9

160 ~

0.6 ~

For each experiment the oocytes transferred to the foster female, and the oocytes incubated in Inedium a or b, were taken from the salne donor female. ~De Boer's solution + progesterone + fetal calf serum, bSerum prepared from 3 ovulating females. ,The ooeytes showed cortex contraction only at tile time where the shed ooeytes showed abortive cleavage {80-110 min instead of 5-15 min after contact with tap water). The controls (no contact with the oviduct) reach the stage attained within 3 h by shed, in vitro matured oocytes only after 20-24 h (see also ref. 3).

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Specialia

Table IL Developmental capacity of oocytes (matured in vitro with progesterone) after shedding by foster females and 'artificial' fertilization

Oocytes shed Activation reaction Blastula stage Hatching tadpoles Feeding tadpoles Metamorphosed frogs

Totals

%

296 158 30 20 13 8

100 53.3 10.8 6.7 4.4 2.7

eggs, s p o n t a n e o u s a c t i v a t i o n , i.e. c o r t e x c o n t r a c t i o n a n d cleavage (in unfertilized eggs cleavage is abortive). The t w o t y p e s of Xenopus laevis laevis oocytes, t h o s e w h i c h had passed through an oviduct within 3 h and those w h i c h h a d b e e n k e p t in m a t u r a t i o n m e d i u m for 3 h, were so t r e a t e d a n d t h e i r b e h a v i o u r c o m p a r e d . O o c y t e s w h i c h h a d p a s s e d t h e o v i d u c t were fertilized following WOLF e t a l ) . In a d d i t i o n t o t h e parallel s t o r a g e of o o c y t e s in m a t u r a t i o n m e d i u m (a), a second c o n t r o l w a s d o n e as follows: 3 o v u l a t i n g females were sacrificed, a n d their serum prepared..Oocytes matured with progesterone were t r a n s f e r r e d into t h i s s e r u m ( m e d i u m b) a f t e r t h e m a t u r a t i o n s p o t h a d a p p e a r e d , a n d i n c u b a t e d for 3 h. AlI e x p e r i m e n t s were realized a t 21-22~ ( t e m p e r a t u r e of t h e solutions used). Results. Tables I a n d II.

EXPERIENTIA 31/11

Discussion. T h e process of m a t u r a t i o n n o t o n l y induces c o m p l e t i o n of meiosis a n d t h e t r a n s f o r m a t i o n f r o m t h e c y t o p l a s m i c s t a t e of a n o o c y t e ( R N A synthesis) t o t h e c y t o p l a s m i c p r o g r a m of an egg ( D N A synthesis), b u t also acts on t h e o o c y t e s m e m b r a n e (for review seeS). The p r e s e n t results suggest t h a t c o r t e x m a t u r a t i o n in vivo d e p e n d s on c o n t a c t of t h e o o c y t e w i t h t h e oviduct, since t h e c o n t r o l s did n o t c o n t a i n fully m a t u r e eggs. This o b s e r v a t i o n is c o r r o b o r a t e d b y t h e p o s s i b i l i t y of raising t h e low m e t a b o l i c a c t i v i t y of a b o d y c a v i t y egg to t h e h i g h level of a shed egg b y t r e a t i n g t h e b o d y c a v i t y eggs w i t h e x t r a c t s from t h e u p p e r m o s t p a r t of t h e o v i d u c t s . Summary. C o n t a c t of p r o g e s t e r o n e m a t u r e d o o c y t e s of Xenopus laevis w i t h t h e o v i d u c t r e d u c e s t h e t i m e n e c e s s a r y to a t t a i n cleavage c a p a c i t y from 24 h to 3 h. Full m a t u r i t y has b e e n d e m o n s t r a t e d b y n o r m a l developm e n t of t h e m a t u r e d eggs a f t e r fertilization.

R. BRUN Station de Zoologie Expdrimentale, University o/Geneva, 154, route de Malagnou, CHL122d Chdne-Bougeries, Gen~ve (Switzerland), 30 J u l y 1975.

8 A. W. SCHUETZ, Biol. Reprod. 10, 150 (1974). 9 A. H. LEGNAME, H. SALOMON DE LEGNAME, S. S. SANCHEZ, A. N. SANCIIEZ,RIERA and S. N. FERNANDEZ, Devl Biol. 29, 283

(1972).

N u c l e a r E n v e l o p e C h a n g e s Related to Cell A c t i v a t i o n in H e l i a n t h u s t u b e r o s u s L. N u c l e a r pore c o m p l e x e s are sites of c o m m u n i c a t i o n b e t w e e n nucleus a n d c y t o p l a s m t h r o u g h w h i c h m a c r o molecular e x c h a n g e is e f f e c t e d l . A l t h o u g h t h e s t r u c t u r a l o r g a n i z a t i o n of t h e p o r e c o m p l e x a p p e a r s to be similar in m o s t cell t y p e s , t h e r e is c o n s i d e r a b l e v a r i a t i o n in t h e n u m b e r of p o r e s in t h e nuclei of d i f f e r e n t species a n d tissues x. F u r t h e r m o r e p o r e f r e q u e n c y and pore n u m b e r p e r nucleus can increase in a given cell t y p e w i t h a n increase in its m e t a b o l i c a c t i v i t y 2-5. The a c t i v a t i o n of p l a n t storage tissues is a c c o m p a n i e d b y m a n y c h a n g e s 6 including a rise in R N A s y n t h e s i s , a n increase in nucleolar size, p o l y s o m e f o r m a t i o n a n d p r o t e i n synthesis. A t h i n - s e c t i o n s t u d y of Daucus carota r o o t cells also r e v e a l e d a d o u b l i n g of nuclear pore f r e q u e n c y T, s u g g e s t i n g t h a t n u c l e a r p o r e n u m b e r m i g h t be an im-

p o r t a n t controlling f a c t o r in t h e process of cell a c t i v a t i o n . I n t h i s p r e l i m i n a r y r e p o r t , we describe t h e results of an i n v e s t i g a t i o n i n t o . n u c l e a r e n v e l o p e u l t r a s t r u c t u r e of d o r m a n t a n d a c t i v a t e d Helianthus tuberosus t u b e r cells, in w h i c h t h e f r e e z e - f r a c t u r e t e c h n i q u e was e m p l o y e d to p e r m i t a m o r e a c c u r a t e analysis of n u c l e a r pore f r e q u e n cies. Materials and method. E x p l a n t s of H. tuberosus t u b e r tissue were p r e p a r e d a n d i n c u b a t e d , as d e s c r i b e d previously s. Nuclei were e x t r a c t e d f r o m d o r m a n t tissue, and tissue i n c u b a t e d for 24 h ( t e r m e d a c t i v a t e d tissue in this report), fixed in 0.1 M c a c o d y l a t e b u f f e r e d 2.5% glutarald e h y d e , g l y c e r i n a t e d , frozen in m e l t i n g freon 12, and f r e e z e - f r a c t u r e d using t h e BULLIVANT-AMEs m e t h o d s. Cleaned replicas were e x a m i n e d w i t h a n A E I E M 6 B

Frequency of nuclear pores in nuclei from Helianthus tuberosus tuber cells

1 W. W. FRANKEand U. SCHEER,in The Cell Nucleus (Ed. H. BUSCH; Academic Press, New York 1974), voL 1, p. 219. G. G. MAUL, J. V~7.PRICE and M. W. LIEBERMAN, J. Cell Biol. 57, 405 (1971). a R. E. SCOTT, R. L. CARTER and W. R. KIDWELL, Nature New Biol. 233, 219 (1971).

Nuclear pores ([xln-e ~- SD)

Sample size

4 G. G. MAUL, H. M. MAUL, J. 2. SCOGNA,M. W. LIEBERMAN, G, S;

Dormant ceils (0 h incubation) Activated cells (24 h incubation)

11.3 i 1.8 11.9 • 1.9

363 tim2 of nuclear envelope from 24 iluclei 191 ~xm'2of nuclear envelope from 34 nuclei ~

9The smaller total area of nuclei sampled in activated cells results from the reduced frequency of large areas of face fractures of the convoluted nuclear envelopes.

STEIN, B. Y. L. Hsu and T. W. BORUN, J. Cell Biol. 55, 433 (1972). 5 j. N. A. LOTT, P. L. LARSONand C. M. WHITTINGTON,Can. J. Bot. 50, 1785 (1972). 6 2. G. JORDAN and J. M. CHAPMAN, J. exp. Bot. 22, 627 (1971). 7 E. G. JORDAN and J. IV[.CHAP~IAN,J. exp. Bot. 24, 197 (1973). S S. BULLIVANT, in Advanced Techniques in Biological Electron Microscopy (Ed. J. K. KOEHLER; Springer-Verlag, Heidelberg 1973), p. 67.