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Nucleolar and chromatin cycles in abies: microsporogenesis in Abies grandis with particular reference… Rattenbury, John Alban 1945

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ITUCISOIAR AMD CHROMATID CYOIES H  ABIES '  M i c r o s p o r o g e n e s i s in-- A b i e s g r a n d i s w i t h p a r t i c u l a r r e f e r e n c e t o the s t r u c t u r e and development of the  n u c l e o l u s and. the t r a n s f e r o f n u c l e o l a r  materia,! t o the chromosomes.  John A l b a n R a t t e n b u r y .  Submitted i n p a r t i a l f u l f i l m e n t o f c r e d i t towards a major i n B i o l o g y and Botany f o r the degree o f Master of A r t s .  The U n i v e r s i t y o f B r i t i s h Columbia October, 1945.  TABLE Off OOHTMIS I. Introduction.  .  .page i  1. O r i g i n o f Problem.  i  2. Acknowledgements...  i  3. Statement o f Problem*.  i  4. D i s c u s s i o n o f Problem*  i i  I I . M a t e r i a l s and Methods.......... ..............» s  1  1. Source o f . M a t e r i a l s .  1  2. Times o f C o l l e c t i o n  1  5. Method o f P r e s e r v a t i o n .  1  4. Embedding, S e c t i o n i n g and S t a i n i n g Procedures  1  5. Drawings and Photographs®....................  2  6. Measurements.................................  2  I I I . D e s c r i p t i o n of Microsporogenesis. T¥» N u c l e o l a r Development and Morphology...  5 .««  4  V. i-Tiicieolar-Ohromosome R e l a t i o n s  6  ¥1. S i z e R e l a t i o n s h i p s o f O e l l P a r t s . . . . . . . . . . . , . . •  7  V I I . Conclusion.  10  V I I I . References.  Iv  IX. P l a t e s .  ...  v  i.  gUOLEOIAR ASP' OHBOM&CT CYCLES IN ABIES  I. Introduction 1. O r i g i n of Problem The problem o r i g i n a t e d as p a r t o f a study of the g e n e t i c s of economic t r e e s being c a r r i e d out under the d i r e c t i o n of Dr. A.H.Hutchinson of the Department of B i o l o g y and Botany at the U n i v e r s i t y of B r i t i s h Columbia. The extent of, the present  r e s e a r c h covers one phase,, i n p a r t ,  o f the r e p r o d u c t i v e c y c l e s i n A b i e s . Other phases, have been p r e v i o u s l y s t u d i e d and w i l l - b e s t u d i e d at a l a t e r  date.  2. Acknowledgements I should l i k e to'acknowledge the a s s i s t a n c e , guidance, and. encouragement r e c e i v e d from Dr. H u t c h i n s o n which have made t h i s r e s e a r c h p o s s i b l e . .  /  I should l i k e to acknowledge the s t a t i s t i c a l a s s i s t a n c e r e c e i v e d from Dr. V.O.Brink o f the Department-of Agronomy i n . this University. I should l i k e to acknowledge the f i n a n c i a l a s s i s t a n c e , r e c e i v e d from the U n i v e r s i t y of B r i t i s h Columbia under a grant for' 'Genetics of Economic Trees'*' S« Statement of Problem E x c e l l e n t p r e p a r a t i o n s were o b t a i n e d of m a t e r i a l of A b i e s g r a n d i s L i n d l e y undergoing microsporogenesis.  These  p r e p a r a t i o n s were s t a i n e d very s p e c i f i c a l l y to show the  ii. presence of h o t h euchromatln and n u c l e o l a r m a t e r i a l , (see under methods). The purpose o f t h i s paper i s to i n d i c a t e the !  extent of these m a t e r i a l s during the v a r i o u s stages of r e d u c t i o n - d i v i s i o n and to. attempt, to harmonize the f i n d i n g s w i t h current t h e o r i e s of chromosome and n u c l e o l a r chemistry  and  p h y s i c s . With t h i s i n view,...the paper has "been d i v i d e d i n t o four s e c t i o n s d e a l i n g w i t h : ( l ) the extent and c h a r a c t e r  of  the stages of r e d u c t i o n - d i v i s i o n ; (2) the morphology and development of the n u c l e o l u s ; (3) the r e l a t i o n s h i p "between the n u c l e o l u s and the chromosomes; (4) the s i z e r e l a t i o n s h i p s of the c e l l p a r t s during the stages before the metaphase* 4. D i s c u s s i o n of Problem The  chemistry  of the nucleus, i s f a r from being  fully  understood. Most workers agree t h a t the p r i n c i p a l components of the n u c l e u s are p r o t e i n s of the protamine and h i s t o n e type p l u s n u c l e i c a c i d s . Stedman and Stedman, 1943,  however p r e -  sent c o n s i d e r a b l e evidence f o r the presence o f another form 1  of p r o t e i n , which they c a l l .'chromosomin , c o n s t i t u t i n g from 50-72^ of the n u c l e a r c o n t e n t s . These workers have suggested, with experimental  v e r i f i c a t i o n , t h a t t h e chromosomin i s  l o c a t e d i n the chromatin and assume t h a t i t p l a y s an import-. . ant p a r t i n the t r a n s m i s s i o n of h e r e d i t a r y  characters.  The n u c l e i c a c i d s have been s t u d i e d i n some d e t a i l , c h i e f l y by means of d i f f e r e n t i a l s t a i n s , u l t r a - v i o l e t absorpt i o n s p e c t r a and enzymatic d e s t r u c t i o n . The b a s i c formula f o r the n u c l e i c a c i d s * (see reviews by ^Painter ,'• 1943;  Gates, 1942),  iii. i s as f o l l o w s :  Phosphoric Phosphoric Phosphoric Phosphoric  acid acid acid acid  -  ribose rioose ribose ribose  sugar sugar sugar sugar  -  adenine (purine) cytosine (pyrimidine) guanine (purine.) u r a c i l or methyl u r a c i l (pyrimidine)  The chemical c o n d i t i o n o f the sugar d i v i d e s the whole group o f n u c l e i c a c i d s i n t o two'main sub-groups: the desoxy* ribose nucleic-acids  which c o n s t i t u t e the s t a i n a b l e p o r t i o n  of the euchromatin and are known a l s o as thymonueleie a s i d s * and the r i b o s e n u c l e i c a c i d s found b o t h i n the cytoplasm and i n the c e n t r a l o x y p h i l p o r t i o n o f the nucleolus'. This group has been c a l l e d the p h y t o n u c l e i e  latter  acids.  . S c h u l t z , 1941, has g i v e n a p i c t u r e o f the c h e m i c a l straicture o f both n u c l e o l u s and chromosomes which, sums up the present-day knowledge as w e l l as possible... Ag cor ding t o him the n u c l e o l u s  c o n s i s t s of a histone-type  o f p r o t e i n t o which  are l i n k e d the r i b o s e n u c l e i c a c i d s t y p i c a l o f the s p e c i e s . The chromosome.consists o f a*protamine thread w i t h n u c l e o p r o t e i n , (combinations o f n u c l e i c a c i d s and p r o t e i n s ) , a t i n t e r v a l s , the whole being sheathed by a more complex p r o t e i n s i m i l a r t o t h a t found i n the n u c l e o l u s . The p r o t e i n sheath i s . the. g r e e n - s t a i n i n g  histone-type  m a t e r i a l o f the l i g h t -  green procedure:. F i i r t h e r d i s c u s s i o n o f the n a t u r e o f the m a t e r i a l s o f the n u c l e u s w i l l be p r e s e n t e d as t h e data are a n a l y z e d . .  J- «  I I . Materials- and Methods 1. Source o f M a t e r i a l s The m a t e r i a l s used f o r t h i s r e s e a r c h were  obtained  e n t i r e l y from a s i n g l e t r e e i n t h e B o t a n i c a l Gardens o f the u n i v e r s i t y o f B r i t i s h Columbia. 2. Times o f C o l l e c t i o n - , The The  c o l l e c t i o n s were made i n two seasons, 1944 and 1945.  1944 c o l l e c t i o n was "begun on February 11 and continued  u n t i l March 27,.''-reduction-division 'being completed about March 20. The 1945 c o l l e c t i o n began on March .10 and continued u n t i l March 30,. r e d u c t i o n - d i v i s i o n being complete about March :22. C o l l e c t i o n s were made a t v a r y i n g i n t e r v a l s - o f 24 hours t o s e v e r a l d.ays. 3. Method o f P r e s e r v a t i o n A l l m a t e r i a l was k i l l e d and f i x e d i n a m i x t u r e o f 2 p a r t s of absolute  a l c o h o l t o 1 part of g l a c i a l a c e t i c a c i d . A f t e r  thorough washing i n 7.0.% a l c o h o l , t h e m a t e r i a l was s t o r e d i n 70/6 a l c o h o l u n t i l embedded. 4. Embedding, S e c t i o n i n g and S t a i n i n g Procedures The. .material was r u n up from 70% a l c o h o l and embedded . f o l l o w i n g the schedule below: ^>D/Q  Q<iCOl3.0l# « e • • • 0 • • * » •  e • a • • • « • • « • • • # 41: ill? S o  'absolute alcohol......... 4: h r s . a b s o l u t e alcohol........-'...............^4 hrs. b()% absolute a l c o h o l + 20% benzene.....2 h r s . 65% a b s o l u t e a l c o h o l 4 35,70 benzene..... 2 h r s . 50% a b s o l u t e a l e o h o i * 507? benzene.... .2 h r s . 35% a b s o l u t e a l c o h o l * 65% benzene -2 h r s . 20/6 a b s o l u t e a l c o h o l + 8 0 % benzene...... 2 h r s . 100% benzene. 2 hrs. 100% benzene.4- wax................overnight.  -;  2. The wax c o n s i s t e d o f standard commercial parawax p l u s about. Ifo o f low m e l t i n g - p o i n t wax, (40-42°o) . The embedding was completed as q u i c k l y as p o s s i b l e , (4-6 h r s . ) t o prevent undue s h r i n k a g e . S e c t i o n i n g was done by means o f a r o t a r y microtome u s i n g a r a z o r - b l a d e . Thickness--o-f s e c t i o n s was 12-15 microns."' The m a t e r i a l was s t a i n e d w i t h t h e S'eulgen's and L i g h t Green procedure o f Semmens and B h a d u r i , 1941. 5. Drawing s and. Pho to graphs The m a t e r i a l was examined by means o f a S e i b e r t b i n o c u l a r compound microscope u s i n g a n o i l - i m m e r s i o n o b j e c t i v e and o i l between s l i d e and condenser. Using t h e o o c u l a r s p r o v i d e d w i t h the microscope  i t was p o s s i b l e t o o b t a i n m a g n i f i c a t i o n s o f  700 and 1500 diameters-. Drawings were made v/ith a camera-lucida using, the o c u l a r s p r o v i d e d or a s p e c i a l o c u l a r o f 50 diameters. 'Photographs were taken w i t h a h o r i z o n t a l camera using^ a t u n g s t e n - a r c lamp t o p r o v i d e u l t r a - v i o l e t wavelengths: i s o l a t e d hy means o f . a Corning #5970 f i l t e r . C o r r e c t i o n s were made i n f o c u s s i n g these s h o r t e r wave-lengths. Eastman P r o c e s s p l a t e s were used f o r the photographs.  These were developed i n a D-76 j  developer. 6. Measurements Diameters o f c e l l s * n u c l e i and n u c l e o l i were measured by means o f a camera-lucida and d i v i d e r s . Where these s t r u c t u r e s were not p e r f e c t l y s p h e r i c a l * an average o f t h e major and minor: axes was used. Volumes were determined by the f o r m u l a f o r t h e volume o f a sphere.  go  I I I . D e s c r i p t i o n of Microsporogenesis The  e a r l y c o l l e c t i o n s may he presumed t o he i n the i n t e r -  phase or r e s t i n g stage. There i s no i n d i c a t i o n of a sudden appearance o f clromonemata, hut r a t h e r a g r a d u a l i n c r e a s e i n t h i c k n e s s and c o n t i n u i t y * ( a p p a r e n t ) , o f t h e t h r e a d s . See P l a t e I , f i g s . 1 & 2. IJeptotene was thought t o "begin about February  26 s i n c e the stages o f prophase proceeded f a i r l y  r a p i d l y from t h i s time on. P I . I , f i g s . 3 & 4. P a i r i n g o f homologues and s y n i z e s i s , ( z y g o t e n e ) , began about the end o f February, through-the  (1944 c o l l e c t i o n ) ,;. and continued  e a r l y p a r t o f M a r c h - about two weeks - when the  f i r s t t h i c k e n i n g o f pachytene was o b s e r v a b l e . P I . I I , f i g s . 5 & 6. A t t h i s stage, r e l a t i o n a l c o i l i n g o f the homologous chromosomes was d e t e c t a b l e and t h i s i s i n d i c a t e d i n f i g . 6 . By the 17th o f March, the d i p l o t e n e chromosomes, c o u l d be r e c o g n i z e d w i t h d i s t i n c t evidence o f four chromatids. P I . I I fig.  7; P I . I l l , f i g . 11 & 12. R e l a t i o n a l c o i l i n g was s t i l l  q u i t e d e f i n i t e . • See P i . I I , f i g . - 8 ; P I . I l l , f i g . 9. By March 20, t h e f i r s t , ( m e i o t i c ) , d i v i s i o n was complete i n some o f the c e l l s , w h i l e o t h e r s were s t i l l a t l a t e d i a k i n e s i S i . P l > IV, f i g . . 14 shows t h e normal miiphase p l a t e seen from p o l a r . view. The. extremely t i g h t r e l a t i o n a l c o i l s were r e a d i l y seen i n the p r e p a r a t i o n s . C o i l i n g appeared c o n s i s t e n t l y t o be i n one d i r e c t i o n , I.e. a c o u n t e r ~ c l o c k w i s e s p i r a l proceeding from t h e p r o x i m a l t o the d i s t a l p o r t i o n . T h i s i s i n a c c o r d w i t h the o b s e r v a t i o n s , recorded the drawings of P I . I I , f i g . 6 and P I . I l l , f i g *  9,  4. The second, { e q u a t i o n a l ) , d i v i s i o n took p l a c e two  days  l a t e r a f t e r a very short i n t e r p h a s e during which the n u c l e o l u s d i d not appear to he comple&tely r e o r g a n i z e d , P I . IY, f i g s . 15, 16, 17 show the f i r s t ; d i v i s i o n t e l o p h a s e , the second d i v i s i o n anaphase, and the second d i v i s i o n telophase  with  the f o r m a t i o n of the q u a r t e t , (spore t e t r a d ) . r e s p e c t i v e l y . The much d i v i d e d s t a t e of the n u c l e o l u s i s observable  i n both  telophases p r e v i o u s to i t s r e e o n s t i t u t i o n a f t e r i t disappears d u r i n g d i p l o t e n e . See P I . I l l ,  f i g s . 12 & 13.  The l a r g e number of chromosqmes, (12 p a i r s ) , i n A b i e s renders i t a r a t h e r poor s u b j e c t f o r some phases of chromosome study, and no attempt has been made as yet to analyse the m a t e r i a l , f o r abnormal behaviour, or to i d e n t i f y the :  chromosomes by t h e i r shape.and s i z e i n t h i s paper. Some evidence of major c o i l s was v i s i b l e :at d i p l o t e n e ; PI. I l l ,  f i g s . 12-..& 13. D u r i n g l a t e r stages, the nattire o f / f  t h e . p r e p a r a t i o n s was  such as to obscure t h i s phenomenon. The  p r e p a r a t i o n s d i d not show the,presence of the chromosome. :sheath a f t e r d i p l o t e n e .  '  »  t  IY. N u c l e o l a r Development and Morphology:. The  s p e c i f i c i t y of l i g h t green and some other s t a i n s f o r  the h i s t o n e component of the n u c l e o l u s has rendered s t r u c t u r e an i n t e r e s t i n g one f o r the The  e a r l i e s t c o l l e c t i o n , (February  this  cytologist. 11, 1944), shows some  d i s p a r i t y i n size- and numbers of n u c l e o l i I n the d i f f e r e n t c e l l s . Some c e l l s appear to have only, one l a r g e n u c l e o l u s while- o t h e r s have as many as f o u r s m a l l e r ones. The  total  •volume, although."by .no means constant,  i s about the same i n  a l l c e l l s as i s i n d i c a t e d by the r e l a t i v e l y s m a l l amount of standard e r r o r o f the mean o f t e n o b s e r v a t i o n s . range i s s c a r c e l y g r e a t e r , t h a n a s i n g l e n u c l e o l u s was  I n f a c t the  t h a t about the mean when only  present i n a l l c e l l s , (March IS,  16  c o l l e c t i o n s ) - . T h i s i s p l o t t e d g r a p h i c a l l y i n PI.IX. The  s t r u c t u r e of the n u c l e o l u s  a l s o v a r i a b l e . The  i n these e a r l y stages i s  o u t e r , ( b a s i p h i l ) , l a y e r , (see  :  McSill,1906  Guenther, 1903;' Cowdry and K i t c h e n , 1930;  Lucas, 1940;  1942), i s c o n s i s t e n t l y s t a i n e d green* The  inner p o r t i o n ,  C o x y p h i l ) , c o n s i s t s o f one or more g l o b u l a r , g r a n u l a r  Gates,  struc-  t u r e s , e i t h e r c o l o r l e s s or pale'pirnr. There seems to be considerable  evidence from the p r e p a r a t i o n s  t h a t the  smaller  c e n t r a l p o r t i o n s e v e n t u a l l y fuse t o form a s i n g l e l a r g e inner sphere, (the r i b o s e n u c l e i c a c i d component - S c h u l t z , 1941). This f u s i o n i s r e p o r t e d by Gates, 1942;  Z i r f e l e , 1928,  1930.  P I . I , f i g s . 1, 2 & 4 show v a r i o u s stages i n the f u s i o n of , the o x y p h i l p o r t i o n s of the n u c l e o l u s . I t i s thought t h a t the h a s i p h i l p o r t i o n i s more l i q u i d and t h a t the f u s i o n i s instantaneous.  P I . I , f i g . 3 i l l u s t r a t e s a c o n d i t i o n found  i n many of the''cells--at t h i s stage,  (February 26). The  two  p a r a l l e l l i n e s w i t h i n the c e n t r a l , o x y p h i l p a r t of the nucleolus  suggest the presence of p o w e r f u l e l e c t r i c a l charges  f o r c i n g some p a r t of the contents of the i n n e r sphere to oppo s i t e s i d e s . The p o r t i o n between the l i n e s appears very c l e a r i n the s t a i n e d m a t e r i a l . PI.V  shows photographs of n u c l e o l i  at v a r i o u s stages of development.  6. V. Nucleolar-Chromosome  fielations  I t i s now g e n e r a l l y accepted t h a t the contents, i n p a r t , of the n u c l e o l u s form the sheath o f t h e chromosomes when the former disappears a t the end o f d i p l o t e n e . { S c h u l t z , 1941; Gates, 1942). Although c o n s i d e r a b l e work has been done on the breaking-up o f t h e sheath -in telophase w i t h subsequent agg r e g a t i o n of the p a r t i c l e s to reform the n u c l e o l u s , { Nebel, 1939; Gates, 1942), the forming o f t h e sheath by d i s i n t e g r a t i o n o f the n u c l e o l u s seems t o have r e c e i v e d l e s s a t t e n t i o n except by e a r l i e r workers, ( Yari Gamp, 1924; Blackman, 1905), the l a t t e r m i s i n t e r p r e t t i n g the process to i n d i c a t e the f l o w of chromatin t o form the chromosomes. Z i r i l e , 1928, 1930; and Schrader, 1940 concluded t h a t the n u c l e o l u s c o n t a i n s no euchre mat i n . P l a t e I I I , f i g s . 12 & 13, taken from d i p l o t e n e , show the (supposed), t r a n s f e r o f n u c l e o l a r m a t e r i a l to the chromo-  J  nemata. I n the p r e p a r a t i o n s , t h e r e c o u l d be d e t e c t e d no l i n e of demarkation between the g r e e n - s t a i n i n g m a t e r i a l o f the n u c l e o l u s and t h a t of the chromosome. I n the f i g u r e s , the p o r t i o n s s t a i n e d green are i n d i c a t e d by l i g h t areas and the euchromatin by dark a r e a s . The n o n - s t a i n i n g , h e t e r o c h r o m a t i c r e g i o n s , ( S c h u l t z , , 1941), are obscured by the green-coloured sheath. A c c o r d i n g t o t h i s a u t h o r , the sheath may be s t r i p p e d o f f by enzymatic d i g e s t i o n u s i n g r i b o n u c l e a s e . F u r t h e r d i g e s t i o n by the same enzyme r e s u l t s i n the removal o f the n u c l e o p r o t e i n s l e a v i n g only the protamine core. 21. I l l ,  f i g s , 9-11  show p o s s i b l e SAT chromosomes but no t r a b a n t s were seen.  7. V I . S i z e R e l a t i o n s h i p s of C e l l P a r t s As an i n d i c a t i o n of the a c t i v i t y of the v a r i o u s p a r t s of the c e l l d u r i n g the i n t e r p h a s e and prophase stages, a number of measurements of c e l l s , n u c l e i and n u c l e o l i were made f o r the. p e r i o d o f February I I to March 17, 1944.  The  restilts  seem to be of- c o n s i d e r a b l e s i n t e r e s t as a means o f approaching the problem of c e l l u l a r metabolism. V a r i o u s workers,  (Gates,  1942; "Schultz, 1941), have i n d i c a t e d t h a t the i n t e r p h a s e far  cell,,  from b e i n g at a " r e s t i n g stage', i s an extremely a c t i v e  system. Schultz', 1941, p o i n t s out t h a t i n a c t i v e l y d i v i d i n g c e l l s the r i b o s e n u c l e i c a e i d c o n c e n t r a t i o n i s r e l a t i v e l y , high. An examination  of the measurements presented g r a p h i c a l l y  i n t h i s paper, ( P l a t e s VI - I X ) , w i l l show t h a t there are very a p p r e c i a b l e changes i n s i z e of the c e l l c o n s t i t u e n t s at t h i s time. Up  to the b e g i n n i n g of the m e i o t i c prophase  -  t h e r e appears to be a d i s t i n c t r e d u c t i o n i n the amount of cytoplasm  i n the c e l l . At the same time t h e r e i s a c o r r e s -  ponding i n c r e a s e i n the nuclear, m a t e r i a l . T h i s suggests t h a t there i s a t r a n s f e r of. m a t e r i a l from cytoplasm t o n u c l e u s . I t was  thought at one time t h a t n u c l e i c a c i d s migrate from the  cytoplasm  to the n u c l e u s , but a c c o r d i n g t o Gates, 1942,  has i t s own  each  separate n u c l e i c a c i d c y c l e . However, a c c o r d i n g  to Caspersson, (see S c h u l t z , 1 9 4 1 ) . p r o t e i n proceeds hy way  synthesis  o f the h i s t o n e s i n the n u c l e o l u s , which  d i f f u s e through the n u c l e a r membrane to form i n the  cytoplasm  r i h o s e n u c l e i c a c i d s which are concerned w i t h the s y n t h e s i s  .  8. of cytoplasmic p r o t e i n s " . T h i s would i n d i c a t e t h a t the n u c l e i c a c i d c y c l e s a r e not independent. S c h u l t z , 1941, presents- evidence t h a t the "....gene....turns i t s s y n t h e s i s ....to t h e i n c r e a s e o f t h e n u c l e i c a c i d component....at the prophase o f m i t o s i s Cor m e i o s i s } ; or c o n v e r s e l y during the i n t e r p h a s e , the p r o t e i n -component i s s y n t h e s i z e d . "  The data  shown f o r the p e r i o d , February 11-26, P l a t e 71, are taken to i n d i c a t e the t r a n s f e r o f m a t e r i a l s from t h e cytoplasm to the nucleus p r e p a r a t o r y to s y n t h e s i s of n u c l e i c a c i d by the genes d u r i n g prophase. I t w i l l be noted t h a t t h e r e i s an apparent decrease i n the n u c l e o l a r volume a t t h i s time which may. i n some way be concerned w i t h the f u s i o n o f the n u c l e o l i . However t h i s may be accounted f o r i n the d i u r n a l v a r i a t i o n which i n some forms may be as much as  BOfo^  (  see Gates, 1942).  From l e p t o t e n e t o zygotene t h e r e i s a marked Increase i n the s i z e o f a l l p a r t s , t h i s i n c r e a s e t a k i n g p l a c e a t about the same r a t e i n a l l three as i n d i c a t e d by t h e slopes o f the l i n e s . The sudden i n c r e a s e seems t o be c o r r e l a t e d w i t h the a b s o r p t i o n o f the i n t e r c e l l u l a r n u t r i t i v e m a t e r i a l and possi b l y w i t h the r e c e i v i n g o f c o n t r i b u t i o n s o f t a p e t a l m a t e r i a l . A f t e r zygotene there i s a sudden decrease i n . s i z e o f a i l p a r t s . T h i s seems t o be i n i t i a t e d a t zygotene by the phenomenon o f s y n i z e s i s when t h e 'spireme" or chromatin  network  c o l l a p s e s t o form t h e bouquet stage. I t seems l i k e l y t h a t the r a p i d s y n t h e s i s o f n u c l e i c a c i d a t t h i s stage i s accompanied by the l o s s o f c o n s i d e r a b l e m e t a b o l i c waste? i n t h e form o f water and d i s s o l v e d s a l t s . T h i s too could account f o r the  9. c o l l a p s e of the spireme as the medium became l e s s v i s c o u s and p o s s i b l e changes i n e l e c t r i c a l charge were effected.The change i s .greatest i n the n u c l e o l u s which may  indicate that t  t h i s s t r u c t u r e I s i n s t r u m e n t a l i n the s y n t h e s i s of  chromatin.  From pachytene to d i p l o t e n e there i s a s l i g h t i n c r e a s e i n a l l . p a r t s which may not he  significant.  *A11 measurements were made i n groups of t e n each t o t a l l i n g one hundred and f i f t y measurements. The graphs i n P l a t e s T i l - i s i n d i c a t e ' theedegree of r e l i a b i l i t y o f the means obt a i n e d . The means are r e p r e s e n t e d by h o r i z o n t a l l i n e s and c i r c l e s . The  s t a n d a r d e r r o r s o f the means are r e p r e s e n t e d by  the l e n g t h s o f the v e r t i c a l r e c t a n g l e s . The ranges of measurements i n the samples are r e p r e s e n t e d by the  vertical  s i n g l e l i n e s . I n P I . VII, i t i s c l e a r t h a t the means are c o n s e c u t i v e l y d i f f e r e n t . e x c e p t f o r the l a s t two, CMareh 13-17). In other words, the r e c t a n g l e s do not o v e r l a p except i n the l a s t two. I n the case of the nucleoplasm, change i n the volume from February  P I . V I I I , the  l l - 2 b cannot be  considered  s t a t i s t i c a l l y v a l i d because o f the o v e r l a p p i n g o f the standard  e r r o r o f the means. The  i n f e r e n c e s p r e v i o u s l y drawn from  t h i s data must be c o n s i d e r e d t e n t a t i v e u n t i l f u r t h e r  obser-  v a t i o n s are made. The  readings.  same i s t r u e f o r the f i n a l two  The n u c l e o l a r changes shown i n P I . IX may  a l l be  considered  v a l i d except the f i r s t change, (February 11-26). E r r o r s o f measurement, o f e s t i m a t i o n o f volume, and v a r i a t i o n s due to other f a c t o r s such as d i u r n a l v a r i a t i o n have not been taken i n t o account; i n these d a t a .  10. VII.  Conclusion  This paper has attempted, i n a p r e l i m i n a r y way, to d i s c u s s the f o l l o w i n g aspects of microsporogenesis  i n Abies g r a n d i s :  1. A "brief d e s c r i p t i o n o f t h e stages o f r e d u c t i o n - d i v i s i o n w i t h drawings and photographs. 2. A study of the s t r u c t u r e and development of'the n u c l e o l u s from the i n t e r p h a s e where i t e x i s t s as s e v e a a l d i s t i n c t n u c l e o l i , through pachytene where f u s i o n i n t o a s i n g l e n u c l e o l u s i s complete, t o d i p l o t e n e where i t d i s i n t e g r a t e s to form the sheath of the chromosomes. 3. A study o f the t r a n s f e r o f n u c l e o l a r m a t e r i a l from n u c l e o l u s to chromosome sheath w i t h drawings as  illustration.  4. A s t a t i s t i c a l study o f changes i n s i z e o f the c e l l p a r t s i n the m e i o t i c prophase and t h e i r s i g n i f i c a n c e i n the metabolism ox t h e c e l l .  iv» BEFEEEN PES Blackman, M.W. 1905. On the karyosphere and n u c l e o l u s i n the spermatocytes o f Scolopendra s u b s i n i p e s . P r o c . Am. Acad. Arts-& S o l . 41:329-544. 1  Cowdry, E.V. and K i t c h e n , S.! . 1930. I n t r a n u c l e a r i n c l u s i o n s i n y e l l o w f e v e r . Am. Jour. Hyg. 11:227-299. Gates, R.R. 1942. N u c l e o l i and r e l a t e d n u c l e o l a r s t r u c t u r e s , Bot. Rev. &( 6) :337-409... Guenther, E. 1903. Ueber den N u c l e o l e n i n r e i f e n d e n E c h i n odermenei und seine Bedeutung. Z o o l . J h r b . Abt. Anat. 19:1-28. a  Lucas, A.M. 1940. The c y t o l o g y of f o x e n c e p h a l i t i s end the e f f e c t s of cent i f a c t i o n on the i n t r a n u c l e a r i n c l u s i o n s . Am. Jour. Path. 16:739-760 M c G i l l , G, 1906., The -behaviour o f .the n u c l e o l i d u r i n g oogenesis of the d r a g o n - f l y , w i t h s p e c i a l r e f e r n c e t o s y n a p s i s . Z o o l . £ahrb. Abt. Anat. 23:207-230. Nebel, B.R. 1939. Chromosome s t r u c t u r e . Bot.Rev. 5_( 11) :563626. P a i n t e r , T.8. 1943. C e l l growth and n u c l e i c a c i d i n the p o l l e n . o f Rheo d i s c o l o r . Bot.Gaz. 105t1):56-58. Schrader, . IP. 1940. The f o r m a t i o n o f t e t r a d s and the m e i o t i c mitoses i n the male o f R h y t i d o l o m i a s e n e l i s Say. Jour. Morph. 67:123-141. S c h u l t z , J . 1941. Evidence o f the n u c l e o p r o t e i n nature o f the gene. C o l d Spring Harbour Symposia on Q u a n t i t a t i v e B i o l o g y . V o l . I X . pp. 55-65, Semmens, C.S. and B h a d u r i , P.N. 1941. S t a i n i n g the n u c l e o l u s . S t a i n Techno1. 16:119-120. Stedman, E. and Stedman, Mrs. E. 1943. Chromosomin, a p r o t e i n c o n s t i t u e n t o f chromosomes. Nature 152:267. Van Camp, G.M. 1924. Le r o l e du n u c l e o l e dans l a caryocinese somatique ( O l i v i a m i n i a t a ) . La C e l l u l e 54:1-50... Z i r k l e , 0. 1928. N u c l e o l u s i n r o o t t i p meristem of Zea mays. Bot. Gaz...86:402-418. Z i r k l e , C. 1930. N u c l e o l i o f the r o o t t i p and cambium o f P i n u s s t r o b u s . C y t o l o g i a 2:85-105.  V.  Explanation of PI?tee P l a t e I. I n t e r p h a s e , ( f i g . 1 & .2), and p r o p h a s e , ( f i g . 3 & 4) , stages, showing n u c l e o l a r s t r u c t u r e . F i g . 1 & 2, February 11, 1944; f i g . 3 & 4, February 26, 1944. M a g n i f i c a t i o n of drawings about 1500 X. P i s t e I I . Fi£. 5, small p o r t i o n of nucleus at zygotene to show p a i r i n g of, hpmologues, (March 4, 1944). F i g . 6, small p o r t i o n of nucleus at pachytene showing beginning of r e l a t i o n a l c o i l i n g , (March 13, 1944). F i g . 7, d i p l o t e n e nucleus with nucleolus 'discharging' i t s contents and nuclear membrane disappearing, (March . 17, 1944). F i g . 8, d i p l o t e n e chromosome showing chromatic, (dark), and green-staining p o r t i o n s , ( l i g h t F i g s . 5-7 about 1500X; f i g . 8 about 6000X. P l a t e I I I . F i g . 9-11, p o s s i b l e SAT chromosomes around the nucleolus at d i p l o t e n e . F i g . 12 & 13, t r a n s f e r of n u c l e o l a r m a t e r i a l to chromosomees, d i p l o t e n e . A l l drawings magnified about 6000X. P l a t e IY. F i g . 14, f i r s t metaphase. F i g . 15, f i r s t telophase. F i g . 16, seoond anaphase. F i g . 17, second telophase. March 20, 1944. A l l drawings about 150QX. P l a t e V. Photographs of microsporocyte development showing n u c l e o l a r s t r u c t u r e and numbers. F i g . 18, February 11, 1944. F i g . 19, February 26,1944. F i g . 20, March 4, 1944. F i g . 21, March 13, 1944. A l l photographs magnified about 800X. P l a t e VI. Changes i n volume during prophase of cytoplasm, nucleoplasm, and n u c l e o l i . P l a t e VII* Volumes of cytoplasm at d i f f e r e n t periods of prophase and s i g n i f i c a n c e of measurements. P l a t e V I I I . Volumes of nucleoplasm at d i f f e r e n t periods of prophase and s i g n i f i c a n c e of measurements. P l a t e IX. Volumes of n u c l e o l i at d i f f e r e n t periods of prophase and s i g n i f i c a n c e of measurements.  Plate I  Plate II  Plate I I I  P l a t e IV  Plate Y  P l a t e VI  P l a t e VII  Plate VIII  P l a t e IX  

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