Effects of testosterone propionate on the reproductive system of the male English sparrow

Citation preview

NORTHWESTERN UNIVERSITY LIBRARY

Manuscript Theses

Unpublished theses submitted for the Master's and Doctor's degrees and deposited in the Northwestern University Library are open for inspection, but are to be used only with due regard to the rights of the authors. Biblio­ graphical references may be noted, but passages may be copied only vith the permission of the authors, end proper credit must be given in subsequent written or published work. Extensive copying or publication of the thesis in whole or in part requires also the consent of the Dean of the Graduate School of Northwestern University. Theses may be reproduced on microfilm for use in place of the manuscript itself provided the rules listed above are strictly adhered to and the rights of the author are in no way jeopardized. This thesis by . C (,. . . . . . has been used by the following persons, whose signatures attest their accept­ ance of the above restrictions. A Library which borrows this thesis for use by its patrons is expected to secure the signature of each user.

NAME AND ADDRESS

DATE

lORTHWESTSRU UNIVERSITY

EFFECTS OF TES'10ST EROHE PROPIOEATE OH THE REPRODUG'TIVE SYSTEM OF THE MALE ENGLISH SPARROW

A 35ISSSRTAT I OH

SUBMITTED TO THE GRADUATE SCHOOL I I PARTIAL FULFILLMENT OF THE REQUIRSMENTS f o r th e degree MASTER OF SCISICE FIELD OF ZOOLOGY

By

Helmuth A l f r e d S t a h l e c h e r , J r .

E vanston, I l l i n o i s A u g u s t , 1950

ProQ uest Number: 10102183

All rights reserv ed INFORMATION TO ALL USERS The quality o f this rep ro d u ctio n is d e p e n d e n t u p o n th e quality o f th e c o p y su b m itted . In th e unlikely e v e n t th a t th e a u th o r did n o t se n d a c o m p le te m anuscript a n d th e re a r e missing p a g e s , th e s e will b e n o te d . Also, if m aterial h a d to b e rem o v e d , a n o te will in d ic a te th e d eletio n .

uest. P roQ uest 10102183 Published by ProQ uest LLC (2016). C opyright o f th e Dissertation is h eld by th e Author. All rights reserved. This work is p ro te c te d a g a in st u n au th o rized co p y in g u n d er Title 17, United S tates C o d e Microform Edition © ProQ uest LLC. P roQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

TABLE OF CONTENTS

I n t r o d u c t i o n ........................................................................................... 1 • Methods a n d M a t e r i a l s . . . ....................................... ..................... 4 . O b s e r v a t i o n s and R e s u l t s 1 . ) F a t d e p o s i t i o n ........................................................................?• 2 . )Body we i g h t ............................................................................... 7 • 3 . ) C l o a c a l l i p h y p e r t r o p h y .................................................. 7. 4 . ) B i l l c o l o r a t i o n ....................................................................8 . 5 . )M acrosoopic p i G t u r e o f r e p r o d u c t i v e t r a c t . . . 1 4 . S . )M i c r o s c o p i c p i c t u r e o f r e p r o d u c t i v e t r a c t . . . 1 8 . D iscu ssio n .

..............

33.

Summary.......................................................................................................3 8. B i b l i o g r a p h y ................................................

39.

V i t a ..............................................................................................................4 1 .

59773$

I am i n d e b t e d t o Dr# A# W o l f s o n o f N o r t h w e s t e r n U n i v e r s i t y f o r h i s e x c e l l e n t a d v i c e and k i n d a s s i s t a n c e i n c o m p l e t i n g t h i s experim ent.

W i t h o u t h i s g u i d a n c e and e n c o u r a g e m e n t t h i s p a p e r

would n o t h a v e b e e n p o s s i b l e . I a l s o w i s h t o t h a n k D r . G. Mickey and D r. 0 . P a r k i o r t h e i r s u g g e s t i o n s and h e l p i n c o m p i l i n g t h i s p a p e r , and t h e B i o l o ^ D e p a rt m e n t o f n o r t h w e s t e r n U n i v e r s i t y f o r t h e r e s e a r c h f a c i l i t i e s g r a n t e d t o me i n r u n n i n g t h i s e x p e r i m e n t .

He lmuth A l f r e d S t a h l e c k e r , J r . N o r t h w e s t e r n U n i v e r s i t y , 1950

EFFECTS OF TESTOSTERONE PROPIONATE ON TEE REPRODUCTIVE SYSTEM OF THE MALE ENGLISH SPARROW By Helmuth A l f r e d S t a h l e c k e r , J r .

A num ber o f w o r k e r s h a v e e x p e r i m e n t e d w i t h t h e e f f e c t s o f e x o g e n o u s a n d r o g e n s on v a r i o u s s p e c i e s o f a n i m a l s , b u t t h e r e s u l t s h a v e b e e n so v a r i e d t h a t t h e m e c h an ism w h e r e b y androgens a c t to i n i t i a t e o r m a in ta in sp e rm a to g e n e sis as yet i s not c le a r l y und ersto o d . N e l s o n (1941) f o u n d t h a t a n d r o g e n t r e a t m e n t I s a b l e t o in itiate rat.

s p e rm a to g e n e s is i n t h e a d u l t hypophysectom ized

T w e n t y - e i g h t d a y s w e re a l l o w e d t o e l a p s e a f t e r t h e

h y p o p h y se c to m y and t h e n one t e s t i s was r e m o v e d .

H isto lo g ical

e x a m i n a t i o n o f t h i s t e s t i s showed d e f i n i t e t e s t i c u l a r r e ­ gression.

The a n i m a l s w e re t h e n i n j e c t e d w i t h 2-3mg. o f

t e s t o s t e r o n e d a i l y f o r 25 t o 40 d a y s .

A fte r t h i s tim e,

removal o f th e rem a in in g t e s t i s r e v e a le d d e f i n i t e

increase

i n s i z e and m a t u r e sp e r m w e r e f o u n d i n t h e lum en o f t h e tu b u le s as w e ll as the ep ididym is. Chu (1 940) o b s e r v e d t h a t i n j e c t i o n o f 2 mg. o f t e s t ­ o s t e r o n e d a i l y i n t o s e x u a l l y m a t u r e p i g e o n s f o r 30 d a y s c a u s e d damage t o t h e t e s t i s .

The same d o s a g e o f t e s t o s t ­

erone i n j e c t e d i n t o hypophysectom ized b i r d s r e s u l t e d i n m aintenance o f gonadal f u n c t i o n .

Chu and You (19 4 6) i n j e c t ­

ed 2 mg. o f t e s t o s t e r o n e d a i l y i n t o p i g e o n s f o r 7 t o 21

d a y s b e g i n n i n g t h e i r t r e a t m e n t 30 d a y s a f t e r h y p o p h y s e c t omy.

The l e f t t e s t i s was rem oved on t h e d a y p r e c e d i n g

the f i r s t

i n j e c t i o n and t h e r i g h t t e s t i s was removed on

t h e day a f t e r t h e l a s t

in jectio n .

H i s t o l o g i c a l exam ination

showed t h e l e f t t e s t i s t o be i n v a r i o u s d e g r e e s o f d e g e n ­ e r a tio n w hile th e r i g h t t e s t i s of the b ir d s in je c te d f o r 21 d a y s had e n l a r g e d t u b u l e s i n a c t i v e s t a g e s o f s p e r m a t o ­ g e n e s i s w i t h s p e r m a t o z o a i n most o f t h e t u b u l e s . B u r g e r (1 944) i n j e c t e d male S t a r l i n g s w i t h 1 . 0 mg. o f t e s t o s t e r o n e f o r 26 t o 36 d a y s .

He f o u n d t h a t t h e a n d r o g e n

p r o d u c e d no s t i m u l a t i n g e f f e c t s on s p e r m a t o g e n e s i s b u t r a t h e r caused a r e g r e s s i o n o f th e t e s t e s which were i n e a r l y s t a g e s of developm ent.

F u r t h e r m o r e , t h e r e g r e s s i o n was more marked

i n b i r d s m a i n t a i n e d a t day l e n g t h s w h i c h n o r m a l l y i n h i b i t s p e r m a t o g e n e s i s t h a n i t was i n b i r d s t r e a t e d w i t h l i g h t r a t i o n s which s t i m u l a t e s p e r m a t o g e n e s i s .

In a l a t e r p ap er,

h o w e v e r , B u r g e r (1945) showed t h a t t e s t o s t e r o n e t r e a t e d S t a r l i n g s , w h i c h w e re i n i t i a l l y

i n s p e r m a t o c y t e s t a g e s and

w e r e m a i n t a i n e d u n d e r i n h i b i t o r y day l e n g t h s developed f u r t h e r in to

(10.5 h o u r s ) ,

sp erm ato g en esis th a n did th e c o n t r o l s

which were n o t i n j e c t e d . P feiffer

(1 947) r e p o r t e d t h a t i n j e c t i o n o f .25m g. o f

t e s t o s t e r o n e s t i m u l a te d complete s p e rm a to g e n e s is in t h e E n g l i s h S p a r r o w , p r o v i d e d t h e t e s t e s had a t t a i n e d t h e s i z e a t which an d rogen b e g in s to be p ro duced i n r e a d i l y m ea su ra b le a m o u n ts

( 2 . 0 - 3 . Omm. i n g r e a t e s t d i a m e t e r ) .

B efore th e t e s t e s

had a t t a i n e d t h i s s i z e i n j e c t i o n o f a n d r o g e n r e s u l t e d i n gonadal re g re ssio n .

Kumaran and b u r n e r (1 94 9 ) f o u n d i n W h it e P l y m o u t h o c k c o c k e r a l s t h a t a d m i n i s t r a t i o n o f t h e male hormone s t i m u l a t e d a marked i n c r e a s e i n t e s t i s w e i g h t due t o i n c r e a s e i n s p e r m a t i d f o r m a t i o n i f t h e a n d r o g e n v/as g i v e n o n l y when t h e s e c o n d a r y s p e r m a t o c y t e s had d e v e l o p e d . I f a d m i n i s t e r e d b e f o r e t h e t e s t i s had a t t a i n e d t h i s s t a g e o f developm ent,

in v o lu tio n of th e sperm atocytes r e s u l te d

le a v in g only th e sperm atogonia. From t h e p r e c e d i n g r e s u l t s i t a p p e a r s t h a t t e s t o s t e r o n e e f f e c t s s p e r m a t o g e n e s i s i n a num ber o f w a y s: it

i n some s p e c i e s

seems t o s t i m u l a t e s p e r m a t o g e n e s i s d i r e c t l y , w h i l e i n

o t h e r s i t may a c t u p o n t h e t e s t e s o n l y a f t e r i t h a s a t t a i n e d a c e r t a i n s t a g e o f d e v e l o p m e n t and h a s t e n s p e r m a t o g e n e t i c com pletion. A p r e l i m i n a r y e x p e r i m e n t was c o n d u c t e d on male E n g l i s h Sparrows i n Ja n u a ry 1949.

3?he r e s u l t s o f t h a t e x p e r i m e n t

in d ic a te d th a t te s to s te r o n e stim u la te d sperm atogenesis d ir e c t ly .

C o n s e q u e n t l y , t h e f o l l o w i n g e x p e r i m e n t was d e s i g n e d t o

o b s e r v e t h e e f f e c t s o f t e s t o s t e r o n e p r o p i o n a t e jfon a d u l t male E n g lis h S parrow s, w ith t h e view o f f u r t h e r e l u c i d a t i n g t h e r e l a t i o n s h i p b e t w e e n t h e t e s t e s and i t s a n d r o g e n .

4 ,

METHODS lilD MATERIA1S A t o t a l o f 18 a d u l t E n g l i s h S p a r r o w s (P a s s e r d o m e s t i o u s ) were u s e d i n t h i s e x p e r i m e n t .

The b i r d s w e r e t r a p p e d i n

E v a n s to n , I l l i n o i s , where t h e y a r e r e s i d e n t t h r o u g h o u t t h e y e a r , d u r i n g D e ce m b er, 1 9 4 9 .

They w e r e i m m e d i a t e l y p l a c e d

i n t o c o n t r o l rooms w h e r e a r t i f i c i a l d a y l e n g t h was m a i n t a i n e d at a constant 8 hour p e rio d .

B a r th o l o m e w (1949) r e p o s t s

t h a t t e s t i c u l a r i n a c t i v i t y , which i s s i g n i f i c a n t f o r t h i s e x p e r i m e n t , c a n b e m a i n t a i n e d i n t h e E n g l i s h S p a r r o w w he re day l e n g t h d o e s n o t e x c e e d 8 h o u r s . I n f e c t i o n s w e r e b e g u n J a n u a r y 2 9 , 1 9 5 0 , w h i o h was a p p r o x ­ i m a t e l y 3 w eeks a f t e r t h e b i r d s h a d b e e n i n c a p t i v i t y .

The

a i t r r o w s fsrere d i v i d e d i n t o 3 m a j o r g r o u p s , e a c h g r o u p c o n [i

sisb m g of six b ird s.

Each g r o u p was t h e n s u b - d i v i d e d i n t o

th ree p a ir s . The e x p e r i m e n t a l g r o u p o f b i r d s v,ras i n f e c t e d d a i l y w i t h 2.5 m g. o f t e s t o s t e r o n e p r o p i o n a t e * i n sesa m e o i l . and M r e c e i v e d a t o t a l o f 1 7 . 5 mg. i n 7 d a y s .

B irds L

B i r d s H and

0 r e c e i v e d 3 5 . 0 mg. o v e r a 14 day p e r i o d , and b i r d s P and R r e c e i v e d 5 2 , 5 mg. o v e r a 21 day p e r i o d .

On t h e d a y f o l l o w ­

in g th e l a s t i n f e c t i o n , t h e r e s p e c t i v e p a i r s were a u t o p s i e d and p r e s e r v e d i n B o u i n ’ s f i x a t i v e .

* The t e s t o s t e r o n e and sesa me o i l w e r e s u p p l i e d t h r o u g h t h e c o u r t e s y o f D r . P . Mohr o f C i b a P h a r m a c e u t i c a l P r o d u c t s .

The s e c o n d g r o u p o f b i r d s was i n f e c t e d d a i l y w i t h p u r e se sa m e o i l .

T h i s g r o u p o f c o n t r o l b i r d s r e c e i v e d t h e same

amount o f se sa m e o i l a s d i d t h e e x p e r i m e n t a l g r o u p .

B irds

G and H w e r e i n f e c t e d f o r 7 d a y s r e c e i v i n g a t o t a l o f . 7 c c . of the o i l .

B i r d I was i n f e c t e d f o r 14 d a y s r e c e i v i n g a

t o t a l o f 1.4 cc.

, and b i r d s J and K w e re i n f e c t e d f o r 21

dafcs t o t a l i n g 2 . 1 c c . o f t h e o i l . last

The day f o l l o w i n g t h e

i n f e c t i o n t h e s e b i r d s w e r e k i l l e d and f i x e d I n B o u i n , s . The l a s t g r o u p o f b i r d s was u s e d a s a s e c o n d c o n t r o l

g r o u p and k e p t u n d e r 8 h o u r s o f day l e n g t h w i t h no e x p e r i ­ m ental t e c h n i q u e s employed. Since re c e n t stu d ie d

( W o lfso n 1 9 4 5 , 1 9 4 7 ) h a v e empha­

s i z e d p e r i o d i c v a r i a t i o n s i n body w e i g h t and f a t d e p o s i t i o n i n m i g r a n t and n o n - m i g r a n t s p e c i e s o f b i r d s , a n a t t e m p t was made t o a s c e r t a i n t h e e f f e c t o f a n d r o g e n o n t h e body w e i g h t and f a t d e p o s i t i o n .

The g r o u p r e c e i v i n g t h e t e s t o s t e r o n e

and t h e g r o u p r e c e i v i n g t h e se sa m e o i l w e r e w e i g h e d a p p r o x i m ­ a te ly every fo u r days. a b o u t once a w e e k .

The s e c o n d c o n t r o l g r o u p was w e i g h e d

The method employed i n w e i g h i n g was t o

e t h e r i z e t h e b i r d s and t h e n p l a c e them u p o n a b a l a n c e .

Accur­

a c y was d e t e r m i n e d t o t h e n e a r e s t t e n t h o f a g r am . O b s e r v a t i o n s on s u b c u t a n e o u s f a t d e p o s i t i o n w e re made i n t h e i n t e r c l a v i c u l a r and a n a l r e g i o n s .

Amounts o f f a t

o b se rv e d were c l a s s i f i e d a s m in im al, m o d e ra te , o r h e a v y . O b s e r v a t i o n s were a l s o made on b i l l c o l o r .

W itschi

and Woods (1936) d i s c u s s t h e e f f e c t s w h i c h i n c r e a s e s i n c o n -

6

.

c e n t r a t io n have upon t h e d a rk e n in g o f t h e d i l l i n t h e sparrow .

D a t a on t h e d e g r e e o f c o l o r a t i o n f o r a l l "birds

w e r e r e c o r d e d i n t h i s e x p e r i m e n t e v e r y 4 d a y s , and w e re c l a s s i f i e d and a n a l y z e d n u m e r i c a l l y . (See SABLE 2 . ) A l l o f t h e "birds v/ere k e p t on a d i e t o f c a n a r y s e e d and w a t e r ,

S h i s was s u p p l e m e n t e d t w i c e a week w i t h a m i x t u r e

o f u - a i n e s 1 Dog Pood and w a t e r . kept in cages 2 2 2 2 1 fe e t h andling.

The i n d i v i d u a l p a i r s were

i n dim ension to f a c i l i t a t e

She t e m p e r a t u r e r a n g e o f t h e room was 6 4 ° t o

throughout th e course o f th e experim ent.

68°P.

7.

OBSERVATIONS AI’ID RESULTS gat D eposition She r e s u l t s i n d i c a t e t h a t

t h e r e was r e l a t i v e l y no

i n c r e a s e o r d e c r e a s e i n t h e amount o f f a t p r e s e n t a t t h e end o f t h e r e s p e c t i v e i n j e c t i o n p e r i o d s .

A ll o f the b ir d s

showed m i n i m a l a m o un ts o f f a t a t t h e b e g i n n i n g o f t h e e x p e r i m e n t and no chang e was o b s e i 5ved a f t e r t r e a t m e n t w i t h sesame o i l o r t e s t o s t e r o n e . Body Weight A ll o f the b ird s v a rie d l i t t l e

in w eight p r i o r to th e

b e g i n n i n g o f i n j e c t i o n s , h e n c e any s i g n i f i c a n t i n c r e a s e o r d e c r e a s e a b ov e t h e mean would be i n d i c a t i v e o f some f u n ­ damental change.

The b i r d s r e c e i v i n g no e x p e r i m e n t a l t r e a t ­

ment showed no s i g n i f i c a n t v a r i a t i o n i n body w e i g h t a t t h e clo se of the experim ent.

(CABLE 1 . , p r e s e n t s a n a n a l y s i s o f

t h e w e i g h t s f o r t h e c o n t r o l g r o u p r e c e i v i n g sesa m e o i l and the e x p e r im e n ta l group t r e a t e d w ith t e s t o s t e r o n e .

EIG-URrj 1 . ,

i n d i c a t e s th e p e rc e n t o f i n c r e a s e o r d e c r e a s e in weight d u r ­ ing th e e x p erim e n tal tr e a tm e n t.

The r e s u l t s

ind icate ra th e r

c o n c l u s i v e l y t h a t none o f t h e b i r d s i n j e c t e d with, t e s t o s t e r ­ one o r se sam e o i l shows any s i g n i f i c a n t

in crease or decrease

i n body w e i g h t . C lo a c a ! Lip H ypertrophy One o f t h e f i r s t marked p h y s i c a l c h a n g e s n o t e d i n t h e

8

.

“b i r d s i n j e c t e d w i t h t e s t o s t e r o n e 'was t h e h y p e r t r o p h y o f t h e elo acal l i p s ,

norm ally, the l i p s of the clo aca are e le v a t ­

ed a b o u t E t'o 3 mm. a b o v e t h e a b d o m i n a l w a l l d u r i n g t h e 'win­ t e r m onths.

D u r i n g t h e s p r i n g m o nth s h y p e r t r o p h y o f t h i s

r e g i o n i s normal i n s e x u a l l y a c t i v e m ales.

A ll of the con­

t r o l b ir d s m aintained t h i s c h a r a c t e r i s t i c of s l i g h t l y e le v a te d lip s,

f h e b i r d s i n j e c t e d w i t h t e s t o s t e r o n e f o r 1 week showed

little

e l e v a t i o n a bove t h e n o r m a l .

Hhose i n j e c t e d w i t h a n d ­

r o g e n f o r 2 weeks were e l e v a t e d f r o m 4 t o 6 mm. a b o v e t h e body w a l l , w h i l e t h o s e i n j e c t e d f o r 3 w e e k s w e re 6 t o 8 mm. in h e ig h t.

FIGURES 2 , 3 , and 4 ,

i l l u s t r a t e t h i s observation

in d e t a i l .

Reasons f o r t h i s h y p e rtro p h y a re d is c u s s e d l a t e r

in th e d e ta ile d a n a l y s i s o f th e h i s t o l o g i c a l c o n d itio n s of the re p ro d u c tiv e t r a c t . R ill C oloration A n o t h e r e x t e r n a l l y v i s i b l e c h a r a c t e r i s t i c o b s e r v e d was t h e e f f e c t w h i e h t h e t e s t o s t e r o n e had u p on t h e b e a k o f t h e sparrow .

Keck (1 93 4 ) f o u n d a n i n t e r e s t i n g c o r r o l a t i o n

b e tw e e n t h e t e s t i s the beak.

s i z e and t h e amount o f p i g m e n t a t i o n i n

Between t h e m onth s o f O c t o b e r and F e b r u a r y , t h e

t e s t e s a r e a t a minimum and t h e b i l l i s a l i g h t g r a y o r h o r n y color.

D u r i n g F e b r u a r y and k a r c h when t e s t i c u l a r s i z e i n ­

c r e a s e s , t h e b i l l t u r n s a v e r y d a r k g r a y and f i n a l l y a c o a l black.

W i t s c h i and h o o d s (1936) r e p o r t e d t h a t b i l l d a r k e n ­

i n g i s d e p e n d e n t u p o n t h e a c t i v e r e l e a s e o f t h e male h o r m o n e . P feiffer

(1947) s u b s t a n t i a t e d t h e s e f i n d i n g s when he f o u n d

to • H

M

a

M

CO CM c - t o co O • ■• • • * « CM H 1 1 + 4 4- +

t o CM CO « ■• • • P3 4 1 1

t o -sf £> o H • • • • • r—1 H H H CM 1 1 1 1 1

04 • H I

CM CO • -•

H CO lO O O sit # * • * , • H H H 1 1 + + 1 -I-

H H to • • • •

O £> tO t o r l « • • • • CM H 1 1 1 1 1

CO * H 1

1

•

1 +

aJ ti

p fx| Erf

o

-H

« M

S3 Ctf

&i

“Cdij to m CO

CM • +

1 4-

1

tO • +

* 1

EH

CM t o • •

P3 [25 O 03 R e-t C/2 o EH C/2 m EH

I

1

£> CM CM t o O a » tO CM H • , • • ■, • • • • • M £> t o C- tt- to tO C- CO D~ CO CM CM CM CM CM CM GM CM CM CM

03

a

at U w

1

1 -*•

4 O

• • , • fsi i— 1 H 1 1

r

1

r* tO t o

• • •

H H H 1 1 1

At CM CO • • -• • #*-1 + -t 1 • jjj

+

• 1

O H « • 1 +

r l lO t o • • •

04 i—1 -, • H 1 1

4- 1 +

c10 t o co O o to • , * • • • , CO CO CO CO CM P3 o ■tp ri< At CM CM CM CM CM CM CM CM

GO • CM CM

D O t o tO O rl rl o • • • • • , « • • P4 tO t o 04 cts o O t o t o CM CM CM CM UT

n o T q .o o C tiT

ptre

tO t o CM CM * , • •

•

S3 •H

q .s o d

P m C-f

I

04 t o t o CM r l cr> t o CM • , • • • , • • • , • CUD t o C" 04 e - CO •“3 tO tO co CM CM CM CM CM CM SM CM CM CM

S A ’O p

i +

0 H O

e- CO • •

O o a> • • • , w H 1

o ra T q .

1

H H CM • • , •

s3 ttf) o •H S3 ■P Of •H r» G) p

Ph

lO CO O 1 1

sc t o c o CM t o 04 CM a5 H H H CM

G) a

CO c - CO CO CM CM CM , • , , • • * , x> o 04 03 E> c - CO CM CM CM CM CM CM CM CM CO CO t o tO tO !> • , • , * • t o tO t o t o t o tO CM CM CM CM CM CM

•

CO 'tP 04 O CO « • • • • co CO 04 CO CO c At CM CM CM CM CM CO CO o 1

1

S t o CO CM t o 0 4

gJ