The effect of ergocornine on the stimulation of progesterone biosynthesis in bovine corpus luteum
tissue
by Charles Leslie Soliday
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in BIOCHEMISTRY
Montana State University
© Copyright by Charles Leslie Soliday (1975)
Abstract:
An in vitro incubation system was used to assess the effects of ergocornine (ECO) on the stimulation of
progesterone synthesis in "bovine corpus luteum tissue. Luteal tissue slices were incubated in
Krebs-Ringer bicarbonate buffer under various experimental conditions: 1) incu- bated controls; 2)
incubations containing ECO; 3) incubations stimulated by LH, PGE2, or cAMP; 4) stimulated
incubations containing ECO. Luteinizing hormone (200 ng/ml) increased (p<0.001) the total
progesterone content of the incubated luteal tissue to 246 ±7 (ug progesterone/g luteal tissue) compared
to the incubated control level of 169 ± 7 ug/g. ECO alone decreased (p<0.01) progesterone synthesis
from an incubated control level of 169 ± 7 to 139 - 7, ug/g. "When ECO (10-4M) was included with
LH in the incubation media, progesterone synthesis was reduced (p<0.01) from an LH-stimulated level
of 246 ± 7 to 188 ± 7 ug/g. The inhibitory effect of ECO on IH stimulation of progesterone synthesis
was verified by decreased acetate-1-14C incorporation into de novo progesterone. In a range of 10-7 to
5 x 10-4M ECO, the inhibitory effect of ECO on LH-stimulated progesterone synthesis was found to be
concentration dependent with 10-4M ECO having maximal effect. When PGE2 (10 ug/ml) was added
to incubated tissue, progesterone synthesis was significantly (p<0.01) stimulated to a level of 204 ± 9
ug/g compared to incubated controls which had a progesterone level of 148 ± 11 ug/g. With the
addition of ECO (10-4M) to the PGE2 stimulated system the progesterone level was significantly
repressed to 148 ± 11 ug/g. Cyclic - AMP also stimulated an increase in progesterone synthesis (215 ±
11 vs. 142 ± 12 ug/g for the control). However, the addition of ECO to this cAMP stimulated system
does not lower progesterone (196 ± 11 ug/g) to a significantly (p<0.01) different level. In light of the
fact that IH and PGE2 are known to stimulate adenylate cyclase in the production of cAMP it was
apparent the ECO may be affecting the activity of this enzyme or cAMP-phosphodiesterase. In luteal
tissue homogenates 10-4M ECO inhibited cAMP-phosphodiesterase activity slightly by 14%.
However, ECO was found to have a larger inhibiting effect on overall adenylate cyclase activity.
In a tissue slice assay the accumulation of 14C - cAMP in 30 min. was stimulated to a 5143 ± 861
cpm/100g tissue level compared to a control level of 1030 ± 89. When 10-4 M ECO was included with
LH the 14C - cAMP accumulation dropped to a 2335 - 466 cpm/lOOg level. In luteal tissue
homogenates LH (20 ug/ml) stimulates the production of cAMP at a rate of 539 ± 4l pmoles/15 min./40
mg of homogenized tissue (e.u.) compared to a control rate of 246 ± 30 e.u. The addition of ECO to the
LH stimulated system results in a significantly (p<0.01) decreased rate of cAMP production (276 ± 8
e.u.). Ergocornine also inhibited PGEg stimulation of adenylate cyclase from 399 ± 25 e.u. to 327 ± 21
e.u. This evidence suggests that the inhibition of LH and PGEg stimulation of progesterone synthesis
by ECO is due to an inhibition of LH and PGE2 stimulation of adenylate cyclase activity by the
alkaloid. THE EFFECT OF ERGOGOENINE
O N THE STIM ULATION OF PROGESTERONE BIOSYNTHESIS
IN BOVINE CORPUS LU TEUM TISSUE
by
CHARLES LESLIE SOLIDAY
A t h e s i s s u b m it t e d i n p a r t ia l
f u lf i ll m e n t o f t h e r e q u ir e m e n ts fo r t h e d e g r e e
of
DO CTO R OF PHILOSOPHY
in
BIOCHEMISTRY
A p p r o v ed :
•
.
__________________
C hairm an^ E x a n fin in g C o m m itte e
H e a d , M a jo r D e p a r tm e n t
G r a d u a te fD e a n
M ONTANA STATE UNIVERSITY
B o z e m a n , M o n ta n a
ACKWOWLEDGmnB
% t hanks go f i r s t o f a l l t o my a d v is o r . D r. L a rry L. Ja c k so n , who
.a t me have th e freedom t o I e a m f o r m y s e lf y e t was alw ays p r e s e n t t o •
h e lp an d a d v is e .
My g r a t i t u d e a ls o g ees t o B r. Edward L. Moody-who
h e lp e d i n t h e a r e a s o f e n d o c rin o lo g y and a n im a l r e p r o d u c tiv e p h y s io lo g y .
A s p e c i a l th a n k s go t o V em IaV o ie who k e p t t r a c k o f th e cows and
e x t r a c t e d th e many c o rp o ra l u t e a n e c e s s a ry f o r my r e s e a r c h .
The s tr u g g le s
o f g ra d u a te s c h o o l w ere made e n d u ra b le by th e commradshlp o f my f e llo w
g ra d u a te s t u d e n t s , Gary B lo m q u ist, S h e rry Far-w ell, F red B elan d , and
C la rk G ross,' whom I th a n k f o r th e many good tim e s we s h a r e d .
I w ould l i k e t o th a n k th e Herman F ra s c h F o u n d atio n f o r t h e i r f i n a n ­
c i a l s u p p o rt w hich e n a b le d me t o do my r e s e a r c h t o com plete my g ra d u a te
e d u c a tio n .
I am v e ry g r a t e f u l t o Judy F a rw e ll f o r ty p in g th e f i n a l d r a f t
o f my t h e s i s .
F i n a l l y , my c o n tin u in g g r a t i t u d e t o my w ife f o r h e r p re s e n c e i n my
l i f e and t o my sons who make i t a l l w o rth w h ile .
TABLE OF COIffiEWTS
CHAPTER '
PAGE
I
HTRODUCTIOEf . . . ..........................................................
I E rg o t A l k a l o i d s ................................■ ........................... ....
I
I
The E f f e c t o f E rg o co rn in e on th e B at R ep ro d u ctiv e
S y s t e m ............................... ................................................................. ....
4
Corpus Luteum T iss u e Development
...........................
8
S te r o i d P ro d u c ts o f th e Corpus Luteum ........................................ .
11
E f f e c t o f G o n adotropins on Corpus Luteum T issu e
in v itr o . . . . . . . . . . . . . . . . . .
14
. . . . . .
..................
C y c lic AMP S tim u la tio n o f L u te a l T iss u e . . . . . . . . . .
15
The E f f e c t o f P r o s ta g la n d in s on P ro g e s te ro n e S y n th e s is
. .
17
The S te ro id o g e n ic Pathw ay i n th e B ovine Corpus Luteum
. .
19
LH S tim u la tio n o f ^ C - a c e t a t e I n c o r p o r a tio n . . . . . . . .
23
Mechanism o f G onadotropin A c tio n on S te ro id o g e n e s is . . » .
26
The E f f e c t o f E rg o t A lk a lo id s on A d e n y late C y clase
. . .
32
STATEMENT OF THE PROBLEM .................................... .... .........................................
35
EXPERIMENTAL MATERIALS AND METHODS........................................ ......................
39
Corpus Luteum T i s s u e .................................... ...........................................
39
T is s u e In c u b a tio n ............................................. . . . . . . . . . .
40
. P ro g e s te ro n e A n a l y s i s .................................... ......................... .... . . .
41
S e p a ra tio n and A n a ly sis o f P r o g e s tin s . . . . . .
P r e p a r a tio n o f Homogenates
. . . .
..................
.................. . . . . . . .
A ssessm ent o f A d e n y late C y clase A c t i v i t y
. . .
.......................
43
43
‘ 44
CHAPTER
PAGE
A ssessm ent o f cAMP-P h o sp h o d ie s t e r a s e A c t i v i t y . . . . . . .
4?
M easurem ent o f C y c lic AMP S y n th e s is i n T issu e S l i c e s
48
. . .
RESULTS AND' DISCUSSIOH......................
51
The E f f e c t o f E rg o c o m in e on LH S tim u la te d P ro g e s te ro n e
S y n th e s is ............................................
51
E f f e c t o f ECO on P r o s ta g la n d in S tim u la tio n
...............................
60
E f f e c t o f ECO on cAMP S t i m u l a t i o n ......................................................
66
The E f f e c t o f ECO on P h o s p h o d i e s t e r a s e ........................................
TO
The E f f e c t o f ECO on A d en y late C y clase A c t i v i t y .......................
74
SUMMARY........................................................................................................................
83
The E f f e c t o f ECO on th e S tim u la tio n o f S te ro id o g e n e s is . .
83
LITERATURE CITED , . . ...........................................................................................
90
L I S T OF TABLES
TABLES
I.
II.
III.
IV .
V.
V I.
PAGE
E f f e c ts o f IH an d ECO on p ro g e s te r o n e B io s y n th e s is . . . .
5^
The e f f e c t o f ECO and LE on t h e r a t i o o f
20 g -d ih y d ro p ro g e ste ro n e t o p r o g e s te r o n e ....................................
$6
The e f f e c t o f ECO on LH s tim u la te d p ro g e ste ro n e b io s y n th e s is a s m easured b y ^ H -a c e ta te in c o r p o r a tio n . . .
59
E f f e c t o f ECO on PGEg s tim u la tio n o f s te r o id o g e n e s is
i n l u t e a l t i s s u e . . . . .................................................
62
E f f e c t o f ECO on cAMP s tim u la tio n o f s te r o id o g e n e s is
in lu te a l tis s u e . . . . . . .
.................................... . . . . .
68
The e f f e c t o f e r g o t a lk a l o i d s on p h o s p h o d ie s te ra s e
a c t i v i t y . ............................................. . . .............................
J2
I
V
L I S T OF FIG U EES
FIGURES
' PAGE
1.
The e r g o t a l k a l o i d s .....................................................................................
2.
S te ro id o g e n ic pathvra.y . ................................................
3
13
3 o The second m essen g er c o n c e p t ................................................................
31
4.
S e p a ra tio n o f n u c l e o t i d e s .......................................................................
46
5«
The e f f e c t o f ECO on IH s tim u la te d and c o n tr o l t i s s u e
s t e r o i d o g e n e s i s .........................................................................
53
E f f e c t o f ECO c o n c e n tr a tio n on LH s tim u la te d
s te r o id o g e n e s is ..............................................................................................
$8
The e f f e c t o f ECO on PGEg s tim u la tio n o f s te r o id o g e n e s is
.
63
8 » The e f f e c t o f ECO on cAMP s tim u la tio n o f s te r o id o g e n e s is
.
67
9.
C onversion of. “^C -ad en in e t o ^C-A TP i n l u t e a l t i s s u e . .
.
rJS
1 0.
C y c lic AMP a c c u m u la tio n i n l u t e a l t i s s u e ......................... . .
.
77
11.
A d en y late c y c la s e a c t i v i t y i n l u t e a l t i s s u e hom ogenates .
.
79
12.
The e f f e c t o f ECO on th e s tim u la to r s o f p ro g e s te ro n e
s y n th e s is i n l u t e a l t i s s u e . . . ...............................
86
The e f f e c t o f ECO on s te r o id o g e n e s is
87
6.
7.
13
ABSTRACT
An i n v i t r o in c u b a tio n system was u s e d t o a s s e s s th e e f f e c t s o f
B rg o c o m in e - (ECO) on th e s tim u la tio n o f p ro g e s te r o n e s y n th e s is i n "bovine
corpus lu te u m ' t i s s u e , L u te a l t i s s u e s l i c e s w ere in c u b a te d i n K reb sR in g er b ic a r b o n a te b u f f e r u n d er v a r io u s e x p e rim e n ta l c o n d i t i o n s : l ) in c u
b a te d c o n t r o l s ; 2 ) in c u b a tio n s c o n ta in in g ECO; 3 ) in c u b a tio n s s tim u la te d
b y IH , RGEg, o r cAMP; 4 ) s tim u la te d in c u b a tio n s c o n ta in in g ECO. L u te in ­
i z i n g hormone (200 n g /m l) in c r e a s e d ( p O .O l) th e t o t a l p ro g e s te ro n e con­
t e n t o f th e in c u b a te d l u t e a l t i s s u e t o 246 ± 7 (ug p r o g e s te r o n e /g l u t e a l
t i s s u e ) compared t o th e "in c u b ate d c o n t r o l l e v e l o f 169 ± 7 u g /g , ECO
a lo n e d e c re a s e d (p < 0 .0 l) p ro g e s te r o n e s y n th e s is from an in c u b a te d c o n tr o l
l e v e l o f 169 ± 7 t o 139 - 7, u g /g . "When ECO (IO -i^M) was. in c lu d e d w ith LH
i n th e in c u b a tio n m ed ia, p ro g e s te r o n e s y n th e s is was re d u c e d (pcO .O l) from
an L K -stim u la te d l e v e l o f 246 ± 7 t o 188 ± 7 u g /g . The i n h i b i t o r y e f f e c t
o f ECO on IH s tim u la tio n o f p ro g e s te r o n e s y n th e s is was v e r i f i e d by
d e c re a se d a c e t a t e - l - ^ C in c o r p o r a tio n i n t o de novo p r o g e s te r o n e . I n a
ra n g e o f 10™7 t o 5 x IO- ^M ECO, th e i n h i b i t o r y e f f e c t o f ECO on IE s tim u la te d p ro g e s te r o n e s y n th e s is was fo u n d t o b e c o n c e n tr a tio n depen­
d e n t w ith 1 0 ECO h a v in g m axim al e f f e c t . VThen PGEg (10 u g /m l) was
added t o in c u b a te d t i s s u e , p ro g e s te r o n e s y n th e s is was s i g n i f i c a n t l y
(pcO .O l) s tim u la te d t o a l e v e l o f 204 ± 9 u g /g compared t o in c u b a te d
c o n tr o ls w hich had a p ro g e s te r o n e l e v e l o f l4 8 ± 11 u g /g . W ith th e a d d i­
t i o n o f ECO (1 0 - 4M) t o t h e PGEg s tim u la te d system th e p ro g e s te ro n e l e v e l
was s i g n i f i c a n t l y r e p r e s s e d t o l4 8 ± 11 u g /g . C y c lic - AMP a ls o s tim u ­
l a t e d an in c r e a s e i n p ro g e s te r o n e s y n th e s is (215 ± 11 v s . 142 ± 12 u g /g
f o r th e c o n t r o l ) . However, th e a d d itio n o f ECO t o t h i s cAMP s tim u la te d
system does n o t lo w e r p ro g e s te r o n e (196 ± 11 u g /g ) t o a s i g n i f i c a n t l y ■
( p O .O l) d i f f e r e n t l e v e l . I n l i g h t o f t h e f a c t t h a t IH and PGEg a r e
known t o s tim u la te a d e n y la te c y c la s e i n th e p ro d u c tio n o f cAMP i t was
a p p a re n t th e ECO may b e a f f e c t i n g th e a c t i v i t y o f t h i s enzyme o r cAMPphosphod i e s t e r a s e . In l u t e a l t i s s u e hom ogenates IO-4 M ECO i n h i b i t e d cAMP
p h o s p h o d ie s te ra s e a c t i v i t y s l i g h t l y b y 14%. However, ECO was found to
have a l a r g e r i n h i b i t i n g e f f e c t on o v e r a l l a d e n y la te - c y c la s e a c t i v i t y .
I n a t i s s u e s l i c e a s s a y th e a c c u m u la tio n o f - 4 C - cAMP i n 30 m in . was
s tim u la te d t o a 5143 ± 86l cpm/lOOg t i s s u e l e v e l compared t o a c o n tr o l
l e v e l o f 1030 ± 89 0 When IO-4 M ECO was in c lu d e d w ith LH th e 14C - cAMP
a cc u m u la tio n dropped t o a 2335 - 466 cpm/lOOg l e v e l . I n l u t e a l t i s s u e
hom ogenates LH (20 u g /m l) s tim u la te s th e p ro d u c tio n o f cAMP a t a r a t e o f
539 ± 4 l p m o le s /l5 m in ./4 0 mg o f hom ogenized t i s s u e ( e . u . ) compared t o a
c o n t r o l r a t e o f 246 ± 30 e .u . The a d d i t i o n o f ECO t o t h e IH s tim u la te d
system r e s u l t s i n a s i g n i f i c a n t l y ( p < 0 .0 l) d e c re a se d r a t e o f cAMP p r o ­
d u c tio n (276 ± 8 e . u . ) . E rg o c o m in e a l s o i n h i b i t e d PGEg s tim u la tio n o f
a d e n y la te c y c la s e from 399 ± 25 e . u . t o 327 ± 21 e .u . T h is ev id en ce
s u g g e s ts t h a t t h e i n h i b i t i o n o f LH and PGEg s tim u la tio n o f p ro g e s te r o n e
s y n th e s is by ECO i s due t o an i n h i b i t i o n o f LH and PGE2 s tim u la tio n o f
a d e n y la te c y c la s e a c t i v i t y by th e a l k a l o i d .
INTRODUCTION.
E rg o t a lk a l o id s have been r e p o r te d to , have a v a r i e t y o f p h y s io lo g i­
c a l e f f e c t s on mammalian s p e c ie s among them th e d i s r u p tio n o f th e fem ale
re p ro d u c tiv e sy ste m .
C a t t l e consuming to x ic le v e l s o f e r g o t have been
.observed t o have p o o r b re e d in g e f f i c i e n c y , a s e v id en c e d by p o o r concep­
tio n r a te o r a b o rtio n .
The mechanism o f t h i s .e rg o t a l k a l o i d a c ti o n i n
c a t t l e has n o t been w e ll c h a r a c t e r i z e d . However, e x te n s iv e e x p e rim e n ta l
I
i n v e s t i g a t i o n s have been con d u cted w ith r a t s t o d e term in e th e mode o f
a c ti o n o f e r g o t a l k a l o i d s .
S in g le i n j e c t i o n s o f one o f th e e rg o t a lk a ­
l o i d s , e r g o c o m in e , g iv e n t o fem ale r a t s
a t c r i t i c a l tim e s d u rin g t h e i r
re p r o d u c tiv e c y c le r e s u l t i n c e r t a i n r e p r o d u c tiv e d y s f u n c tio n s .
The
p rim a ry s i t e o f t h i s e r g o t a l k a l o i d e f f e c t i s th o u g h t t o b e a t th e
p i t u i t a r y g la n d w here th e a lk a l o id s have an i n h i b i t o r y a c t i o n on th e
r e l e a s e o f hormones n e c e s s a ry t o s tim u la te p ro g e s te ro n e b io s y n th e s is i n
th e corpus lu te u m .
A lthough th e co rp u s lu teu m has rem ain ed a s u sp e c te d
s i t e o f a sec o n d a ry e r g o t a l k a l o i d a c t i o n , th e d i r e c t e f f e c t o f e rg o t
a l k a l o i d on p r o g e s te r o n e s y n th e s is i n l u t e a l t i s s u e h as n o t been p r e ­
v io u s ly t e s t e d .
I t w i l l be th e p u rp o se o f t h i s t h e s i s t o i n v e s t i g a t e a .
p o s s ib le d i r e c t e f f e c t o f an e r g o t a l k a l o i d on corpus lu teu m t i s s u e .
E rg o t A lk a lo id s
The te rm " e r g o t" i s a common name f o r s p e c ie s o f f u n g i o f th e genus
C la v ic e p s w hich p a r a s i t i z e th e g r a in h ead s o f v a rio u s c u l t i v a t e d g r a in s
2
and w ild g r a s s e s .
E rg o t o c c u rs t o some e x te n t e v ery y e a r on c e r e a l
g ra s s e s su ch a s w h eat, b a r l e y , and r y e and i n p a s tu r e g r a s s e s .
A fte r
i n f e c t i n g th e open f l o r e t th e fungus f ila m e n ts ra m ify th ro u g h o u t th e
g r a s s o v a r i a l t i s s u e e v e n tu a lly com pacting i n t o a d a rk p u r p le , h a rd
s t r u c t u r e c a l l e d th e s c le r o ti u m .w hich form s i n p la c e o f th e g r a in k e r n e l .
These s c l e r o t i a a re composed o f l i p i d s , c a r b o h y d ra te s , in o r g a n ic c o n s t i ­
tu e n ts and a l k a l o i d s .
Among th e a lk a l o id s a r e a group o f am ine, amino
a c id , and c y c lo p e p tid e d e r iv a tiv e s o f l y s e r g i c a c id (F ig . I ) t h a t have
pronounced p h y s io lo g ic a l e f f e c t s on a n im a ls in g e s tin g them /I n d iv i d u a l a n im als d is p la y a w ide v a r i a t i o n o f r e a c tio n s to th e
in g e s tio n o f e r g o t a lk a l o id s i n . t h e i r f e e d g ra in s and g r a s s e s .
Some o f
th e g e n e r a l symptoms o f e r g o t p o is o n in g o f c a t t l e , sw in e , sh eep and
p o u ltr y a r e :
l ) gangrene o f th e e x tr e m i t i e s j 2) s e v e re c o n v u ls io n s ;
3) u l c e r a t i o n o f th e g a s t r o i n t e s t i n a l t r a c t ; 4 ) p o o r b re e d in g e f f ic ie n c y ;
and 5 ) d e c re a s e d l a c t a t i o n ( f o r a re v ie w s e e K in g sb u ry , 1 9 6 4 ).
I n g e s tio n
o f c o m p a ra tiv e ly l a r g e r amounts o f e r g o t r e s u l t s i n th e c o n v u lsiv e ty p e
e rg o tis m ,- i n w hich c o n tr a c tio n o f a r t e r i o l e m a s c u la tu re and smooth
m uscle o f th e g a s t r o i n t e s t i n a l t r a c t a r e im p o rta n t i n p ro d u c in g th e
c h a r a c t e r i s t i c symptoms and l e s i o n s .
C ontinuous in g e s t io n o f s m a ll
amounts o f e r g o t d a i l y r e s u l t s in . t h e gangrenous ty p e e r g o tis m .
Ih e
s tim u la to r y e f f e c t o f e r g o t a lk a l o id s on sm ooth m uscle c a u se s v aso co n ­
s t r i c t i o n r e s u l t i n g i n a g r e a t r e s t r i c t i o n o f b lo o d s u p p lie d to th e
e x tre m itie s .
I n g e s tio n o f e r g o tiz e d f e e d a ls o seems t o d i s r u p t th e
3
F ig u re I .
The E rg o t A lk a lo id s
CHo
R =
I
3
— NH— CH
e r gone v in e
CH2 CXi
l y s e r g i c a c id n u c le u s
E rgotam ine group
erg o tam in e
Ri
E rg o to x in s group
e rg o c ris tin e
rI
CHo
I
3
CKo
— CH2— CH— CH-j
e rg o s in e
1 3
R l= —~ CH2 - CH- - Clio
e rg o k ry p tin e
CHo
R l=
I
CHo
3
CH-CH-j
e rg o v a lin e
I
3
Ri = - C H - C H 3
e rg o c o rn in e
k
c y c li c r e p r o d u c tiv e hormone l e v e l s i n fem ale a n im als r e s u l t i n g i n p o o r
c o n c e p tio n r a t e s ( D innusson e t a l . , 1971) and d e c re a se d l a c t a t i o n (P eace
and Shaw, 1967) .
A lthough th e mode o f a c ti o n o f th e s e a lk a l o id s has n o t
been f u l l y c h a r a c t e r i z e d , g r e a t i n s i g h t s have b een made i n d e te rm in in g
some o f th e mechanisms by w hich e r g o t a lk a l o id s cau se t h e i r e f f e c t s .
One a r e a o f a c t i v e r e s e a r c h h a s b een th e a c ti o n o f e r g o t a lk a lo id s i n
th e d is r u p tio n o f r a t r e p r o d u c tiv e f u n c t i o n s .
:
■
'
The E f f e c t o f E rg o c o rn in e on th e R at R e p ro d u ctiv e System
An e x te n s iv e i n v e s t i g a t i o n h a s b e en done on th e mechanism o f a c ti o n
o f e r g o t a lk a lo id s , on th e fem ale r e p r o d u c tiv e sy stem o f th e r a t .
A
s in g le i n j e c t i o n o f e rg o c o m in e m e th a n e s u lfo n a te i n t h e f i r s t s i x days
a f t e r f e r t i l i z a t i o n w i l l p r e v e n t im p la n ta tio n o f th e b l a s t o c y s t
(V aravudhi e t a l . , 1966) o r cau se r e a b s o r p tio n o f an im p la n te d f e tu s
(K isch and S h e le sn y a k , 1968) .
!An i n j e c t i o n o f exogenous p ro g e s te ro n e
o r p r o l a c t i n i n t o an e rg o c o m in e t r e a t e d r a t , w i l l n e u t r a l i z e th e e f f e c t s
o f th e a l k a l o i d ( S h e le sn y a k , 1958)*
S in c e i t i s p ro g e s te r o n e t h a t m ain­
t a i n s t h e u t e r i n e w a ll f o r im p la n ta tio n and p re g n a n cy , i t w ould seem
t h a t a d e c re a s e d l e v e l o f p ro g e s te r o n e i s th e end r e s u l t o f an e rg o ­
c o m in e i n j e c t i o n .
I n th e o v a ry , th e c o rp u s lu te tu a i s th e t i s s u e re s p o n ­
s i b l e f o r th e p r o d u c tio n o f p r o g e s te r o n e .
I n th e r a t , p r o l a c t i n
(Lam precht 'e t a l . , 1969) and l u t e i n i z i n g hormone (IH ) ( L oew it e t a l . ,
1969) a r e r e q u ir e d t o m a in ta in th e c o rp u s luteum and s tim u la te
5
p ro g e s te ro n e s y n t h e s i s .
I t has "been fo u n d t h a t e rg o c o m in e p re v e n ts any c y c li c in c r e a s e s
i n serum p r o l a c t i n l e v e l s and a ls o s i g n i f i c a n t l y re d u c e s th e serum p r o ­
la c tin le v e l.
P i t u i t a r y p r o l a c t i n and IH l e v e l s v e re fo u n d to be
re d u c e d a f t e r e rg o c o m in e tr e a tm e n t (W uttke e t a l . , 1971) •
E x p e rim en ta l
r e s u l t s i n d i c a t e t h a t t h e e rg o c o m in e a c ti o n i s m e d ia te d th ro u g h th e
hypothalam us by in c r e a s in g p r o l a c t i n i n h i b i t o r y hormone ana d e c re a s in g •
l u t e i n i z i n g r e l e a s i n g f a c t o r , and t h a t e rg o c o m in e can d i r e c t l y act- on
p i t u i t a r y c e l l s t o p r e v e n t p r o l a c t i n r e l e a s e •(Malven and Hbge, 1971; '
Lu e t a l . , 1971) •
T h e re fo re , th e p rim a ry mechanism f o r th e e r g o t a lk a ­
lo i d s a c t i o n on th e hypothalam us and; a n t e r i o r p i t u i t a r y r e s u l t i n g i n Io v
serum l e v e l s o f p r o l a c t i n and IH w hich a r e n e c e s s a ry f o r th e m ain ten an ce
o f th e c o rp u s lu teu m and s tim u la tio n o f p ro g e s te ro n e s y n th e s is from th e
1
co rp u s lu teu m t o m a in ta in th e u t e r i n e w a ll.
F a th e r r e s e a r c h w ith r a t s jo ffe re d a p o s s ib le e x p la n a tio n f o r th e
d e c re a se d p r o g e s te r o n e a c ti v ity j due t o e rg o c o m in e in d u c ed low p r o l a c t i n
le v e ls .
P r o g e s te ro n e
i s c o n v e rte d t o a r e l a t i v e l y i n a c t i v e form ,
2 0 a -d ih y d ro p ro g e s te ro n e , by th e enzyme 2 0a -h y d ro x y s te ro id dehydrogenase
i n o v a ria n t i s s u e (W iest and F o rb e s , 196^ ) .
The a c t i v i t y o f t h i s enzyme
f l u c t u a t e s w ith th e c y c l i c r e l e a s e o f IH and p r o l a c t i n from th e p i t u i t a r y
g la n d (L in d n e r and Zanigrod, 1967) .
From, a d d i t i o n a l e x p e rim e n ta l e v i­
d e n ce , i t a p p e a rs t h a t a d u a l c o n t r o l sy stem e x i s t s w here IH s tim u la te s
s te r o id o g e n e s is to w ard p ro g e s te r o n e s y n th e s is by d e c r e a s in g
6
2 0 a - h ydroxyst e r o i d dehydrogenase a c t i v i t y (W iest e t a l v
1968) .
S in c e
e rg o c o m in e a d m in is tr a tio n r e s u l t s i n d e c re a s e d p r o l a c t i n l e v e l s , p seu d o p re g n a n t r a t s show in c r e a s e d 2 0 a -h y d ro x y s te ro id d ehydrogenase a c t i v i t y
( Lam precht e t a l . ,
1969) and in c r e a s e d o v a ria n c o n te n t o f
2 0 a -d ih y d ro p ro g e s te r o n e r e l a t i v e t o p ro g e s te r o n e (L in d n e r and S h e le sn y a k ,
1967) when i n j e c t e d w ith th e a l k a l o i d .
Throughout th e i n v e s t i g a t i o n o f d e c re a s e d p ro g e s te r o n e l e v e l s i n
r a t s due t o e r g o c o m in e , a d i r e c t e f f e c t o f th e a l k a l o i d on s t e r o i d o ­
g e n e s is i n th e corpus luteum rem ain ed a s u s p e c te d s i t e o f a c t i o n .
The
i n t e r p r e t a t i o n o f th e e x p e rim e n ta l r e s u l t s o f K ra ic e r and S tr a u s s (1970)?
who found t h a t e rg o c o m in e a d m in is te r e d a t a c r i t i c a l tim e w i l l b lo c k
o v u la tio n i n th e r a t , i s i n d i c a t i v e o f su ch an e f f e c t o f th e a l k a l o id on
corpus luteum f u n c tio n .
The tim in g o f t h i s e rg o c o m in e i n j e c t i o n i s n o t
a s c r i t i c a l a s t h a t o f o t h e r o v u la tio n b lo c k in g dru g s — a tr o p in e ,
d ibenam ine, b a r b i t u r a t e s , and q h lo rp ro m az in e w hich a r e know t o i n h i b i t
j
n e u r o s e c r e tio n o f th e o v u la tio n in d u c in g hormone r e l e a s i n g f a c t o r s
(K ra ic e r and S t r a u s s ,
1 9 7 0 ).
S in c e e rg o c o m in e does n o t e x h i b i t th e
same te m p o ra l s p e c i f i c i t y , i t does n o t o p e r a te i n th e same way a s do
th e s e o th e r d ru g s .
B ecause i t a c t s e a r l i e r th a n th e o t h e r d ru g s , e rg o ­
c o m in e may b lo c k some p ro c e s s w hich ta k e s p la c e w e ll b e f o r e th e o v u la ­
t o r y peak o f I S .
The a l k a l o i d a c ti o n c o u ld b e due t o i n h i b i t i o n o f th e
f a c i l i t a t o r y a c ti o n o f p ro g e s te r o n e i n o v u la tio n - in d u c tio n .
The a l k a l o i d
may d i r e c t l y i n h i b i t a c y c li c c o rp o ra l u t e a s e c r e tio n o f s m a ll amounts o f
I
p r o g e s te r o n e w hich have b een i n d i c a t e d t o in f lu e n c e th e tim in g of th e
p r e o v u la to r y p u ls e o f g o n a d o tro p in s e c r e t i o n (Acker and A llo ite a u , 1968) .
A d i r e c t e f f e c t o f e rg o c o m in e on th e co rp u s luteum would "be d i f f i ­
c u l t t o d e t e c t i n th e i n v iv o e x p e rim e n ta l d e s ig n s u s e d i n th e p r e ­
v io u s ly m en tio n ed i n v e s t i g a t i o n s "because o f th e p r i o r e f f e c t o f e rg o ­
c o m in e on th e p i t u i t a r y g la n d .
By c o n tr o lin g g o n a d o tro p ic hormone
l e v e l s , th e p i t u i t a r y c o n tr o ls c o rp u s lu teu m f u n c tio n th u s o v e r­
shadow ing th e o b s e r v a tio n o f any d i r e c t e f f e c t o f e rg o c o m in e on th e
c o rp u s lu te u m .
The a c t i o n o f e rg o c o m in e i n b lo c k in g in c r e a s e d p ro ­
g e s te ro n e l e v e l s may n o t be e n t i r e l y a t th e p i t u i t a r y l e v e l .
The r e p r o ­
d u c tiv e d is f u n c tio n s c au se d by e rg o c o m in e may b e d u e, a t l e a s t i n p a r t ,
t o th e i n t e r f e r e n c e o f n o rm al s te r io d o g e n i s is r e s u l t i n g i n th e lo s s o f
an o p tim a l s t e r o i d en v ironm ent e s s e n t i a l f o r n orm al r e p ro d u c tiv e f u n c tio n .
To t e s t th e d i r e c t e f f e c t o f e rg o c o m in e on th e co rp u s lu teu m , th e
t i s s u e n eed s t o b e p la c e d i n an i s o l a t e d environm ent f r e e o f o u ts id e
in f l u e n c e s .
The a ss e ss m e n t o f p ro g e s te r o n e s y n th e s is from an in v i t r o
co rp u s luteum t i s s u e s l i c e in c u b a tio n w ould p ro v id e th e d e s ir e d s i t u a ­
tio n .
C orpora l u t e a from cows w ould p ro v id e th e q u a n tity o f s p e c ia liz e d
t i s s u e n eed ed f o r i n v i t r o t i s s u e s l i c e in c u b a tio n s .
B efo re s u g g e s tin g
p o s s ib le e x p erim en ts t o t e s t f o r a d i r e c t e f f e c t o f e rg o c o m in e on th e
c o rp u s lu te u m , th e t i s s u e and i t s f u n c tio n n eed s t o b e d e s c r ib e d .
-
-
8
Corpus Luteum T is s u e DeveIo~pment
The b o v in e o v a ry i s a h e te ro g e n e o u s t i s s u e composed o f s e v e r a l
e n d o c r in o lo g ic a lly a c t i v e s t r u c t u r a l s u b u n its V nlch undergo f u n c tio n a l
changes d u rin g th e cow 's 21 day e s tr o u s c y c le .
These s t r u c t u r a l su b ­
u n i t s in c lu d e th e f o l l i c l e s , c o rp o ra I u t e a if and th e s p e c ia liz e d c e l l s
o f th e n o n g e rm in a l e le m e n ts .
The n o rm al e n d o c rin e f u n c tio n o f th e o v ary
i s dep en d en t on t h e c y c le o f f o l l i c u l a r developm ent, o v u la tio n , corpus
luteum fo rm a tio n , and c o rp u s luteu m regression which are all a p p a r e n tly
u n d e r th e c o n tr o l o f g o n a d o tro p ic s u b s ta n c e s d e riv e d p re d o m in a n tly from
th e a n t e r i o r p i t u i t a r y , p la c e n ta , and u te r u s (r e v ie w s : ' S av ard e t a l . ,
1965 J L o b el and Levy, 1968) .
The names o f th e p i t u i t a r y g o n ad o tro p in s
a r e d i s c r i p t i v e o f th e changes th e y b r in g a b o u t i n th e o v a r ie s o f e x p e r­
im e n ta l a n im als — f o l l i c l e - s t i m u l a t i n g hormone (FSE), l u t e i n i z i n g
hormone (LH), and l u t e o t r o p i c hormone (LTH).
A t any g iv e n tim e , th e o v ary h a s a p o p u la tio n o f f o l l i c l e s i n
v a r io u s s ta g e s and horm onal a c t i v i t y .
The f o l l i c l e s c o n s i s t o f th e
ovum aro u n d w hich th e two c e l l t y p e s , g a n u lo sa and th e c a , a r e a rra n g e d
i n a c o n c e n tr ic f a s h i o n .
D uring i t s d ev elo p m en t, th e th e c a la y e r
becomes v a s c u la r iz e d an d th e f o l l i c l e becomes c y s t i c and f i l l s w ith
flu id .
A m atu re f o l l i c l e o v u la te s upon a g o n a d o tro p in s i g n a l , and
th e re m a in in g f o l l i c u l a r t i s s u e tra n s fo rm s t o a corpus lu teu m th ro u g h
p r o l i f e r a t i o n and v a s c u l a r i z a t i o n o f th e g r a n u lo s a l and t h e c a l c e l l s .
These two parenchym al c e l l u l a r ty p e s o f t h e p r e - o v u la to r y f o l l i c l e
a r e in te r m in g le d and th e m o rp h o lo g ic a l d i s t i n c t i o n betw een them becomes
b lu rre d .
However, th e b u lk o f th e l u t e a l c e l l s a p p e a r t o b e d e riv e d
from th e g ra n u lo s a .
T h e c a l c e l l s can b e i d e n t i f i e d a lo n g th e edges, o f
th e tr a b e c u le a w hich c o n ta in th e la r g e b lo o d v e s s e l s .
T h is s t r u c t u r a l
change i s accom panied by a s tr ik in g 'h o r m o n a l change from a f o l l i c l e
t h a t p re d o m in a te ly p ro d u ces e s tr o g e n s i n m icrogram q u a n t i t i e s t o a
co rp u s lu teu m t h a t p ro d u c e s m illig ra m q u a n t i t i e s o f p ro g e s te r o n e .
Hy t h e n in e t h day p o s t- o v u la tio n , a f u l l sp ectru m o f a s many as
s i x d i f f e r e n t m o rp h o lo g ic a l c e l l ty p e s (F o ley and G re e n s te in , 1 9 5 8 ),
a r e p r e s e n t i n th e corpus lu te u m .
These l u t e a l c e l l s may e x h i b i t a
v a r ie ty o f d if f e r e n t c h a r a c t e r i s t i c in h is to lo g ic a l p re p a ra tio n s , b u t
th e h is to c h e m ic a l p r e p a r a tio n s d e m o n stra te th e l u t e a l c e l l s t o p o s s e s s
th e same en zy m atic p r o f i l e .
T h e re fo re , th e c l a s s i f i c a t i o n o f l u t e a l
c e l l s a c c o rd in g t o s i z e and shape may b e c o n v e n ie n t f o r d e s c r ip ti v e
p u rp o s e s , b u t a p p e a rs t o have l i t t l e fo u n d a tio n w ith r e s p e c t t o c e l l - .
u la r fu n c tio n a l a b i l i t y .
The f i r s t s ig n o f c y c li c c o rp o ra l u t e a d e g e n e ra tio n i s i n f i l t r a t i o n
o f lym phocytes w hich b e g in a b o u t day lU and in c r e a s e s t e a d i l y t o th e end
o f th e c y c le .
U n t i l day 1 7, th e en zy m atic a c t i v i t i e s i n th e l u t e a l c e l l s
a p p e a r s tr o n g , b u t t h e r e i s an in c r e a s e i n l i p i d d r o p l e t s .
By day 18,
th e w a lls o f th e s m a ll b lo o d v e s s e ls have th ic k e n e d and a c o n s t r i c t i o n
o f th e v a s c u la r netw ork h a s o c c u re d .
F o llo w in g th e c o lla p s e o f th e
v a s c u la r system w i t h i n th e corpus lu teu m t i s s u e , t h e t i s s u e shows a
10
d e c lin e i n enzyme a c t i v i t y and an in c r e a s e i n f r e e l i p i d s .
A f te r th e
J e x t o v u la tio n , th e l u t e a l c e l l s s h r in k r a p i d l y u n t i l o n ly la r g e b lo o d
v e s s e ls a r e l e f t su rro u n d e d by m acrophages h e a v ily la d e n w ith l i p o f u c h s i n .
As h a s b een d e s c r ib e d , corpu s lu teu m h as a c h an g in g , l im it e d
f u n c t i o n a l l i f e sp a n , th e r e f o r e th e s ta g e o f developm ent m ust be
d e fin e d f o r m e ta b o lic s t u d i e s .
M easurem ents o f th e p ro g e s te r o n e con­
c e n t r a t i o n o f th e p e r i p h e r a l venous b lo o d o f cows d u rin g th e e s tr o u s
c y c le r e v e a l a slow in c r e a s e o f p ro g e s te r o n e t o a b o u t day 9 p o s t ­
o v u la tio n w here i t p eak s a t some l e v e l b e f o r e d r a m a tic a lly d e c lin in g
a t a b o u t day 18 o r 19 (Gomes e t a l . , 1963) .
In a d d itio n t o d i f f e r e n t
co rp u s lu teu m t i s s u e l e v e l s o f p ro g e s te r o n e (Gomes e t a l . , 1 9 63); th e r e
a l s o a p p e a rs t o b e m arked d if f e r e n c e s i n th e i n v i t r o s tim u la to r y
re s p o n s e o f l u t e a l t i s s u e ta k e n a t d i f f e r e n t s ta g e s o f th e e s tr o u s
c y c le (A rm strong e t a l . , 1964a; A rm strong and B lack , 1966) .
The
re s p o n se t o IH i s l e a s t i n t i s s u e ta k e n 2 t o 7 days p o s t - e s t r o u s
compared t o t i s s u e o b ta in e d ' 8 t o 18 days p o s t - e s t r o u s .
when
The a b i l i t y o f
th e t i s s u e t o s y n th e s iz e p ro g e s te r o n e i n v i t r o i s m axim al 4 -1 3 days
p o s t - e s t r o u s and th e n g r a d u a lly d e c lin e s t o day 19 w here no d e te c ta b le
s y n th e s is o c c u rs (A rm strong e t a l . , 1 9 6 4 a ).
C onversion o f t r i t i n t e d
c h o l e s t e r o l t o r a d i o a c t i v e p ro g e s te r o n e i s g r e a t e r a t day 11-13•
H is to ­
l o g i c a l s tu d ie s show t h a t from f o l l i c l e r u p tu r e u n t i l day 7 ; th e l u t e a l
c e l l s a r e r a p i d l y p r o l i f e r a t i n g and in c r e a s in g i n s i z e .
These c e l l s a re
c a p a b le o f p r o g e s tin s y n th e s is d u rin g t h i s tim e , b u t i t s n o t u n t i l a f t e r
11
a c t i v e m i t o t i c d i v i s i o n t h a t t h e f u l l y form ed l u t e a l c e l l s a re c a p a b le
o f m axim al p r o g e s tin s y n th e s is c a p a c ity an d exogenous
g o n a d o tro p in
s tim u la tio n ( I o b e l and Levy, 1968) . .
When com paring th e two p a ra m e te rs o f IH s tim u la to r y a b i l i t y and
p ro g e s te r o n e s y n th e s is a b i l i t y , th e in f lu e n c e o f endogenous g o n a d o tro p in
on th e e x p e rim e n ta l t i s s u e a p p e a rs t o b e an im p o rta n t f a c t o r .
P r o g e s t­
e ro n e fo rm a tio n may be m axim al from endogenous g o n a d o tro p in d u rin g th e
e a r l y developm ent o f th e corpus lu teu m and n o t c a p a b le o f f u r t h e r s tim ­
u l a t i o n by a d d itio n o f exogenous IH .
S te r o i d P ro d u c ts o f th e Corpus Luteum
The s t e r o i d p ro d u c ts o f th e in d i v i d u a l t i s s u e com partm ents o f an
o v ary have been q u a n t i t a t i v e l y m easured th ro u g h i n v i t r o m eth o d s.
The
in c o r p o r a tio n o f r a d io a c ti v e s te r o id o g e n ic p r e c u r s o r s h as p ro v id e d a
q u a n t i t i v e m ethod t o c h a r a c te r iz e th e sp ectru m o f s t e r o i d s form ed i n
th e t i s s u e and a ls o in d ic a te d t h e r e l a t i v e p r o p o r tio n o f e a c h .
The
in f lu e n c e o f g o n a d o tro p in s ,on th e q u a n t i t a t i v e fo rm a tio n o f t h e s t e r o i d
p ro d u c ts h a s a l s o been d e m o n stra te d .
The i n v i t r o sy stem employed to
s tu d y b o v in e c o rp o ra l u t e a f u n c tio n in v o lv e s th e in c u b a tio n o f t i s s u e
s l i c e s i n K reb s-R in g er b ic a r b o n a te b u f f e r a t pH J . h f o r 2 -3 h o u rs as
d ev elo p ed by S u a re z , S o ta and Demare ( i 9 6 0 ) , and A rm strong (1 9 6 4 ).
t h i s system r a d i o a c t i v e p r e c u r s o r s ,and g o n a d o tro p ic s tim u la to r s a re
To
added and th e r e s u l t i n g s t e r o i d p ro d u c ts a r e e x tr a c te d from th e t i s s u e ,
s e p a ra te d "by chrom atography, and a n a ly z e d .
A ccording t o S av ard e t a l . (1965) , th e c o rp o ra l u t e a o f v a rio u s
mammalian s p e c ie s may b e d iv id e d i n t o two g ro u p s:
th o s e w hich p ro d u ce
o n ly p ro g e st-in s and th o s e p ro d u c in g b o th p r o g e s tin s an d e s tr o g e n s .
' A lthough th e b o v in e co rp u s luteum d e v elo p e s from a p re o v u la to r y f o l l i c l e
w hich p ro d u ces e s tr o g e n s b e f o r e i t s r u p tu r e ( l o b e l and Levy, 1968) , i t
f a l l s i n t o th e group t h a t s y n th e s iz e s o n ly p r o g e s t i n s . ' "When ^ C - I a c e t a t e i s in c lu d e d
i n bovine
CL
t i s s u e in c u b a tio n s , o n ly two r a d i o ­
a c t i v e s t e r o i d s . a r e re c o v e re d (S av ard and B u r d u lis , 1961) b o th o f w hich
a re p ro g e s tin s .
These have b een c h a r a c te r iz e d a s p ro g e s te ro n e and
2O3- h y d r o x y -p re g n a n - 3 - one ( 2 0 g -d ih y d ro p ro g e s te ro n e ) ( F ig . 2 ) .
e s tr o g e n f r a c t i o n s a r e d e v o id o f r a d i o a c t i v i t y .
The
When ^ 0 - 4 - t e s t o s t e r o n e
was u sed a s s u b s t r a t e , a g a in no r a d i o a c t i v i t y was re c o v e re d w ith e s t r o ­
gen c a r r i e r s ( S avard and B u r d u lis , 1961) .
In cu b a-tio n s w ith
-4 -
p ro g e s te ro n e r e s u l t i n o n ly one r a d i o a c t i v e p r o d u c t, 20g -d ih y d ro p ro g e s t­
e ro n e (Hayeno e t a l . , 19$1, an d S av ard and B u rd u lis , 1961) .
There was
no r a d i o a c t i v i t y i n th e chrom atogram s i n t h e re g io n s o c c u p ie d by
17-h y d ro x y p ro g e ste ro n e and
-a n d ro s te n e d io n e .
T h e re fo re , i t a p p e a rs t h a t th e b o v in e co rp u s lu teu m does n o t con­
t a i n th e enzym es, 17-h y d ro x y la s e and t h e a ro m a tiz in g enzyme complex.
13
F ig u re 2 .
The s te r o id o g e n ic pathway
CH3—C—SC0A
■ »■ >
>
C h o le s te ro l
E stro g e n s
Pregnenolone
19 -h y d ro x y
te s to s te ro n e
C=O
T e s to s te ro n e
P ro g e ste ro n e
HC--OH
I ? -hydroxy
p ro g e s te ro n e
20 -hydroxy
p ro g e ste ro n e
t o c o n tin u e s te r o id o g e n e s is from p ro g e s te r o n e t o androgen and e s tro g e n s
a s does t h e o t h e r o v a ria n t i s s u e s .
E f f e c t o f G on ad o tro p in s on Corpus Luteum T iss u e i n v i t r o
The t i s s u e s l i c e in c u b a tio n sy stem h a s b een u t i l i z e d t o t e s t f o r
s tim u la to r y a c t i v i t y o f s u b s ta n c e s on p ro g e s te r o n e s y n t h e s i s .
The
g o n a d o tro p in o r o t h e r s u b s ta n c e o f s u s p e c te d g o n a d o tro p ic a c t i v i t y i s
added t o t i s s u e in c u b a tio n s and th e t i s s u e l e v e l o f p ro g e s te r o n e a f t e r
in c u b a tio n i s com pared t o c o n tr o l in c u b a tio n t i s s u e l e v e l s .
G onadotropins w ere f i r s t r e p o r te d t o cau se a s i g n i f i c a n t in c r e a s e
i n p ro g e s te r o n e
s y n th e s is i n t i s s u e s l i c e s by Mason e t a l . ( 1961) .
In
a d e t a i l e d s tu d y . Mason e t a l . ( 1962) w ent on t o d e m o n stra te t h a t human .
c h o rio n ic g o n a d o tro p in (HCG), h o rs e and b o v in e p i t u i t a r y g o n a d o tro p in
e x t r a c t , and a h ig h ly p u r i f i e d sh eep p i t u i t a r y l u t e i n i z i n g hormone
s i g n i f i c a n t l y enhanced p ro g e s te ro n e s y n t h e s i s .
The s tim u la to r y e f f e c t
o f th e s e g o n a d o tro p in s a p p e a rs t o b e s p e c i f i c r a t h e r th a n due to any
n o n - s p e c if ic p r o t e i n .
T is s u e t h a t was re s p o n s iv e t o IH was shown t o b e
i n a c t i v e t o b o v in e serum album in and a d r e n o c o r tic o tr o p ic hormone (ACTE).
S te ro id o g e n e s is i n t h e a d r e n a l g la n d i s s tim u la te d by ACTE.
Mason e t a l . ( 1962) c o n tin u e d t o d e m o n stra te th e s p e c i f i c i t y o f
th e g o n a d o tro p in p r o t e i n by t e s t i n g th e a c t i v i t y o f d e n a tu re d IE .
Sheep
TH -was exposed t o hydrogen p e ro x id e a t room te m p e ra tu re and when added
t o th e i n v i t r o t i s s u e in c u b a tio n f a i l e d t o s tim u la te p ro g e s te ro n e
15
s y n th e s is o v e r c o n t r o l v a l u e s .
A lth o u g h p r o l a c t i n i s an a c c e p te d t r o p i c
hormone f o r th e c o rp u s luteum o f th e r a t (A stvood, 1 9 6 4 ), i t h as no
a c t i v i t y i n v i t r o on p r o g e s te r o n e s y n th e s is i n th e "bovine c o rp u s lu te u m .
.F o lli c l e - s t i m u l a t i n g hormone (FSH) and grow th hormone (GE), i n i t i a l l y
d e m o n stra te d p ro g e s te r o n e s y n th e s is s tim u l a t i o n , how ever, su b se q u e n t
i n v e s t i g a t i o n by Mason e t a l . (1964a) c o n clu d e d , a s s u s p e c te d , t h a t
th e s e hormone p r e p a r a t io n s were c o n ta m in a te d by t r a c e am ounts o f LH.
' A rm strong and c o -w o rk e rs (1964a) co n firm ed t h a t h ig h ly p u r i f i e d b o v in e
LH i s a c t i v e i n s tim u la tin g p ro g e s te r o n e .s y n th e s is i n v i t r o i n l u t e a l
tis s u e .
The in c u b a tin g b o v in e s l i c e p r e p a r a t io n i s e x tre m ely s e n s i t i v e to
s m a ll q u a n t i t i e s o f LH.
The minimum e f f e c t i v e dose o f LH i s betw een
0 .0 1 and 0 .0 2 ug p e r gram o f t i s s u e o r 0 .0 0 2 and 0 .0 0 4 u g p e r m i l l i l i t e r
o f in c u b a tio n m edia a s r e p o r te d b y Mason and S avard (1 9 6 4 a ).
These low
d ose l e v e l s o f LH c a u s in g s tim u la tio n i n th e i n v i t r o sy stem a re con­
s id e r e d t o b e i n th e p h y s io lo g ic a l ra n g e .
C y c lic AMP S tim u la tio n o f L u te a l T issu e
G uided by th e work o f Haynes e t a l . ( i 960) , im p lic a tin g cAMP a s a
m e d ia tin g a g e n t o f ACiJH s tim u la tio n o f s te r o id o g e n e s is i n th e a d re n a l
c o r te x . Marsh and S a v a rd (1964) i n v e s t i g a t e d th e p o s s i b i l i t y t h a t t h i s
c y c li c n u c le o tid e m ig h t a l s o a c t a s a- m e d ia to r o f th e s tim u la to r y a c ti o n
o f LE on s te r o id o g e n e s is i n in c u b a tin g s l i c e s o f b o v in e c o rp o ra l u t e a .
16
I n t h i s e a r l y s tu d y cAMP a t a c o n c e n tr a tio n o f 0 .0 0 2 M c a u se d a s l i g h t
in c r e a s e i n p ro g e s te r o n e s y n th e s is w hich was much s m a lle r th a n th e
in c r e a s e b ro u g h t a b o u t by IE .
A c o n c e n tr a tio n s tu d y u s in g , g ra d u a te d
d o ses from 0 .0 0 2 M t o 0 .0 4 M cAMP d e m o n stra te d t h a t m axim al s tim u la tio n
o f p ro g e s te ro n e s y n t h e s i s , a s m easured s p e c tr o p h o to m e tr ic a lly and by
■ ^ C -a c e ta te in c o r p o r a tio n , o c cu red a t 0 .0 2 M cAMP (M arsh and S av ard ,
1966) .
The l e v e l o f p ro g e s te r o n e p ro d u ced b y m axim al cAMP s tim u la tio n
i s com parable t o t h a t cau sed by s a t u r a t i n g amounts o f LH.
H a ll and
K o ritz ( 1965b ) a l s o fo u n d t h a t h ig h c o n c e n tr a tio n s o f cAMP a r e r e q u ir e d
t o in c r e a s e th e
c o n v e rsio n o f
in c u b a tin g l u t e a l s l i c e s .
- c h o l e s t e r o l i n t o p ro g e s te r o n e i n .
Marsh and S av ard ( 1966) d e m o n stra te d th e
s p e c i f i c i t y o f th e cAMP re s p o n se b y showing t h a t th e s t r u c t u r a l l y
r e l a t e d n u c l e o t i d e s , 3 ' -AMP, 5 ' -AMP o r ATP a t 0 .0 2 M, do n o t e f f e c t
p ro g e s te ro n e s y n th e s is i n th e i n v i t r o sy ste m .
The c o n c e n tr a tio n o f exogenous cAMP n eed ed f o r m axim al s tim u la tio n
o f p ro g e s te r o n e s y n th e s is i n t i s s u e s l i c e s f a r exceeds endogenous l e v e l s
o f cAMP ( 8 .5 nanom oles p e r gram) fo u n d i n th e co rp u s lu teu m (M arsh e t a l . ,
1966) .
High c o n c e n tr a tio n s o f exogenous cAMP have a ls o b e en needed to
s tim u la te l i v e r and a d r e n a l c e l l s (S u th e r la n d and B a l l, i 9 6 0 ) .
S in ce
hom ogenates o f th e s e l a t e r t i s s u e s r e q u i r e much s m a lle r c o n c e n tra tio n s
o f cAMP t o e f f e c t s t i m u l a t i o n , i t was c o n clu d e d t h a t th e h ig h c o n c e n tra ­
t i o n s o f exogenous cAMP was needed due t o th e i n a b i l i t y o f th e n u c le o i
■ !
t i d e t o e a s i l y p e n e t r a t e th e c e l l membrane. In hom ogenates o f c o rp o ra
17
l u t e a , Marsh and S av ard ( 1966) w ere u n a b le t o d e m o n stra te a s tim u la tio n
o f p ro g e s te ro n e s y n th e s is w ith e i t h e r h ig h o r low c o n c e n tr a tio n s o f
cAMP.
Tbe E f f e c t o f P r o s ta g la n d in s on P ro g e s te ro n e S y n th e s is
I n a number o f s p e c ie s i t was found t h a t th e p r o s ta g la n d i n , PGFga,
when a d m in is te r e d s u b c u ta n e o u s ly , r e s u l t e d i n a d e c re a s e o f p r o g e s tin
b lo o d l e v e l s a lo n g w ith a d e c re a s e i n f e r t i l i t y ( P h a r r i s s . e t a l . , 1968,
1969; B la tc h le y and Donovan, 1969) .
P a r a d o x ic a lly , when p ro s ta g la n d in
was added t o an i n v i t r o l u t e a l t i s s u e in c u b a tio n , a s tim u la tio n o f
p ro g e s te ro n e s y n th e s is o v e r c o n tr o l in c u b a tio n s was o b se rv e d (Bedwani
and H o rto n , 1968; P h a r r i s s e t a l . , 1968) .
S p e ro ff and Barnwell (1970)
c h a r a c te r iz e d th e e f f e c t s o f p r o s ta g la n d in s on p ro g e s te r o n e s y n th e s is
i n b o v in e c o rp o ra l u t e a s l i c e s .
(d ecen d in g o r d e r o f p o te n c y :
A U o f th e p r o s ta g la n d in s th e y exam ined
PGEg> PGE1=* PGFgct=* PGA1 ) in c r e a s e d p r o ­
g e s te ro n e s y n th e s is compared t o c o n t r o l in c u b a tio n s a s d e te rm in e d by
s p e c tro p h o to m e tric a s s a y o f t o t a l t i s s u e p ro g e s te ro n e an d by in c o r p o r ­
a t i o n o f ^ C - a c e t a t e i n t o p ro g e s te r o n e s y n th e s iz e d de n o v o .
The peak
o f PGEg s tim u la tio n o c c u re d a t a dose o f a p p ro x im a te ly 1 .0 u g /m l.
The i n v i t r o s tim u la to r y a c t i v i t y o f PGFga i s c o n tr a d ic to r y w ith
th e a n t i - f e r t i l i t y and I u t e o I y t i c i n v iv o e f f e c t s o f t h i s p ro s ta g la n d in
when la r g e d o ses a r e g iv e n t o r a t s ( P h a r r is s and VJyngardon, 196 9) ,
g u in e a p ig s ( B la tc h le y and Donovan, 1969) , r a b b i t s ( P h a r r i s s , 1 9 7 0 ),
18
sheep (McCracken e t a l . , 1970) , h a m ste rs (G utknecht e t. a l . , 1971}, and
cows (L ouis e t al,3 1973; 197^-)•
t h a t PGFg
The e v id e n c e s u p p o rtin g th e co n cep t
i s th e u t e r i n e l u t e o l y t i c a g e n t r e s p o n s ib le f o r th e o n s e t o f
c o rp u s luteum r e g r e s s io n i s v e ry c o n v in c in g .
In sh e e p , PGF2 a i s
r e le a s e d i n r e l a t i v e l y h ig h c o n c e n tr a tio n s from th e u te r u s a t th e tim e
o f l u t e a l r e g r e s s io n and th o u g h t t o b e t r a n s f e r r e d from th e u te r o o v a ria n v e in to th e o v a ria n a r t e r y by a c o u n te r - c u r r e n t mechanism
(McCracken e t a l ., 1 9 7 2 ).
'When an i n t r a u t e r i n e d e v ic e (IUD) i s p la c e d
i n th e u te r u s o f s h e e p , th e corpu s lu teu m does n o t d ev elo p n o rm a lly
(Hawk, 1968) and th e e n d o m e tria l PGF2a l e v e l in c r e a s e d (W ilson e t a l . ,
1 9 7 2 ).
In a r e p o r t by Spilm an and Duby (1972) u s in g th e IUD m odel sy ste m ,
th e fo llo w in g o b s e r v a tio n s w ere made; l ) PGF^ct was in c r e a s e d i n th e
endom etrium a t th e s i t e o f th e . IUD and th e u t e r i n e v e in p la sm a , b u t
2 ) when an i n h i b i t o r o f p r o s ta g la n d in s y n th e s is ( in d o m e th a e in ) was
I
added, th e in c r e a s e s i n PGFga w ere a b o lis h e d , 3 ) co rp u s lu teu m d e v elo p ­
m ent was i n h i b i t e d by a n IUD, Tout 4 ) th e i n h i b i t i o n o f c o rp o ra l u t e a by
th e IUD was b lo c k e d by in d o m e th a e in .
T h is e v id en c e i n d i c a t e s , in s o f a r
a s in d o m eth aein i s t r u l y a s p e c i f i c i n h i b i t o r o f p r o s ta g la n d in s y n th e s is ,
t h a t th e u t e r i n e l u t e o l y t i c f a c t o r in d u c e d by an IUD i s a p r o s ta g la n d in .
A lthough PC-F2 a h as b e e n shown t o b e l u t e o l y t i c i n v iv o , v a rio u s
i n v e s t i g a t o r s have r e p o r te d in c r e a s e d s y n th e s is o f p r o g e s tin s i n r a t ,
m ouse, r a b b i t , cow, rh e s u s monkey, an d human o v a ria n t i s s u e s in c u b a te d
w ith PGF2 a . (L a u d e rd a le , 197U ).
A d d itio n a l c h a r a c t e r i z a t i o n o f t h i s
19
s tim u la to r y e f f e c t s tr o n g ly i n d i c a t e s t h i s e f f e c t t o a ls o b e o f p h y s io ­
lo g ic a l s ig n ific a n c e .
S tim u la tio n by p r o s ta g la n d in s c lo s e ly p a r a l l e l s
t h e p r o p e r t i e s o f IH s tim u la tio n a s c h a r a c te r iz e d b y S p e ro f f and Eamwell
(1970) i n th e fo llo w in g e x p e rim e n ts: ( l ) The in c r e a s e d l ^ C -P ro g e ste ro n e
ph
*
form ed de novo from C- a c e t a t e i n th e p re s e n c e o f p r o s ta g la n d in s has
a b o u t t h e same s p e c i f i c a c t i v i t y a s t h e ^ C - p r o g e s te r o n e form ed by
g o n a d o tro p in s tim u la tio n ; (2 ) The tim e -re s p o n s e c u rv e s f o r PGEg and IH
a r e s i m i l a r ; ( 3 ) There i s no a d d i t i v e e f f e c t when s a t u r a t e d doses o f
p r o s ta g la n d in a r e added t o in c u b a tio n s w ith s a t u r a t i n g d o ses o f e i t h e r
HCG o r IH; (4 ) C yclohexim ide e q u a lly b lo c k s th e s te r o id o g e n ic re sp o n se
t o e i t h e r PGEg o r IH .
The S te ro id o g e n ic Pathw ay i n th e B ovine Corpus Luteum
Mich o f o u r u n d e rs ta n d in g o f th e pathw ay o f p ro g e s te r o n e s y n th e s is
an d i t s t r o p i c s tim u la tio n h a s r e s u l t e d from s tu d ie s o f th e in c o r p o r ­
a t i o n o f r a d i o a c t i v e p re c u rso rs! su ch a s ^ C - a c e t a t e ,
C-m e v a lc n a te ,
1^ C -S q u a le n e ,.and 1^C- o r ^H-chol e s t e r o l i n b o v in e t i s s u e (Mason e t a l . ,
1962; S av ard and C asey, 1964; Mason an d S a v a rd , 1964a; S av ard e t a l . ,
1965; H e llig and S a v a rd , 1965, 1966; H a ll and K o r itz , 1 9 6 5 a ,b ).
p o r a tio n o f
l4
In c o r -
C - a c e ta te h a s p ro v id e d t h e b e s t t o o l o f e s t a b l i s h i n g th e
pathw ay from a c e t a t e t o c h o l e s t e r o l and p ro g e s te r o n e and t h e gonado­
t r o p i n s tim u la tio n o f t h i s pathw ay.
M ev alo n ate-S -1^C was u s e d t o l a b e l
s q u a le n e , c h o l e s t e r o l , and p ro g e s te r o n e i n b o v in e co rp u s lu teu m s l i c e s
(/
20
( H e llig and S a v a rd , 1965a ) , b u t t h e in c o r p o r a tio n i s l e s s e f f i c i e n t th a n
DUld have b een e x p e c te d from l i v e r and y e a s t s t u d i e s .
IH added i n v i t r o
in c r e a s e s th e in c o r p o r a tio n o f ^ C -m e v a lo n a te . i n t o p ro g e s te ro n e b u t n o t
i n t o c h o l e s t e r o l (E e l l i g and S a v a rd , 1965a ) .
A ttem pts t o in tr o d u c e
exogenous 1^C -S qualene a s p r e c u r s o r i n l u t e a l t i s s u e f a i l e d (Mason and
S a v a rd , 1964; S a v a rd e t a l . , 1965) , l i k e l y due t o th e i n s o l u b i l i t y o f
th e h y d ro carb o n t o th e aqueous in c u b a tio n m ed ia.
However, th e p r e c u r s o r
r o l e s o f s q u a le n e w ere d e m o n strated u s in g an a n a e ro b ic in c u b a tio n to
a ccu m u late 1^C -S qualene from exogenous 1^C- a c e t a t e and th e n a d m ittin g
oxygen t o t h e sy stem t o a llo w f o r th e d is a p p e a ra n c e o f 1^C -S qualene to
form in c r e a s e d r a d i o a c t i v e s t e r o l s (H e llig and S a v a rd , 1966) .
In c o rp o r­
a t i o n o f r a d i o a c t i v e c h o l e s t e r o l i n t o p ro g e s te ro n e h as d e m o n strated i t s
o b lig a to r y r o l e i n t h e b io s y n th e tic p a th w ay ' (Mason and S a v a rd , 1964;
R a il and K o r itz , 1965a , b ) .
However, c e r t a i n e x p e rim e n ta l d i f f i c u l t i e s
m ust b e re c o g n iz e d when th e in c o r p o r a tio n o f r a d io a c ti v e c h o le s te r o l
i n t o p ro g e s te r o n e i s u se d a s a m easure o f g o n a d o tro p in s tim u la tio n o f
s te r o id o g e n e s is .
I n i t i a l l y , t h e r e i s a problem o f g e t t i n g c h o l e s t e r o l ,
w hich i s in s o lu b le i n in c u b a tio n m ed ia, a c r o s s c e l l membranes in to th e
b i o s y t h e t i c p r e c u r s o r p o o l.
Once a c r o s s th e c e l l membrane t h i s r a d io ­
a c t i v e exogenous c h o l e s t e r o l i s s u b s t a n t i a l l y d i l u t e d by, t h e v a s t l y
g r e a t e r q u a n tity o f endogenous c h o l e s t e r o l a lr e a d y c o n ta in e d by th e
tis s u e .
Td f u r t h e r c o m p lic a te l a b e l i n g by c h o l e s t e r o l , t h e r e a p p ea r
t o b e v a r io u s "m e ta b o lic p o o ls " (S av a rd e t a l . , 1965; A rm strong, 1966)
21
o f c h o l e s t e r o l i n t h e t i s s u e i n t o w hich th e exogenous c h o l e s t e r o l maye n te r.
W hile a t l e a s t one o f th e s e "m e ta b o lic p o o ls " a c t i v e l y s u p p lie s
p r e c u r s o r f o r p ro g e s te r o n e s y n th e s is th e o th e r p o o ls have o th e r m eta­
b o lic f a te s .
The p r o p o r tio n s o f exgenous c h o l e s t e r o l t h a t e n te r i n t o
th e v a r io u s "m e ta b o lic p o o ls " i s an unknown when th e r e s u l t s o f c h o l e s t ­
e r o l l a b e l i n g e x p e rim e n ts a r e i n t e r p r e t e d .
The tim e c o u rs e l a b e l i n g o f ^ C - a c e t a t e i n t o th e in d i v i d u a l com. p o n e n ts o f th e n o n s a p o n if ia b le f r a c t i o n s u g g e s t t h a t th e b io s y n th e tic
pathw ay i n th e c o rp u s l u t e m i s a s f o ll o w s :
Cgg and Cgo s t e r o l s —
1966) .
s q u a le n e — l a n o s t e r o l
c h o l e s t e r o l —e - p ro g e s te r o n e ( H e llig and S av ard ,
F ig u re 2 i s an i l l u s t r a t i o n o f th e s e q u 'e n c ia l a p p e a ra n c e o f
14 C l a b e l i n th e s e compounds i n th e p ath w ay .
The dynamic a s p e c ts o f
l a b e l i n g i n t h i s tim e -c o u rs e e x p erim en t i s e v id e n t from s u r g e - lik e
a p p ea ra n ce s o f r a d i o a c t i v i t y among th e in te r m e d ia te s i n s te p w is e f a s h io n .
There i s a r a p i d an d l i n e a r r a t e o f r a d i o a c t i v e in c o r p o r a tio n o f 1^Ca c e t a t e i n t o s q u a le n e f o r th e f i r s t h o u r o f in c u b a tio n and th e n le v e l s
o f f and d e c re a s e s when m o n ito re d f o r 4 h o u rs .
in c u b a tio n ,
i4
A f te r 15 m in u te s o f
C la b e l e d l a n o s t e r o l a p p e a rs , and n o t u n t i l a f t e r a 30
m in u te la g tim e does la b e l e d c h o l e s t e r o l a p p e a r.
The p r e c u r s o r r e l a ­
t io n s h ip s a r e e v id e n t by com paring th e s lo p e o f th e r a t e o f la b e l in g
o f each.
The p r e c u r s o r re a c h e s i t s maximum r a t e o f
l4
•
C in c o r p o r a tio n
j u s t b e f o r e th e n e x t compound i n t h e pathw ay b e g in s t o in c r e a s e i t s
r a t e o f i n c o r p o r a tio n .
When t h e in c u b a tio n s w ere c a r r i e d o u t t o 4 h o u rs ,
th e r a d i o a c t i v i t y o f l a n o s t e r o l and th e C^q and
s te r o l fra c tio n s
d e c lin e d w h ile c h o l e s t e r o l l a b e l i n g c o n tin u e s in c r e a s in g (H e llig and
S a v a rd , 1966) .
T h is m ost l i k e l y i s due t o th e d e p le tio n o f th e la b e le d
a c e t a t e p o o l b e f o r e th e c e s s a tio n o f tu r n o v e r o f th e in te rm e d ia ry p o o ls .
In com paring s p e c i f i c a c t i v i t i e s o f s q u a le n e , c h o l e s t e r o l , and
p r o g e s te r o n e , s q u a le n e c o n s i s t e n t l y h a s a g r e a t e r s p e c i f i c a c t i v i t y
th a n p ro g e s te ro n e i n d i c a t i n g th e p r e c u r s o r r o l e o f sq u a le n e ( H e llig
and S a v a rd , 1966) .
These s tu d ie s show th e s p e c i f i c a c t i v i t y o f th e
t o t a l c h o l e s t e r o l was c o n s i s t e n t l y l e s s th a n e i t h e r sq u a le n e o r p r o ­
g e s te ro n e ,
c h o le s te ro l.
T his e v id e n c e s u p p o rts t h e c o n c e p t o f "m e ta b o lic p o o ls " o f
S in c e th e s p e c i f i c a c t i v i t y o f p ro g e s te ro n e can n o t be
g r e a t e r th a n i t s p r e c u r s o r c h o l e s t e r o l , th e c h o l e s t e r o l u t i l i z e d in
p ro g e s te ro n e s y n th e s is w ould have t o come from a " s te r o id o g e n ic p o o l"
o f r e l a t i v e l y h ig h e r s p e c i f i c a c t i v i t y th a n p r o g e s te r o n e . . T h is im p lie s
t h a t th e d i s t r i b u t i o n o f r a d io a c ti v e c h o l e s t e r o l i s n o t u n ifo rm ly e q u i l ­
i b r a t e d w ith th e p re fo rm ed c h o l e s t e r o l o f th e t i s s u e , t h e r e f o r e , new ly
s y n th e s iz e d c h o l e s t e r o l m ust rem ain te m p o r a r ily i n a co m p artm e n talize d
s t a t e (A rm strong, 1967; G rio l-B o sc h and Romanoff, 1966) .
Exogenous la b e l e d c h o l e s t e r o l h a s b een e x te n s iv e ly u s e d i n e l u c i ­
d a tin g th e s te r o id o g e n ic pathw ay:
2 0 , 2 2 - d ih y d r o x y c h o le s te r o l—s-
c h o l e s t e r o l —t- 20- h y d r o x y c h o le s te r o l
-p re g n e n o lo n e an d is o c a p r o ic ald eh y d e
( Tamaoki and P in c u s , 196I ; I c h i i e t a l . , 19633 H a ll and K o r itz , 1 9 6 4 ).
The tr a n s f o r m a tio n s o f ^ C - 2 6 - c h o l e s t e r o l and ^ C - 4 - c h o l e s t e r o l have
23
r e s u l t e d i n th e d e m o n stra tio n o f 2 0 -h y d ro :x y c h o le s te ro l and 2 0 , 2 0dihydroxychol e s t e r o l a s k ey in te r m e d ia te s i n th e c le a v a g e o f th e c h o l­
e s t e r o l s id e c h a in .
The 20-hydro x y l a t i o n s te p h a s b een s tu d ie d i n a
c e l l - f r e e sy stem by H a ll an d K o ritz (1964) who c o n firm ed i t s s u b - c e l l u l a r
l o c a l i z a t i o n and i t s HADP re q u ire m e n t.
They su g g e s te d i t t o b e th e r a t e -
l i m i t i n g r e a c t i o n i n t h e s te r o id o g e n ic p ath w ay .
■LH S tim u la tio n o f
l4
C - a c e ta te I n c o r p o r a tio n
The in c o r p o r a tio n o f r a d io a c ti v e p r e c u r s o r s h as b een u t i l i z e d to
s tu d y th e s i t e o f LH a c t i o n on th e s te r o id o g e n ic p ath w ay .
Bovine co rp u s
luteum t i s s u e s tim u la te d by LH i n v i t r o c o n s i s t a n t l y h a s b o th an in c r e a s e d
in c o r p o r a tio n o f
i4
C - a c e ta te and in c r e a s e d fo rm a tio n o f m icrogram quan­
t i t i e s o f p r o g e s te r o n e (S av ard and C asey, 1964; S av ard e t a l . , 1965;
Marsh and S a v a rd , 1966) .
The s p e c i f i c a c t i v i t y o f LH s tim u la te d p r o ­
g e s te ro n e i s h ig h e r b u t o f th e same o r d e r o f m agnitude when compared t o
th e s p e c i f i c a c t i v i t y o f th e in c r e a s e d p ro g e s te r o n e o f c o n tr o l in c u b a ­
tio n s .
S in c e c h o l e s t e r o l i s th e o b lig a to r y p r e c u r s o r o f p ro g e s te r o n e ,
th e in c r e a s e d s p e c i f i c a c t i v i t y o f LH s tim u la te d p ro g e s te r o n e sh o u ld b e
a d i r e c t r e f l e c t i o n o f th e s p e c i f i c a c t i v i t y o f th e c h o l e s t e r o l from
w hich i t i s fo rm ed .
As p ro p o se d by S av ard e t a l . , ( 1969) , t h i s im p lie s
t h a t LH h a s two s i t e s o f a c t i o n :
l ) s tim u la tio n o f th e c o n v e rsio n o f
a c e t a t e and th e o th e r p r e c u r s o r s i n t o c h o l e s t e r o l , and 2 ) s tim u la tio n
o f c h o l e s t e r o l tr a n s f o r m a tio n t o p r o g e s te r o n e .
2h
WHen Mason and S a v a rd (1964a) an d M arsh e t a l . ,
( 1966) examined
t h e tr a n s f o r m a tio n o f -j H -Y -C h o le ste ro l, th e y found a s i g n i f i c a n t p o r ti o n
o f th e la b e l e d p r e c u r s o r was in c o r p o r a te d i n t o p ro g e s te ro n e i n t i s s u e
s l i c e i n c u b a t io n s .
IH added t o th e sy stem in c r e a s e d th e in c o rp o ra tio n ,
o f l a b e l i n t o p r o g e s te r o n e , how ever, t h e in c r e a s e o f m ic ro gram q u a n t i t i e s
o f p ro g e s te r o n e was g r e a t e r .
T his r e s u l t i n g lo w er s p e c i f i c a c t i v i t y o f
IH s tim u la te d p r o g e s te r o n e i n d i c a t e s IH fa v o r s s y n th e s is from p re c u r s o r s
o th e r th a n la b e l e d c h o l e s t e r o l .
T h is e v id e n c e s u p p o r ts .th e S avard e t a l . ,
( 1969) h y p o th e s is t h a t IH s tim u la to r y a c t i o n o f p ro g e s te r o n e b io s y n th e s is
i s n o t c o n fin e d t o th e tr a n s f o r m a tio n o f c h o l e s t e r o l b u t a ls o in v o lv e s
s tim u la te d in c o r p o r a tio n o f p r e - c h o l e s t e r o l p r e c u r s o r s .
O ther i n v e s t i g a t o r s , A rm strong and H a ll i n p a r t i c u l a r , a rg u e t h a t
t h i s second a c t i o n o f IH on p r e - c h o l e s t e r o l p r e c u r s o r s i s u n n e c e s sa ry .
B ased on t h e i r e x p e rim e n ta l d a ta (A rm strong, 1966; H a ll, 1966.; A rm strong
e t a l . , 1964a; M ajor e t a l . , 1967 ), th e y c o n ten d t h a t th e d e p le tio n o f
th e s te r o id o g e n ic c h o l e s t e r o l p o o l u n d e r LH s tim u la tio n i s s u f f i c i e n t t o
prom ote new s y n th e s is o f c h o l e s t e r o l from a c e t a t e due t o ' th e removal, o f
a n e g a tiv e fe e d b ack a c ti o n from th e mass o f c h o l e s t e r o l .
I n f a c t . H a ll
and K b ritz ( 1965a ) r e p o r te d r e s u l t s from ^ E - c h o le s te r o l in c o r p o r a tio n
i n t o IH s tim u la te d p r o g e s te r o n e c o n tr a r y t o th o s e o f Mason and S av ard
(1964b) and M arsh e t a l . ,
( 1966) .
U sing th e same i n v i t r o b o v in e t i s s u e
s l i c e in c u b a tio n b u t a s l i g h t l y d i f f e r e n t method o f e n te r in g exogenous
T
;
^ E - c h o le s te r o l t o th e m e d ia , th e s p e c i f i c a c t i v i t i e s o f IH s tim u la te d
I
25
p ro g e s te r o n e i n H a ll and K o r i t z 1s e x p erim en ts w ere n e v e r l e s s th a n
c o n tr o l v a l u e s o
lfe rsh and S a v a rd ( 1966) compared " ^ C -a c e ta te in c o r p o r a tio n in to
p ro g e s te r o n e s y n th e s is s tim u la te d b y cAMP t o LH s tim u la te d and c o n tr o l
in c u b a tio n o f b o v in e c o rp u s luteum t i s s u e .
The c y c li c n u c le o tid e
s tim u la te d a n in c r e a s e d fo rm a tio n o f m icrogram s o f p ro g e s te r o n e in th e
same m anner a s TH.
However, th e s p e c i f i c a c t i v i t y o f th e ' p ro g e s te ro n e
from t h i s cAMP s tim u la te d in c o r p o r a tio n o f
- a c e t a t e i s a b o u t th e same
a s th e c o n tr o l t i s s u e s p e c i f i c a c t i v i t y u n lik e th e h ig h e r s p e c i f i c a c t ­
i v i t y o b se rv e d w ith LH s tim u l a t i o n . . These r e s u l t s i n d i c a t e t h a t th e
a c ti o n o f cAMP i s c o n fin e d t o tr a n s f o r m a tio n o f c h o l e s t e r o l i n t o p r o ­
g e s te r o n e , w h e re a s, th e in c r e a s e d s p e c i f i c a c t i v i t y from LH s tim u la tio n
i n d i c a t e s an LH s i t e o f a c ti o n b e f o r e c h o l e s t e r o l i n a d d itio n to th e
s tim u la tio n o f c h o l e s t e r o l tr a n s f o r m a tio n i n t o m icrogram q u a n t i t i e s o f
I
p r o g e s te r o n e . T h e re fo re , LH a p p e a rs t o have an a d d i t i o n a l s i t e o f
I
a c t i o n e a r l i e r th a n c h o l e s t e r o l i n th e b io s y n t h e t i c pathw ay a s w e ll a s
p a r a l l e l a c ti o n w ith cAMP.
VThen % - c h o l e s t e r o l was u s e d a s a r a d i o ­
a c t i v e p r e c u r s o r , LH and cAMP d e m o n stra te d th e same i n c r e a s e . i n s p e c i f i c
a c t i v i t y (H a ll and K o r itz , 1965a ) com pared t o c o n tr o l in c u b a tio n s .
I n t h e i r s tu d y o f p r o s ta g la n d in s tim u la tio n o f p r o g e s te r o n e sy n ­
t h e s i s i n b o v in e l u t e a l t i s s u e , S p e ro f f and Eamwell (1970) a ls o com­
p a re d ^ +O a c e t a t e in c o r p o r a tio n u n d e r th e c o n d itio n s o f p r o s ta g la n d in
s tim u la tio n , LH s tim u l a t i o n , and c o n t r o l s y n th e s is o f p r o g e s te r o n e .
26
They fo u n d th e s p e c i f i c a c t i v i t y o f t h e new ly form ed p ro g e s te ro n e , u n d e r
Ifi s tim u la tio n o r p r o s ta g la n d in s tim u la tio n t o h e o f th e same o r d e r o f
m ag n itu d e , th u s d e m o n stra tin g a p o s s ib le p a r a l l e l s tim u la tio n o f p r o ­
g e s te ro n e b io s y n th e s is by p r o s ta g la n d in and IH .
Mechanism o f G o nadotropin A c tio n on S te ro id o g e n e s is
A f te r t h e f i r s t d e m o n stra tio n o f t h e i n v i t r o s tim u la tio n o f p r o ­
g e s te ro n e s y n th e s is by g o n a d o tro p in (Mason e t a l . , 1962 ), i t seemed
n a t u r a l f o r th e i n v e s t i g a t o r s t o c o n tin u e t o fo llo w t h e example o f ACTH
s tim u la tio n o f c o r tic o s t e r o i d o g e n e s i s i n th e a d r e n a l c o r te x .
The con­
c e p t o f t r o p i c hormone a c ti o n o f ACTH a s advance by Haynes (1957 > 1958;
i 9 6 0 ) was t h a t ACTH i n i t i a l l y a c t s on a d e n y la te c y c la s e t o in c r e a s e th e
cAMP l e v e l w hich i n t u r n a c t i v a t e s a p h o s p h o ry la se enzyme o f th e a d re n a l
c o r te x .
The a c t i v a t e d p h o s p h o ry la se a c t s on gly co g en to p ro d u ce g lu c o se
I-POjt and g lu c o s e - 6 -PO^.
O lu c o se -6 -PO^ i s m e ta b o liz e d v i a th e p e n to s e -
p h o sp h a te s h u n t r e s u l t i n g i n in c r e a s e d amounts o f th e n u c le o tid e JSADPH.
T h is n u c le o tid e s tim u la te s c o r t i c o s t e r o i d s y n th e s is th r o u g h " its a c ti o n a s a c o f a c to r f o r many o f th e s te p s i n th e s te r o id o g e n ic pathw ay.
Jh th e i n i t i a l s t u d i e s o f g o n a d o tro p in s tim u la tio n - b y Mason e t a l . ,
( 1962) i t was fo u n d t h a t HADP + g lu c o s e - 6 -PO^ cau sed in c r e a s e p r o g e s te r ­
one s y n t h e s i s .
I n a su b se q u e n t r e p o r t y IH was shown to s tim u la te p h o s ­
p h o ry la s e o f th e b o v in e c o rp u s luteum (M arsh and S a v a rd jl 1964 ), th u s
C '
re m a in in g c o n s i s t e n t w ith th e Haynes h y p o th e s is .
3
However, e x p erim en ts
i'
27
■where LH was added t o in c u b a tio n s i n t h e p re s e n c e o f s a t u r a t i n g amounts
>
o f EADPH d e m o n stra te an a d d i t i v e e f f e c t by th e s e s tim u la to r s (S avard
e t a l . , 1963) .
T h is w ould n o t have o c c u rre d i f EADPH w ere th e l i m i t i n g
fa c to r.
I n ^ C - a c e t a t e in c o r p o r a tio n s t u d i e s , LH and MADPH w ere found t o
have d i f f e r i n g e f f e c t s on th e in c o r p o r a tio n o f r a d i o a c t i v i t y (S av ard and
C asey, 196^ ) .
The LH s tim u la tio n r e s u l t e d i n th e fo rm a tio n o f de novo
p ro g e s te r o n e o f ro u g h ly th e same s p e c i f i c a c t i v i t y a s in c u b a te d c o n t r o l s .
Qn th e o t h e r h an d , exogenous MADPH r e s u l t e d i n fo rm a tio n o f p ro g e s te ro n e
o f much lo w er s p e c i f i c a c t i v i t y .
I t i s o b v io u s from th e s e r e s u l t s t h a t
th e p ro g e s te ro n e s y n th e s iz e d i n th e p re s e n c e o f MADPH c o u ld n o t have
come from th e same p o o l o f la b e l e d p r e c u r s o r s a s t h e p ro g e s te r o n e form ed
i n th e c o n tr o l o r i n th e LH s tim u la te d s l i c e s .
-3
"When H - 7 - c h o le s te r o l
was added a s a r a d i o a c t i v e p r e c u r s o r , MADPH in c r e a s e d th e c o n v e rsio n o f
t h i s p r e c u r s o r t o a f a r g r e a t e r e x te n t th a n d id LH (Mason and S av ard ,
1 9 6 4 a ).
A gain th e p ro g e s te r o n e s y n th e s iz e d by LH s tim u la tio n had t o have
come from a d i f f e r e n t p r e c u r s o r p o o l th a n th e p ro g e s te r o n e s y n th e s iz e d
by MADPH s ti m u l a t i o n .
I t was co n clu d ed (S av ard and C asey, 1964j Mason
and S a v a rd , 1964b) from th e s e e x p e rim e n ta l r e s u l t s t h a t b e c a u se o f th e
d i s t i n c t q u a l i t i a t i v e d if f e r e n c e s i n th e p r e c u r s o r p o o ls o f p ro g e s te ro n e
s y n th e s iz e d i n th e p re s e n c e o f th e two a g e n ts , th e s tim u la to r a c tio n s
o f LH and MADPH on s te r o id o g e n e s is a r e independent^.
28
A no th er b ro a d c o n c e p t c o n c e rn in g t h e mechanism o f hormone a c ti o n
on i t s t a r g e t t i s s u e in v o lv e s th e s y n th e s is o f p r o t e i n s and th e a s s o ­
c i a t e d g e n e t i c a l l y lin k e d r e g u la to r y mechanism (KarI s o n , 1963) .
As
a p p lie d t o th e a c ti o n o f g o n a d o tro p in on th e co rp u s lu te u m , t h i s im p lie s
t h a t IE s tim u la te s s t e r o i d o g e n i s i s th ro u g h a s tim u la tio n o f t h e s y n th e s is
o f c e r t a i n k ey enzymes i n th e s te r o id o g e n ic p ath w ay .
To t e s t t h i s
h y p o th e s is S av ard e t a l . , ( 1965) in tr o d u c e d an i n h i b i t o r o f p r o t e i n
s y n th e s is , purom ycin, t o t h e t i s s u e s l i c e in c u b a tio n sy ste m .
When •
purom ycin was added t o th e in c u b a tio n m edia a lo n g w ith IE , th e ■s tim u la ­
to r y a c ti o n o f IE was c o m p le te ly i n h i b i t e d .
A nother known i n h i b i t o r ,
a ctin o m y cin D, had th e same e f f e c t o f s u p p re s s in g IE s tim u la tio n o f p r o ­
g e s te ro n e s y n th e s is a s purom ycin.
A ctinom ycin D i n d i r e c t l y i n h i b i t s
p r o t e i n s y n th e s is th ro u g h i t s i n h i b i t i n g a c t i o n on MA s y n th e s is (B re u er
and D a v is, 1 9 6 4 ).
The I n h i b i t o r y e f f e c t o f th e s e two a n t i b i o t i c s on
IH s tim u la tio n im ply t h a t IE may c au se in c r e a s e d p ro g e s te r o n e s y n th e s is
th ro u g h a s tim u la tio n o f th e p r o t e i n s y n th e s is o f key enzymes i n th e
s te r o id o g e n ic p ath w ay .
The i n h i b i t o r y e f f e c t o f t h e p r o t e i n s y n th e s is i n h i b i t o r s i s a ls o
se e n w ith cAMP and PGE2 s tim u la tio n o f p r o g e s te r o n e s y n t h e s i s .
Purom ycin
i n h i b i t s t h e in c r e a s e d p ro g e s te r o n e s y n th e s is when in c u b a te d w ith t i s s u e
s l i c e s i n th e p re s e n c e o f 0 .0 2 M cAMP i n t h e same m anner i t i n h i b i t e d IE
s tim u la tio n (M arsh e t a l . , 1966) .
When cy clo h ex im id e was in c lu d e d i n
29
in c u b a tio n s c o n ta in in g LH o r PGE2 , t h e s tim u la tio n o f p ro g e s te r o n e sy n ­
t h e s i s by b o th a g e n ts was e q u a lly b lo c k e d (S p e r o f f and Barnwell, 1 9 7 0 ).
F u r th e r s i m i l a r i t i e s betw een th e s te r o id o g e n ic s tim u la tin g a g e n ts ’
a r e se e n i n th e r e s u l t s o f in c o r p o r a tio n s t u d i e s o f r a d i o a c t i v e p r e ­
c u rso rs .
The in c o r p o r a tio n o f
- a c e t a t e i n t o de novo p ro g e s te ro n e
s y n th e s is i s s tim u la te d by PGE2 i n th e same m anner a s LH ( S p e ro ff and
Barnwell, 1 9 7 0 ).
C y c lic AMP s tim u la te d th e in c o r p o r a tio n o f b o th 1^C-
a c e t a t e and % - 7 - c h o l e s t e r o l i n t o p ro g e s te r o n e r e s u l t i n g i n s im ila r
s p e c i f i c a c t i v i t i e s t o LH s tim u la tio n (M arsh e t a l . , 1966)»
T his a ls o
s tr o n g ly i n d i c a t e s t h a t th e e f f e c t o f cAMP, l i k e t h a t o f LH c an n o t b e
I
m e d ia te d th ro u g h KADPH.
S in c e th e s e t h r e e a g e n ts , LH, cAMP, and p r o s ta g la n d in , a l l seem t o
e f f e c t p ro g e s te r o n e b io s y n th e s is i n th e same m anner, t h e i r a c tio n s m ust
i n some way b e r e l a t e d t o e ac h o t h e r .
s tim u la tio n a r e n o t a d d i t i v e .
T h e ir e f f e c t s on s te r o id o g e n ic
!'When LH and cAMP (Marsh and S av ard , 1966)
o r LH and PGEg (S p e ro f f and Barnwell, 1970) a r e added c o l l e c t i v e l y t o
t i s s u e in c u b a t io n s , th e p ro g e s te r o n e l e v e l i s n o t any g r e a t e r from com­
b in e d s tim u la to r s th a n from each s t i m u l a t o r a lo n e .
The p o s s i b i l i t y o f
a m e d ia to ry r o l e f o r cAMP i n th e LH s tim u la to r y re s p o n s e was i n v e s t i ­
g a te d by m e asu rin g th e changes i n endogenous cAMP c o n c e n tr a tio n due t o
t h e a d d itio n o f LH t o in c u b a tin g t i s s u e (M arsh e t a l . , 1966) . . I t was
fo u n d t h a t LH in c r e a s e d endogenous cAMP by 100 f o l d , and t h i s re sp o n se
j
i n b o v in e l u t e a l t i s s u e was s p e c i f i c f o r LH compared t o o th e r horm ones.
30
Marsh and c o -w o rk ers ( 1966) a l s o fo u n d t h a t endogenous cAMP re a c h e d
maximum c o n c e n tr a tio n 7*5 m in u te s a f t e r a d d itio n o f IE , w h e re a s, an
in c r e a s e i n p ro g e s te r o n e s y n th e s is was n o t o b se rv e d u n t i l 15-30 m in u tes
i n t o th e s tim u la te d in c u b a t io n .
T h is tim e - r e l a t i o n s h i p i s i n d i c a t i v e o f
a cAMP m e d ia to r r o l e i n IE s tim u la to r y a c t i o n .
F u r th e r i n v e s t i g a t i o n by Marsh (1970a) i n t o th e r e l a t i o n s h i p betw een
cAMP and IE fo u n d t h a t IE s tim u la te s t h e enzyme, a d e n y la te c y c la s e , w hich
p ro d u ces cAMP from ATP.
I t was fo u n d t h a t IE had. no e f f e c t on
p h o s p h o d ie s te ra s e w hich c o u ld have a c c o u n te d f o r in c r e a s e d endogenous
cAMP th ro u g h an i n h i b i t i o n o f i t s a c t i v i t y .
T his m e d ia to r r o l e o f cAMP i s common f o r many o t h e r hormones i n
o th e r t i s s u e s (re v ie w s , R obison, 1971, 1972) and h as been term ed " th e
second m essenger c o n c e p t" ( F ig . 3 )•
The hormones r e l e a s e d from t h e i r
c e l l s o f o r i g i n a r e th e f i r s t m essen g ers w hich i n t e r a c t w ith th e s p e c i f i c
r e c e p to r s o f t h e i r t a r g e t t i s s u e .
T h is i n t e r a c t i o n r e s u l t s i n a d e n y la te
c y c la s e a c t i v a t i o n , th e r e b y , in c r e a s in g th e i n t r a c e l l u l a r l e v e l o f cAMP.
The cAMP i s th e second m essen g er w hich i n t e r a c t s w ith one o r more system s
w ith in a c e l l t o p ro d u ce th e e f f e c t a s s o c ia te d w ith t h e ■p a r t i c u l a r horm one.
The p o s s i b i l i t y t h a t p r o s ta g la n d in s tim u la tio n o f p ro g e s te ro n e
s y n th e s is may a ls o be m e d ia te d by cAMP, was i n v e s t i g a t e d by Marsh ( 1970a,
1 9 7 1 ).
The p r o s ta g la n d in s s tim u la te a d e n y la te c y c la s e fo llo w in g th e same
g e n e r a l o r d e r o f a c t i v i t y o b se rv e d i n t h e i r s tim u la to r y e f f e c t on s t e r o ­
id o g e n e s is .
PGE2 and PGE1 r e s u l t e d i n more th a n a 100$ s tim u la tio n o v e r
31
E ndocrine
Hormone
5 ’-AMP
( f i r s t m essenger)
(second m essen g er)
p h y s io lo g ic a l re sp o n se s
(in c lu d in g s tim u la tio n o f
s te r o id o g e n e s is )
F ig u r e
3.
The se c o n d m e s s e n g e r c o n c e p t .
32
/
''--V
c o n t r o l a c t i v i t y , w h e re a s, PG F ^ and PGA^ r e s u l t e d i n a b o u t a 50$ s tim u ­
la tio n .
The f u n c t i o n a l s ig n if ic a n c e o f t h i s e f f e c t o f p r o s ta g la n d in s on
l u t e a l tis s u e i s u n c e rta in .
Marsh ( 1971) d e m o n stra te d t h a t LH and PGE^
added to g e th e r d o u b led th e s tim u la tio n o f a d e n y la te c y c la s e compared to
s tim u la tio n by e i t h e r one o f th e a g e n ts added s i n g ly .
T h is w ould in d i c a t e
t h a t th e two e f f e c t s a r e s e p a r a te phenomena and t h a t PGE
a t o r o f th e e f f e c t o f LH on a d e n y la te c y c la s e .
i s n o t a m edi­
There a r e in d iv id u a l
r e c e p to r s i t e s f o r g o n a d o tro p in s and p r o s ta g la n d in i n b o v in e corpus
luteum c e l l
phenomena.
membranes (Bao, 1973) a l s o i n d i c a t i v e o f two s e p a r a te
However, K uehl e t a l . , (1970) c o n clu d ed t h a t th e LH e f f e c t
on a d e n y la te c y c la s e i s m e d ia te d by PGE^ i n t h e i r s tu d ie s w ith mouse
o v a rie s .
They fo u n d t h a t a p r o s ta g la n d in a n ta g o n i s t, 7 -o x a -1 3 -p ro s ty n o ic
a c i d , n o t o n ly b lo c k s PGE^ s tim u la tio n o f a d e n y la te c y c la s e , b u t a ls o
b lo c k s LH s tim u la tio n o f a d e n y la te c y c la s e .
A lthough t h i s o b s e rv a tio n
i n d i c a t e s a m e d ia to ry r o l e o f p r o s ta g la n d in on LH s tim u la tio n , th e y
o b se rv e d a d d itiv e e f f e c t s o f LH and p r o s ta g la n d in on
l4
C-cAMP a c c u m u la tio n .
The E f f e c t o f E rg o t A lk a lo id s on A d en y late C yclase
The s tim u la to r y a c ti o n o f LH and PGE^ on p ro g e s te r o n e s y n th e s is ,
h a s been shown t o b e m e d ia te d by t h e i r s tim u la to r y e f f e c t on a d e n y la te
c y c la s e .
E rg o t a lk a l o i d s have a h i s t o r y o f b lo c k in g s tim u la to r y re s p o n se s
o f a d e n y la te c y c la s e by o th e r a g e n ts i n v a r io u s t i s s u e s i
f
33
The e a r l y s tu d ie s on e p in e p h r in e . D ale i n 1906 r e p o r te d t h a t
e rg o to x in e an d erg o ta m in e b lo c k e d th e p r e s s o r e f f e c t s o f e p in e p h rin e
on sy m p a th e tic n e rv e s tim u la tio n w ith o u t b lo c k in g th e v a s o d i l a t o r e f f e c t
o f th e horm one.
T his b eg an th e work w hich s e p a r a te d c a te c h o la m in e
re s p o n se s i n t o th e a lp h a o r b e ta a d re n e rg ic r e c e p to r c l a s s i f i c a t i o n s .
D ihydro- d e r i v a t i v e s o f e r g o t a l k a l o i d s w ere fo u n d t o b e even more p o te n t
a lp h a a d re n e rg ic b lo c k in g a g e n ts th a n t h e i r p a r e n t compounds ( E o th lin ,
1947).
D ihydroerg o tam in e a t h ig h c o n c e n tr a tio n h as b een fo u n d t o have
numerous p h y s io lo g ic a l re s p o n s e s i n a d d i t i o n t o i t s a c t i o n a s an a lp h a
a d re n e rg ic a n t a g o n i s t .
Most o f th e s e a d d i t i o n a l re s p o n s e s seem t o in v o lv e
cAMP a s a key in te r m e d ia te w hich i s a c h a r a c t e r i s t i c o f a b e ta a d re n e rg ic
e f f e c t ( S u th e r la n d , 1968) .
I n r a t s , d ih y d ro e rg o tam in e b lo c k s e p in e p h rin e
an d cAMP in d u c ed h y p e rg ly c em ia (N o rth ro p and P a rk s , 1 9 6 4 ).
The b lo c k ag e
o f th e h y p erg ly cem ic e f f e c t o f cAMP i s i n t e r p r e t e d a s an i n d i c a t i o n t h a t
t h e s i t e o f a c ti o n o f th e a l k a l o i d i s beyond a d re n e rg ic r e c e p to r s and
t h a t d ih y d ro e rg o ta m in e may b lo c k th e a c t i v a t i o n o f p h o sp h o ry la se by cAMP
o r in c r e a s e th e r a t e o f i n a c t i v a t i o n o f th e c y c li c n u c e o tid e .
In th e dog,
i t was d e m o n stra te d t h a t 0 .1 mM d ih y d ro e rg o tam in e b lo c k e d c ate c h o lam in e
a c t i v a t i o n o f a d e n y la te c y c la s e o f t h e h e a r t and l i v e r (Murad e t a l . , 1 9 6 2) .
E rgotam ine i n h i b i t s th e a d r e n a l i n - p o t e n t i a t e d p r o d u c tio n o f cAMP i n r a t
l i v e r hom ogenates (B e r th e t e t a l . , 19 5 7 )•
G lucagon, a d r e n a lin , and
n o r a d r e n a lin in c r e a s e c y to p la sm ic p h o sp h o p y ru v ate c a rb o x y la s e
/
a c tiv ity
th ro u g h th e in d u c tio n o f cAMP w hich i s i n h i b i t e d by th e e r g o t a l k a l o i d
(Yeung and O liv e r , 1968) .
upon l i p o l y s i s .
D ihydro erg o tam in e h as shown v a r i a b l e e f f e c t s
S c r ia b in e and c o -w o rk ers ( 1968) r e p o r te d t h a t d ih y d ro -
e rg o tam in e p re v e n ts c a te c h o la m in e s tim u la tio n o f l i p o l y s i s i n r a t a d ip o s e
t i s s u e fra g m e n ts (B ooker an d C a lv e r t, 1967) and i s o l a t e d f a t c e l l s (F a in ,
1970)-
I n an i n v e s t i g a t i o n u s in g c e l l s i s o l a t e d from b o th brown and
w h ite r a t a d ip o se t i s s u e . Ward and F a in ( l 9 ? l ) fo u n d t h a t d ih y d ro erg o tam in e
• in f lu e n c e s th e i n t r a c e l l u l a r l e v e l o f cAMP by i t s e f f e c t on two d i f f e r e n t
enzym es.
The e r g o t a l k a l o i d i n h i b i t s p h o s p h o d ie s te ra s e r e s u l t i n g i n
in c r e a s e d l e v e l s o f cAMP and i t a ls o i n h i b i t s th e a c t i v a t i o n by c a te c h o la ­
m ines o f a d e n y la te c y c la s e r e s u l t i n g i n d e c re a s e d l e v e l s o f cAMP.
I
I
STATEMENT OF THE PROBLEM
The a c ti o n o f LH on th e c o rp u s lu teu m h a s n o t b een s u f f i c i e n t l y
c h a r a c te r iz e d t o th e p o i n t where we f u l l y u n d e rs ta n d th e i n t e r a c t i o n o f
t h e hormone w ith i t s r e c e p to r and t h e fo llo w in g m e d ia to ry s te p s tow ard
in c r e a s e d s t e r o i d o g e n e s i s .
One a p p ro a ch t o th e e l u c i d a tio n o f th e
mechanism b e h in d LH s tim u la tio n o f p r o g e s te r o n e s y n th e s is h a s been to
b re a k t h e system down i n t o i t s component p a r t s and a tte m p t t o f u l l y
c h a r a c te r iz e each p a r t .
C orpora l u t e a s l i c e s and hom ogenates have
p ro v id e d i s o l a t e d m odel sy stem s w here th e c o n c e n tr a tio n s o f endogenous
c o n t r o l l i n g c h em ica l components may b e m easured o r changed by exogenous
a d d itio n s .
By th e a d d itio n o f one c h em ica l component and th e m easure o f
th e re s p o n se t o t h i s a d d itio n by q u a n t i t a t i n g th e e f f e c t on o th e r com­
p o n e n ts , th e i n t e r - r e l a t i o n s h i p s betw een th e v a rio u s components o f th e
system may b e d e te rm in e d .
There a r e i n d ic a tio n s t h a t e r g o t a lk a lo id s
c o u ld d i r e c t l y in f lu e n c e th e i n t e r a c t i o n s betw een some o f th e im p o rta n t
m e d ia to rs i n th e LH s tim u la tio n o f p r o g e s te r o n e s y n th e s is .
One o f th e c h e m ic a l m e d ia to rs i n s te r o id o g e n e s is i n in c u b a te d c o rp o ra
l u t e a s l i c e s and hom ogenates h as b e en fo u n d t o be cAMP.
I n v e s tig a t o r s
have shown many tim e s t h a t e r g o t a l k a l o i d s b lo c k horm onal s tim u la tio n o f
cAMP i n s e v e r a l d i f f e r e n t ty p e s o f t i s s u e .
One example i s th e e rg o tam in e
i n h i b i t i o n o f th e a d r e n a l i n - p o t e n t i a t e d p ro d u c tio n o f cAMP w hich would
n o rm a lly in d u c e an in c r e a s e i n c y to p la sm ic p h o sp h o p y ru v ate c a rb o x y la se
a c t i v i t y (Yeung and O liv e r , 1968) .
I t c o u ld be p o s t u l a t e d t h a t e r g o t
\
36
a lk a l o id s may i n h i b i t IH - p o t e n t i a t e d p ro d u c tio n o f cAMP i n a s im ila r
f a s h io n i n th e corpus lu te u m .
A lthough a p rim a ry e f f e c t o f e r g o t a lk a l o id s on an im a l r e p ro d u c tiv e
d y s fu n c tio n s a p p e a rs t o r e s u l t from d i r e c t a c ti o n on th e p i t u i t a r y g la n d ,
t h e r e i s a l s o some i n d i c a t i o n o f a d i r e c t e f f e c t o f th e a l k a l o i d on th e
co rp u s lu te u m .
The p u rp o se o f t h i s r e s e a r c h has been t o d e te rm in e i f th e
p h y s io lo g ic a ll y a c t i v e e r g o t a l k a l o i d , e rg o c o m in e , d i r e c t l y e f f e c t s IH
*
s tim u la tio n o f p ro g e s te r o n e b io s y n th e s is i n th e co rp u s lu te u m .
The
in c u b a te d l u t e a l s l i c e system employed b y p re v io u s i n v e s t i g a t o r s p ro v id e s
t h e n e c e s s a ry i s o l a t e d c o n d itio n s r e q u ir e d t o i n v e s t i g a t e any d i r e c t
e f f e c t s e rg o c o m in e may have on s tim u la tio n o f s te r o id o g e n e s is .
The cow
p ro v id e s th e q u a n tity o f l u t e a l t i s s u e n eed ed f o r th e r e p e t i t i o n s o f
d i f f e r e n t e x p e rim e n ta l c o n d itio n s r e q u ir e d t o d e f in e th e sy stem and demon­
s t r a t e any e f f e c t o f e rg o c o m in e on t h a t sy stem .
The f i r s t s te p i n t h e i n v e s t i g a t i o n w ould b e t o e s t a b l i s h a l u t e a l
t i s s u e in c u b a tio n system c a p a b le o f show ing in c r e a s e d p ro g e s te r o n e s y n th e s is
w ith t h e a d d itio n o f IH t o th e in c u b a tio n m ed ia.
S tim u la tio n o f p r o g e s t e r ­
one s y n th e s is may b e m easured by d e te rm in in g " s p e c tro p h o to m e tric a lly th e
q u a n tity o f p ro g e s te r o n e e x tr a c te d from t h e IH t r e a t e d t i s s u e and m edia
and com paring i t t o th e q u a n tity o f p ro g e s te r o n e e x tr a c te d from a c o n t r o l
in c u b a tio n .
tio n o f
3
P ro g e s te ro n e s y n th e s is may a ls o b e m easured by th e in c o r p o r a -
H -a c e ta te i n t o de novo p r o g e s te r o n e .
'
Qnce IH s tim u la tio n i s
37
e s t a b l i s h e d j e rg o c o rn in e may be added t o th e in c u b a tio n t o see i f i t h as
an e f f e c t on p ro g e s te ro n e s y n th e s is *
S te r o id o g e n e s is i n l u t e a l t i s s u e h a s been shown t o be s tim u la te d by
p ro s ta g la n d in s and cAMP*
The e f f e c t o f e rg o c o rn in e on s tim u la tio n by
th e s e two a g e n ts sh o u ld be te s te d *
S in ce PGEg s tim u la te s p ro g e s te ro n e
s y n th e s is i n th e same manner as d o es LH, a s im ila r e f f e c t o f e rg o c o rn in e
on s tim u la tio n by b o th a g e n ts m ight be e x p e c te d .
However, i t sh o u ld be
k e p t i n mind t h a t s e p a r a te a d e n y la te c y c la s e r e c e p to r s i t e s f o r th e gonad­
o tr o p in and th e p r o s ta g la n d in have been d e m o n stra te d .
Exogenous cAIvIP
s tim u la te s s te r o id o g e n e s is m im icking th e r i s e o f endogenous cAMP due to
' LH o r PGEg s tim u la tio n o f a d e n y la te c y c la s e ,
% d e te rm in in g th e e f f e c t
o f e rg o c o rn in e on exogenous cAMP s tim u la tio n o f p ro g e s te ro n e s y n t h e s i s ,
th e s i t e o f a lk a l o id a c ti o n sh o u ld be n a rro w e d .
I f e rg o c o rn in e e f f e c t s
th e s tim u la to r y a c ti o n o f .cAMP in th e same way i t e f f e c t s LH s tim u la tio n ,
t h e , r e s u l t s would i n d i c a t e th e a l k a l o id a c ti o n to be beyond LH s tim u la tio n
o f a d e n y la te c y c la s e .
However, i f e rg o c o rn in e does n o t e f f e c t exogenous
cAMP s tim u la tio n i n th e same manner a s i t s e f f e c t on LH s t i m u l a t i o n , th e
r e s u l t s w ould in d i c a t e a p o s s ib le e f f e c t o f th e a l k a l o id on th e a d e n y la te
c y c la s e sy stem .
To t e s t a p o s s ib le e f f e c t o f e rg o c o rn in e on a d e n y la te c y c la s e , th e
a lk a l o id my be added t o a d e n y la te c y c la s e a s s a y system s s tim u la te d by LH,
A denylate c y c la s e a c t i v i t y may be m easured i n t i s s u e hom ogenates, and th e
a cc u m u la tio n o f cAMP from a d e n y la te c y c la s e may be m easured i n t i s s u e
s lic e s .
S in ce e r g o t a lk a l o id s have been im p lic a te d i n an i n h i b i t o r y
'
a c t i o n on a d e n y la te c y c la s e s tim u la tio n i n o th e r t i s s u e s , t h i s would seem
t o h e a p o s s ib le s i t e o f e rg o c o m in e a c t i o n i n co rp u s lu teu m t i s s u e .
The
e r g o t a lk a l o i d s have a l s o been shown t o e f f e c t th e a c t i v i t y o f cAMPp h o s p h o d ie s te r a s e .
A p o s s ib le e rg o c o m in e a c tio n on p h o s p h o d ie s te ra s e
a c t i v i t y sh o u ld a ls o be d e te rm in e d .
The r e s u l t s from th e e x p erim en ts o u tlin e d above sh o u ld d em o n strate
t h e s u s p e c te d d i r e c t e f f e c t o f e rg o c o m in e on th e s tim u la tio n o f
p ro g e s te r o n e b i o s y n t h e s i s .
The i n i t i a l e x p erim en t w i l l show i f such
an e f f e c t e x i s t s , and th e fo llo w in g ex p erim en ts w i l l be u se d t o c h a r a c t e r ­
i z e t h i s p o s s i b l e d i r e c t e f f e c t o f e rg o c o m in e on th e s tim u la tio n o f
s te r o i d o g e n e s i s .
EXPERIMENTAL MATERIALS AND METHODS
Corpus Luteum T iss u e
T iss u e f o r t h e i n v i t r o in c u b a tio n s tu d ie s was p r e p a r e d from b o v in e
c o rp o ra l u t e a .
The c o rp o ra l u t e a w ere e x t r a c t e d from cows th ro u g h a n ■
i n c i s i o n i n th e a n t e r i o r w a ll o f t h e v a g in a d o r s o l a t e r a l t o th e c e r v ix .
S in c e .th e c o rp o ra l u t e a a r e a t m axim al s tim u la to r y c a p a c ity betw een days
10-13 o f th e Cowi S e s t r u s c y c le (A rm strong and B la ck , 19663 In s k e e p , e t a l .
1967) , c a r e f u l r e c o rd s o f th e e s t r u s c y c le s o f th e d o n a tin g cows w ere k e p t,
and c o rp o ra l u t e a w ere ta k e n o n ly d u rin g t h i s th r e e day p e r i o d .
A f te r
ta k in g a c o rp u s lu te u m , t h e cow was a llo w e d a t l e a s t one f u l l , u n d is tu rb e d
c y c le b e f o r e a n o th e r c o rp u s luteum was ta k e n .
Each cow c o u ld p ro v id e up
to . f o u r c o rp o ra l u t e a b e f o r e a ccu m u lated s c a r t i s s u e p re v e n te d th e e x t r a c ­
t i o n o f a d d i t i o n a l c o rp o ra l u t e a .
Im m ediately a f t e r e x t r a c t i o n , t h e c o rp u s lu teu m was p la c e d in 0 .9 $
NaCl s o l u t i o n ( s a l i n e ) on i c e and ru s h e d t o th e c o ld room (4°C) f o r
f u r th e r p re p a ra tio n .
Once i n th e c o ld room th e e x tra n e o u s c o n n e c tiv e
t i s s u e was p e e le d from th e co rp u s lu teu m b e f o r e c u t t i n g i t i n t o q u a r t e r s . ,
Each q u a r te r was s l i c e d c a r e f u l l y w ith a r a z o r b la d e and th e s l i c e s
random ly p la c e d i n a p e t r i d is h c o n ta in in g c o ld s a l i n e o v e r a c i r c l e o f
f i l t e r p ap er.
Each s l i c e was a g a in c u t i n t o a t l e a s t t h r e e p ie c e s and
random ly p la c e d i n a seco n d p e t r i d is h o f c o ld s a l i n e .
Through t h i s
ran dom izing p ro c e s s i t was hoped t h a t r e l a t i v e l y homogenious sam ples
!.
'i
c o u ld b e ta k e n from th e t o t a l t i s s u e . I n d iv i d u a l t i s s u e sam ples were
/
ko
b l o t t e d on f i l t e r p a p e r b e f o r e w eig h in g them t o 175 + 5 mg sam ples f o r
in c u b a t io n .
T issu e I n c u b a tio n - . ■
The te c h n iq u e s o f i n v i t r o c o rp u s lu teu m t i s s u e in c u b a tio n a s d e s c r ib e d
by S u a re z , S oto and Demare ( i 9 6 0 ) and f u r t h e r d ev elo p ed b y Mason e t a l . ,
(1 9 6 4 ), and S e i f a r t an d H an sel ( 1968) p ro v id e a re a d y t o o l f o r t e s t i n g
th e e f f e c t s o f v a r io u s a g e n ts i n v i t r o on co rp u s lu teu m f u n c t i o n .
The
t i s s u e s l i c e sam ples w ere p la c e d i n 5 nil o f K reb s-H in g er b ic a r b o n a te b u f f e r
(U m breit e t a l . , . 1957) a t pH rJ.4 c o n ta in in g g lu c o se (2 m g/m l).
P r o g e s t­
ero n e s y n th e s is s tim u la to r s (LH, 200 n g /m l, HIH-IH-BSj PGE2 , 10 u g /m l,
p ro v id e d by D r. J . E. P ik e , U pjohn, I n c . ) w ere added w here a p p r o p r ia te
a f t e r a 10 m in u te p r e - in c u b a tio n o f t i s s u e i n m edia c o n ta in in g th e t e s t
s u b s ta n c e , e rg o c o m in e (75 uM, e rg o c o m in e hydrogen E ia le a te , p ro v id e d by
Sandoz P h a rm a c e u tic a ls , I n c . ) , when c a l l e d f o r .
S tim u la tio n o f th e l u t e a l
t i s s u e w ith 0 .0 2 M cAMP (A d e n o sin e -3 ' : 5 1 cy clic-m o n o p h o sp h o ric a c id ,
sodium s a l t . Plenum S c i e n t . R e s ., I n c . ) d id n o t le n d i t s e l f t o a p r e - .
in c u b a tio n due t o th e la r g e q u a n tity o f cAMP r e q u ir e d f o r s tim u la tio n .
I n some e x p erim en ts I uC i % - a c e t a t e (sodium s a l t , s p e c . a c t . 0.0 5 ci/mM,
ICN P h a rm a c e u tic a ls , I n c . ) was added t o t h e in c u b a tio n s t o m easure de novo
p ro g e s te r o n e s y n t h e s i s .
The in c u b a tio n s w ere c a r r i e d o u t i n 25 ml
e rle n m ey e r f l a s k s p la c e d i n a D ubnoff m e ta b o lic s h a k e r a t 37°C u n d er
95$ Og and 5$ COg.
A f te r two h o u rs , th e in c u b a tio n s w ere te r m in a te d by
f r e e z i n g th e in c u b a tio n sam ples on d ry i c e and s to r e d a t -20°C .
4^*
Each co rp u s lu teu m p ro v id e d enough t i s s u e f o r t h r e e t o f o u r e x p e r­
im e n ta l g roups c o n s is tin g o f f o u r o r f i v e d i f f e r e n t in c u b a tio n c o n d itio n s
p e r e x p e rim e n ta l g ro u p .
The e x p e rim e n ta l g roups c o n ta in e d . an in c u b a te d .
c o n t r o l , in c u b a te d t i s s u e W ith e r g o c o r a in e , an in c u b a tio n s tim u la te d by
IH o r PGE2 o r cAMP, an in c u b a tio n c o n ta in in g one o f th e s tim u la to r s p lu s
e rg o c o m in e .
The in c u b a tio n s w ere s t a r t e d s e q u e n tia lly , th ro u g h th e
e x p e rim e n ta l group b e f o r e c o n tin u in g on to th e seco n d e x p e rim e n ta l g ro u p ,
e tc .
T his mode o f i n i t i a t i n g in c u b a tio n s te n d s t o h e lp e lim in a te any,
b i a s due t o s l i g h t lo s s o f t i s s u e re s p o n se w ith tim e .
T h e ,tim e from when
th e c o rp u s luteum was ta k e n from th e cow u n t i l th e s t a r t o f th e l a s t
in c u b a tio n was k e p t u n d e r two h o u rs'.
P ro g e s te ro n e A n a ly sis
P ro g e s te ro n e a n a ly s is o f th e in c u b a te d t i s s u e fo llo w e d th e p ro c e d u re s
d e v elo p e d by S e i f a r t and H an sel '(1969).-
B efo re b e g in n in g th e e x tr a c ti o n
and a n a ly s is o f p r o g e s te r o n e , a s ta n d a r d amount o f "**4 C -4 -p ro g e s te ro n e
(a p p ro x . lcA cpm, s p e c . a c t . $ 2 .8 mCi/mM, C alato m ic , I n c . ) was added to
each in c u b a tio n sam ple t o p ro v id e a m easure o f ch em ical re c o v e ry a f t e r
c o m p le tio n o f th e a n a l y s i s .
A f te r th a w in g , th e .t i s s u e and m edia were
added t o a ro u n d b o tto m f l a s k and e x t r a c t e d i n TC m l o f b o i l i n g e th a n o l
f o r one h o u r, th e s u p e r n a ta n t was poure'd o f f and f i l t e r e d , and th e r e s id u e
r e f lu x e d f o r any a d d i t i o n a l 30 m in u te s i n $0 m l o f e th a n o l.
The combined
f i l t r a t e s w ere e v a p o ra te d on a r o t a r y e v a p o r a to r u n d e r vacuum i n a 60°C
w a te r b a t h .
F iv e sam ples w ere s p o tte d on each s i l i c a g e l p l a t e ( th ic k n e s s
42
0 .5 mm) c o n ta in in g 2 .5 mg f l u o r e s c e n t z in c s i l i c a t e / g s i l i c a g e l .
The
p l a t e s w ere f i r s t d e v elo p e d i n a s o lv e n t system o f h e x an e : e th y ! a c e t a t e
( 5 :2 ) and th e n a f t e r d r y in g , d e v elo p e d i n a second s o lv e n t system o f
"benzene: e th y ! a c e t a t e ( 3 : l ) i n th e same d im en sio n .
T h is r e s u l t e d i n t h e '
o ran g e pig m en ts m ig r a tin g t o th e to p o f th e p l a t e w h ile th e p ro g e s te r o n e ,
v i s u l a i z e d u n d er a U.V. lam p, was a t an Rf o f a b o u t 0 . 4 .
The a re a o f th e
p l a t e c o n ta in in g th e p ro g e s te r o n e was s c ra p e d o f f i n t o c e n tr if u g e tu b e s
and th e p ro g e s te r o n e e x tr a c te d from th e s i l i c a g e l by t h r e e , 2 m l washes
w ith d i e t h y l e t h e r .
A f te r e v a p o ra tio n u n d e r a stre a m o f II^, th e e x tr a c te d
p ro g e s te r o n e was f u r t h e r p u r i f i e d on s i l i c a g e l p l a t e s (T h ick n ess 0.2 5 mm)
d ev elo p ed i n a s o lv e n t system o f is o p ro p y l e t h e r : e th y ! a c e t a t e ( 5 : 2 ) .
The
p ro g e s te r o n e a r e a s o f th e p l a t e s w ere s c ra p e d i n t o sam ple v i a l s to w hich
3 m l o f m e th a n o l w ere ad d ed .
One column on each p l a t e was l e f t b la n k and
an e q u iv a le n t p ro g e s te r o n e a r e a was s c ra p e d from t h i s column to s e rv e a s
a b la n k f o r t h a t s e t o f p ro g e s te r o n e sam ples d u rin g sp ec tro p h c to m e tric
I
' •
a n a l y s i s . The sam ple v i a l s w ere th o ro u g h ly m ixed and c e n tr if u g e d b e fo re
s p e c tro p h o to m e tric q u a n t i t a t i v e a n a ly s is on a C a ry -l4 s p e c tro p h o to m e te r
a t a w av elen g th o f 240 nm.
A liq u o ts o f e ac h sam ple w ere e v a p o ra te d and
th e n d is s o lv e d i n 0 .4 $ PRO i n to lu e n e f o r a sse ssm e n t o f r a d i o a c t i v i t y
by a Beckman IS 100 l i q u i d s c i n t i l l a t i o n s p e c tro m e te r.
A ll sam ples were
c o r r e c te d t o 100$ re c o v e ry and p ro g e s te r o n e was e x p re s s e d p e r gram o f
lu te a l tis s u e
if
:
■
>3
A tim e e f f e c t on th e t i s s u e , m easu red from th e tim e i t was s l i c e d
u n t i l i t was p la c e d i n th e in c u b a to r , was c o r r e c te d f o r b y u se o f
c o v a ria n c e a n a ly s is ( S t e e l and T o rre y , i 9 6 0 ) .
S e p a ra tio n and A n a ly sis o f P r o g e s tin s
A l i q u i d - l i q u i d chrom atography sy stem was d ev elo p ed t o q u a n t i t a t e
t h e r e l a t i v e p e rc e n ta g e s o f p r o g e s te r o n e and 20 3 -d ih y d ro p ro g e ste ro n e
w hich a c c o u n t f o r th e t o t a l p r o gest i n s o f t h e corpus lu te u m .
A f te r
t r y i n g d i f f e r e n t s o lv e n t system s and flo w r a t e s , i t was fo u n d t h a t
p ro g e s te r o n e and 2 0 3 -d ih y d ro p ro g e ste ro n e can b e s e p a r a te d on a 2 mm X I m
C o r a s il C^g column e lu te d w ith M eth an o l: BLO (6 6 :3 4 ) a t a 0 .9 m l/m in .
flo w r a t e em ploying th e W aters A s s o c ., ALC 202 l i q u i d ch ro m ato g rap h .
The l u t e a l t i s s u e e x t r a c t was i n j e c t e d on th e column, and q u a n t i t a t i o n
was d e te rm in e d by d is c i n t e g r a t i o n o f t h e r e c o r d e r t r a c e .
P r e p a r a tio n o f Homogenates
j
I
H om ogenization o f b o v in e c o rp o ra l u t e a f o r th e a sse ssm e n t o f
a d e n y la te c y c la s e a c t i v i t y and cA M P-phosphodiesterase a c t i v i t y fo llo w e d
th e p ro c e d u re s o f S u th e rla n d , e t a l . , ( 1962) , and M arsh, ( l9 7 0 a ,b ) .
A f te r o b ta in in g a co rp u s luteum a s p r e v io u s ly d e s c r ib e d , i t was ta k e n
t o th e c o ld room (4 °C ), c u t i n t o q u a r t e r s , and th e c a p s u la r t i s s u e
rem oved.
A pproxim ately 2 .5 g o f l u t e a t i s s u e was s l i c e d and m inced w ith
a r a z o r b la d e b e f o r e p la c i n g i t i n th e g la s s ,m ortar o f a P o tte r - E lv e h j em
h o m o g en izatio n a p p a r a tu s .
A f te r th e a d d itio n o f 10 m l o f c o ld 0 .0 2 M
44
g O y cy lg ly cln e (pH 7 .4 ) c o n ta in in g O .O l M MgSO1^, th e h o m o g en izatio n was
a cco m p lish ed by no more th a n sev en up and down s tr o k e s o f th e m o to rd riv e n T e flo n p e s t l e i n th e g la s s m o r ta r .
The m o rta r was su rro u n d e d by
an i c e w a te r b a th d u rin g th e h o m o g e n iz atio n .
The hom ogenate was s t r a i n e d
th r o u g h ' c h ee se c l o t h t o remove c o n n e c tiv e t i s s u e and k e p t on i c e f o r
.
im m ediate u s e .
A ssessm ent o f A d en y late C y clase A c tiv ity
The m easurem ent o f a d e n y la te c y c la s e a c t i v i t y i n l u t e a l t i s s u e
hom ogenates was c a r r i e d o u t a s d e sc rib e d ..b y Marsh ( l9 7 0 a ,b ) .
One t e n t h
o f a m l o f hom ogenate s u sp e n sio n was added t o 0 .$ 1 ml o f a s o lu tio n
c o n ta in in g 25 um oles o f t r i s
(pH 7 * 4 ), 2 4 .4 um oles th e o p h y llin e , 1 .2
um oles ATP, 10 uC i ^H-ATP ( % -2 -A d e n o sin e 5 1- tr ip h o s o p h a te te tra s o d iu m ,
s p e c . a c t . 15 Ci/mm, Schwarz/M ann) , 1 .8 um oles Mg, 1 .5 4 um oles HaCl,
0 .1 2 mg b o v in e serum a lb u m in , and th e a p p r o p r ia te t e s t s u b s ta n c e .
The
t e s t s u b s ta n c e s w ere added i n th e fo llo w in g c o n c e n tr a tio n s ; LH (10 u g /m l) ,
PGEg (10 u g /m l) , and e rg o c o m in e (100 uM).
The r e a c t i o n m ix tu re was
in c u b a te d w ith s h a k in g i n a D ubnoff m e ta b o lic in c u b a to r , i n a i r , a t 37°0
f o r 10 m in u te s .
The r e a c t i o n was te r m in a te d by im m ersing th e in c u b a tio n
tu b e s i n a b o i l i n g w a te r b a th f o r t h r e e m in u te s .
B lanks w ere made by
b o i l i n g th e r e a c t i o n m ix tu re b e f o r e in c u b a tio n .
The fo rm a tio n o f cAMP
3
- 3
'
was a s s e s s e d by m e asu rin g th e c o n v e rs io n o f j H-ATP i n t o H-cAMP. '
The p u r i f i c a t i o n o f cAMP was a c h ie v e d th ro u g h jbhe c o m b in atio n o f
io n -e x c h an g e chrom atography and p r e c i p i t a t i o n w ith z in c s u l f a t e and
I
barium h y d ro x id e a s f i r s t d e s c r ib e d by K rish n a e t a l . , (1968) and th e n
by Forn and K rish n a (1 9 7 1 ).
The cAMP was found t o be 99$ pure a f t e r
t h i s a n a l y t i c a l p ro c e d u re as checked by io n -ex ch an g e and p a p er chroma­
to g ra p h y and e l e c t r o p h o r e t i c te c h n iq u e s , a s w e ll a s by c r y s t a l i z a t i o n to
c o n s ta n t s p e c i f i c a c t i v i t y .
x
The cAMP a s s a y p ro c e d u re s t a r t s w ith th e a d d itio n o f 0 .1 ml o f a
s o lu tio n (0 .2 mg/ml) o f c a r r i e r cAMP t o th e hom ogenate.
C e n tr if u g a tio n
o f th e homogenate p ro d u ces a s u p e r n a ta n t f l u i d w hich was chrom atographed
on a Dowex 50W-X8 (200-400 mesh) column ( 0 .4 X 4 . ^ cm ), p re p a re d by
p i p e t t i n g 2 ml o f a I i l s l u r r y o f r e s i n and w a te r i n t o a g la s s wool
pugged, d is p o s a b le p i p e t .
The column was e lu te d w ith w a te r and 2 ml
f r a c t i o n s were c o l l e c t e d .
The r e s u l t s o f a t r i a l ru n o f 't h e "ion-exchange
s e p a r a tio n of ATP, ADP, cAMP a re shown i n F ig . 4 .
e l u t e s o u t i n th e t h i r d f r a c t i o n .
About 75$ ° f t h e . cAMP
A fte r e l u t i o n from th e column, th e
cAMP f r a c t i o n was t r e a t e d w ith 0 .2 ml each o f 5$ ZnSO^ and 2 .5 $ B afO H ^.
I t h as been o b serv ed by K rish n a e t a l . , (1968) t h a t th e a d d itio n o f
ZnSO^ and Ea(HO)2 q u a n t i t a t i v e l y p r e c i p i t a t e s ATP, ADP, 5*-AMP, a d e n in e ,
and in o r g a n ic p h o sp h ate w h ile more t h a t 9 9 .9 $ o f th e cAMP rem ain s i n
th e s u p e r n a ta n t.
The r e s u l t i n g p r e c i p i t a n t i s c e n tr if u g e d and th e
s u p e r n a ta n t i s t r a n s f e r r e d to a n o th e r tu b e f o r a second BaSO^ p r e c i p i ­
ta tio n .
A fte r c e n t r i f u g a t i o n , an a l i q u o t o f th e r e s u l t i n g s u p e rn a ta n t
was m easured f o r a b s o r p tio n a t 260 nm to c a lc u la te th e re c o v e ry o f cAMP.
i
■
A 1 .0 ml a l i q u o t o f s u p e r n a ta n t was added t o 15 ml o f c o c k t a i l D(4 g PPO
4
46
Absorbance a t 260 ran
2 . 00 -
1 . 00 -
cAMP
12
14
16
18
20
ml o f e l u t a n t
F ig u re 4 . S e p a ra tio n o f ATP, cAMP, and AMP on a Dowex $0W X8 column
an d 100 g n a p h th a le n e i n I l i t e r o f d io x a n e ) f o r l i q u i d s c i n t i l l a t i o n
c o u n tin g by a Beckman ISlOO s c i n t i l l a t i o n s p e c tro m e te r.
A ssessm ent o f cAMP-P h o s p h o d ie s te ra s e A c tiv ity
The a s s a y f o r eA M P -phosphodiesterase c o n s i s t s o f a two s ta g e
enzym atic i s o t o p i c p ro c e d u re a s d e v elo p e d by Thompson and Appleman
(1 9 7 1 )•
P h o s p h o d ie s te ra s e i n th e hom ogenate h y d ro liz e d ^H-cAMP to
3H -51-AMP.
to
A sn ak e venom n u c le o tid a s e f u r t h e r h y d r o liz e d ^H -5’ -AMP
E -a d e n o s in e .
An io n -e x c h an g e r e s i n was u s e d t o b in d a l l o f th e
3H-CAMP----------- ---------------->- 3H -5' -AMP ------------------ >- ^H-Adenosine
hom ogenate
n u c le o tid a s e
p h o s p h o d ie s te ra s e
ciiarg ed n u c le o tid e s le a v in g % -a d e n o s in e a s t h e o n ly la b e l e d compound
t o be c o u n te d .
The hom ogenate in c u b a tio n f o r p h o s p h o d ie s te ra s e was th e same as
t h a t d e s c r ib e d f o r a d e n y la te c y c la s e e x c e p t f o r a change i n s u b s t r a t e
and th e e lim in a tio n o f th e o p h y llin e .
I n p la c e o f ATP, 0 .0 6 umoles o f
cAMP and 0 .3 uCi o f 3H-CAMP (A denosine- 8 - 3H-3 1, 5 1- c y c l i c p h o s p h a te ,
s p e c . a c t . 2 1 .8 Ci/mM, I CE, I n c . ) w ere added p e r 0 .5 1 m l o f in c u b a tio n
m ed ia.
The r e a c t i o n was i n i t i a t e d b y th e a d d itio n o f 0 .1 m l o f
hom ogenate and in c u b a te d w ith sh a k in g i n a D ubnoff. m e ta b o lic in c u b a to r
i n a i r , a t 37°C f o r 15 m in u te s b e f o r e te r m in a tin g by im m ersion i n a
b o i l i n g w a te r b a th f o r t h r e e m in u te s .
48
A f te r th e b o i l e d in c u b a tio n s w ere c o o le d , th e y w ere p u t back i n t o
th e m e ta b o lic in c u b a to r and 0 .1 m l ( l mg/ml) o f snake venom n u c le o tid a s e
( Ophiophagus h annah, Sigma Chem.) ad d ed .
T his r e a c t i o n was in c u b a te d f o r
30 m in u te s a t 37°C b e f o r e te r m in a tin g by t h e a d d itio n o f I ml o f a 1 :3
s l u r r y o f AG- 1-X2 (200-400 mesh) i n w a te r .
A f te r c e n t r i f u g a t i o n a
1 .0 m l a l i q u o t was added t o 1$ m l o f c o c k t a i l D f o r a sse ssm e n t o f r a d i o ­
a c t i v i t y by l i q u i d s c i n t i l l a t i o n c o u n tin g .
M easurement o f C y c lic AMP S y n th e s is i n T iss u e S li c e s
C y c lic AMP fo rm a tio n was m easu red a s d e s c rib e d p r e v io u s ly by
Humes e t a l . , ( 1969) and Kuo and DeRenzo ( 1969) .
T h is ra d io m e tric
a s s a y p e rm its th e i n v i t r o m easurem ent o f a d e n y la te c y c la s e a c t i v a t i o n
i n th e i n t a c t c e l l by t h e d e te r m in a tio n o f new ly s y n th e s iz e d cAMP.
The b o v in e co rp u s luteum t i s s u e was c o l l e c t e d and s l i c e d as p r e ­
v io u s ly d e s c r ib e d f o r th e p r e p a r a t io n o f t i s s u e s l i c e in c u b a tio n .
T iss u e
sam ples o f a p p ro x im a te ly 175 mg w ere i n i t i a l l y in c u b a te d f o r one h o u r i n
1 .5 m l o f K re b s-R in g e r b ic a r b o n a te b u f f e r (pH 7*4) w ith o n e - h a lf th e
p r e s c r i b e d Ca++ c o n c e n tr a tio n and c o n ta in in g I uC i o f 1^C -S-U denine
(52 mCi/mM, Schwarz /M ann) .
The 10 m l e r lenm eyer in c u b a tio n v e s s e ls w ere
f lu s h e d w ith 95$ 0 : 5$ CO- and s to p p e r e d b e fo re in c u b a tio n a t 37°C i n
th e D ubnoff m e ta b o lic in c u b a to r .
The p u rp o s e o f t h i s one h o u r p r e ­
in c u b a tio n i s t o p ro d u ce i n t r a c e l l u l a r 1^C-ATP from th e 1^C-a d e n in e .
A f te r th e one h o u r p r e - in c u b a tio n , 0 .5 m l o f \20 mM th e o p h y llin e
and I mg/ml b o v in e serum album in was added t o each in c u b a tio n v e s s e l
49
a lo n g w ith t h e a p p r o p r ia te s tim u la tin g a g e n ts an d e r g o c o m in e .
Each
in c u b a tio n v e s s e l was a g a in f lu s h e d w ith 95# Og : 5# COg b e f o r e f u r t h e r
in c u b a tio n f o r v a r io u s tim e p e r io d s .
The in c u b a tio n s w ere te rm in a te d
by f r e e z i n g th e t i s s u e and m edia on dry. i c e .
The sam ples w ere th e n
s to r e d a t -20°C u n t i l a n a ly s is f o r "^C-cAMP.
B efo re a n a l y s i s , 0H-CAMP (3 X K r cpm) was added t o th e in c u b a tio n
sam ples t o m easure re c o v e ry a lo n g w ith 0 .5 m l o f 0.0 5 M t r i g b u f f e r
(pH 7 . 5 ) c o n ta in in g 1 .2 5 mM cAMP a s c a r r i e r and 0 .0 1 M th e o p h y llin e .
The in c u b a tio n sam ples w ere th e n hom ogenized by th e P o tt e r - E lv e h jem
a p p a r a tu s ,
A f te r th e hom ogenate was p r e c i p i t a t e d w ith 0 .2 m l o f 20#
t r i c h l o r a l a e e t i c a c id and c e n tr if u g e d , th e s u p e r n a ta n t was p r e c i p i t a t e d
w ith 0 .1 m l o f 5# ZhSO^ and 0 .1 m l o f 2 . 56# Ba(OH)^, c e n tr if u g e d , and
th e n p r e c i p i t a t e d a g a in w ith BaSO, and c e n tr if u g e d .
The s u p e r n a ta n ts
w ere t r a n s f e r r e d t o 10 m l e rle n m ey e r f l a s k s and e v a p o ra te d i n a vacuum
oven a t 60°C.
The sam ples were; ta k e n up i n 0 .5 m l o f w a te r and p la c e d
on a 0 .4 X 4 .5 cm, Dowex 5OW-X8! (200-400 mesh) column w hich was e lu te d
b y w a te r .
The 2 m l f r a c t i o n c o n ta in in g t h e cAMP was added t o 15 ml o f
c o c k t a i l D f o r a ss e ss m e n t o f 1^C and % a c t i v i t y by l i q u i d s c i n t i l l a t i o n
c o u n tin g .
The
-cAMP p ro d u ced by t i s s u e a d e n y la te c y c la s e was
c o r r e c te d t o 100# re c o v e ry from th e re c o v e ry o f %-cAMP.
The in c o r p o r a tio n o f
-a d e n in e i n t o ^C-ATP was a l s o a s s e s s e d
i n t h i s t i s s u e s l i c e sy ste m . • The l u t e a l t i s s u e was p r e p a r e d and in c u V
;
b a te d i n th e same m anner a s th e p r e - in c u b a tio n s te p d e s c r ib e d above.
/
50
However, t h e p r e - in c u b a tio n s w ere s to p p e d a t v a r io u s tim e i n t e r v a l s byf r e e z i n g on d ry i c e . .
A denine and ATP w ere e x tr a c te d from t h e t i s s u e a s d e s c r ib e d above
em ploying th e h o m o g en izatio n p ro c e d u re , and th e r e s u l t i n g s u p e r n a ta n t
was iy o p h i l i z e d .
Each r e s id u e was r e d is s o lv e d i n 0 .2 m l o f a s o lu tio n
c o n ta in in g 1 .2 5 mM ATP and 1 .2 5 %iM a d e n in e .
A $0 u l a l i q u o t o f each
sam ple was s p o tte d on c e l l u l o s e (M tf-c e llu lo se powder 300G, 250 m icrons
t h i c k ) t h i n - l a y e r p l a t e s f o r s e p a r a tio n o f th e a d e n in e and ATP. ■A f te r
d e v e lo p in g th e c e l l u l o s e p l a t e s u s in g th e s o lv e n t sy stem o f b u ta n o la c e to n e - a c e tic acid-5% MH^OH-water (4 .5 :1 » 5 :1 :1 :2 , by volum e) (Marsh and
LeMfeiire, 1 9 7 4 ), th e p l a t e was view ed u n d e r a U.V. l i g h t an d th e a p p ro ­
p r i a t e a r e a s ' c o rre sp o n d in g t o ATP an d a d e n in e s ta n d a rd s w ere s c ra p e d
i n t o s e p a r a te v i a l s .
Each sam ple was e lu te d w ith 1 .5 m l o f w a te r and
a 1 .0 m l a l i q u o t was added t o 15 m l o f c o c k t a i l D f o r l i q u i d s c i n t i l l ­
a t i o n c o u n tin g .
RESULTS MD DISCUSSION
The i n v i t r o in c u b a tio n system p ro v id e s a means o f i n v e s t i g a t i n g th e
d i r e c t e f f e c t o f an a g e n t su ch a s e r g o t a l k a l o i d on p ro g e s te r o n e p ro d u c ­
t i o n i n i s o l a t e d c o rp u s luteum t i s s u e .
The e n t i r e s te r o id o g e n ic p ro c e s s
i s c o n se rv e d i n t h e t i s s u e s l i c e in c u b a tio n w hich in c lu d e s ; l ) th e endo­
genous p r e c u r s o r s o f p r o g e s te r o n e , 2 ) t h e enzym atic pathw ay t o c o n v e rt
th o s e p r e c u r s o r s t o p r o g e s te r o n e , and 3 ) th e c o n tr o l a p p a ra tu s by w hich
hormones s tim u la te th e en zy m atic pathw ay t o in c r e a s e th e p ro d u c tio n o f
p r o g e s te r o n e .
T a n g ib le q u a n t i t i e s o f p r o g e s te r o n e , w hich can be m easured
s p e c tr o p h o to m e tr ic a lly a r e p ro d u ced by a s l i t t l e a s 100 mg o f in c u b a te d
tis s u e .
The p ro g e s te r o n e b io s y n t h e t i c pathw ay may b e s tim u la te d by th e
a d d itio n o f LH (Mason e t a l . , ' 1962) , p r o s ta g la n d in s (S p e ro f f and Ramwell,
1 9 7 0 ), o r cAMP (M arsh and S a v a rd , i 9 6 0 ) t o th e in c u b a tio n m ed ia.
The
r e s u l t i n g p ro d u c tio n o f m e asu ra b le in c r e a s e s i n p ro g e s te r o n e t i s s u e l e v e l s
s tim u la te d by t h e s e a g e n ts , p ro v id e s one way o f i n v e s t i g a t i n g th e r e g u l a ­
t o r y mechanism o f s t e r o id o g e n e s is .
’
r a d i o a c t i v e l y la b e l e d by exogenous
The endogenous p r e c u r s o r p o o l may b e
l4
3
C- a c e t a t e o r uH -C h o le s te ro l t o p r o ­
v id e th e i n v e s t i g a t o r a n o th e r means o f m o n ito rin g th e s te r o id o g e n ic
p ro c e ss.
TIie E f f e c t o f E rg o c o rn in e on LH S tim u la te d P ro g e s te ro n e S y n th e s is
When e rg o c o m in e (ECO) was added t o th e in c u b a tio n m edia o f th e
i n v i t r o in c u b a tio n sy stem , i t had th e e f f e c t o f s u p p re s s in g p r o g e s te r ­
one b i o s y n t h e s i s .
The co rp u s luteu m h as b een a p r e v io u s ly s u s p e c te d s i t e
52
o f ECO a c tio n ' ( D ra ic e r and S t r a u s s , 1970; S h e le sn y ak , 1958; Z eilm aker
an d C a rls o n , 1962; L obel e t a l . , 1966) , b u t th e d i r e c t e f f e c t o f ECO
on l u t e a l t i s s u e had n o t b een d e m o n stra te d .
As can b e see n i n F ig u re 5 ,
ECO r e s u l t s i n d e c re a s e d p ro g e s te r o n e s y n th e s is compared t o c o n tr o l in c u b a
tio r is a s d e te rm in e d from th e s p e c tro p h o to m e tric m easurem ent o f th e t o t a l
p ro g e s te ro n e c o n te n t o f t h e in c u b a tio n s a m p le s .
IM in cu b ated l u t e a l
t i s s u e sam ples w ere fo u n d t o c o n ta in 35-50 ug o f p ro g e s te r o n e p e r g o f
lu te a l tis s u e .
The a d d itio n o f IH t o th e t i s s u e in c u b a tio n r e s u l t s i n a
s tim u la te d l e v e l o f p r o g e s te r o n e s y n th e s is com parable to t h a t o b serv ed by
o th e r i n v e s t i g a t o r s ( S e i f a r t and H a n se l, 1968; Marsh an d S a v a rd , 1966';
V eenhuizen e t a l . , 1 9 7 2 ).
When ECO was added a lo n g w ith IE , th e s tim u la ­
t o r y a c ti o n o f t h e hormone was s u p p re s s e d .
The a c t u a l p ro g e s te r o n e v a lu e s and t h e i r s t a t i s t i c a l s ig n if ic a n c e
a r e e x p re s s e d i n T able I .
There i s a s i g n i f i c a n t d if f e r e n c e ( P < 0 .0 l) ,
I
a s d e te rm in e d by th e s t u d e n t ’s j t e s t , betw een th e IH s tim u la te d c o n d itio n
I
an d t h e IH p lu s ECO c o n d itio n no m a tte r how th e d a ta i s e x p re s s e d .
E rg o c o m in e a t 75 uM s u p p re s s e d 80$ o f th e p ro g e s te r o n e b io s y n th e s is
p ro d u c e d by m axim al IH s tim u la tio n (200 ug H /m l).
The p ro g e s te ro n e con­
t e n t i s lo w e re d by t h e a d d itio n o f ECO t o th e IH in c u b a te d c o n d itio n to
a l e v e l t h a t does n o t d i f f e r s i g n i f i c a n t l y (p<0 . 0l ) from th e in c u b a te d
c o n tro l.
VThen th e d a ta was t r e a t e d by c o v a ria n c e a n a l y s i s , a s i g n i f i c a n t
d if f e r e n c e (p^O .O l) i s o b se rv e d betw een in c u b a te d t i s s u e and t i s s u e
i
'
in c u b a te d w ith ECO i n th e ab sen ce o f exogenous IE . C o v arien ce a n a ly s is
53
Total Progesterone Level
Experimental Condition
Flgxire 5 . The e f f e c t o f ECO on LH s tim u la te d and c o n tr o l t i s s u e
s te r o id o g e n e s is . ( I ) in c u b a te d c o n t r o l , (E) in c u b a te d w ith 75 uH
ECO, (L ) in c u b a te d w ith 2OOng LH/m l, (LE) in c u b a te d w ith LH and
ECO.
T a b le I .
E ffe c ts
o f LH a n d ECO o n p r o g e s t e r o n e b i o s y n t h e s i s
T o ta l p ro g e s te ro n e
]no. o f
CL
no. of
sam p les
In c u b a te d
c o n tro l
ECO
in
lu te a l
tis s u e
u g /g tis s u e *
LH
LH + ECO
C o v arian ce
c o rre c te d
3
10
1 6 9 .2 + 6 .8a
1 3 9 .5 + 6 .8b
2 4 6 .3 + 6 .8C
1 8 7 .9 + 6 .8a
U n c o rre c te d
3
10
1 5 6 .5 + 8 .Oab
1 3 8 .3 + 8 .Oa
2 2 0 .3 + 8 .Oc
1 7 5 .1 + 8 .Ob
C o v arian ce
c o r r e c te d
7
21
1 5 8 .6 + 5 .5 a
2 1 4 .4 + 5 .5b
1 7 2 .1 + 5 .5a
U n c o rre c te d
7
'21
1 4 2 .2 + 5 .7a
2 0 0 .4 + 5 .7b
1 6 0 .1 + 5 .7a
*A11 v a lu e s a r e l e a s t s q u a r e means ± SE
a b c v a lu e s w ith d i f f e r e n t s u p e r s c r i p t s d i f f e r s i g n i f i c a n t l y by p<c6»01
-
55
a tte m p ts t o c o r r e c t th e d a ta f o r d e c re a s e d p ro g e s te r o n e b io s y n th e s is due
t o th e e f f e c t o f tim e o f t i s s u e p r e p a r a t io n b e fo re th e s t a r t o f in c u b a tio n
(Moody and H a n se l, 1969)«
As can b e se e n i n T able I th e c o v a ria n c e
a n a ly s is r e s u l t s i n s l i g h t l y h ig h e r p ro g e s te r o n e v a lu e s and d e c re a se i n
th e s ta n d a r d e r r o r o f th e mean.
VZhether o r n o t th e s l i g h t d e p re s s iv e e f f e c t o f ECO on p ro g e s te ro n e
s y n th e s is i n t i s s u e in c u b a tio n s i s due t o th e e f f e c t o f ECO on endogen­
ous hormone s tim u la tio n i s a p o i n t o f s p e c u la tio n .
A ccording t o Efiswender
e t a l . , ( 1969) th e b lo o d serum l e v e l s o f IH i n th e cow a r e 12-60 n g /m l on
t h e day o f e s tr o u s and th e n f a l l o f f t o a 1 . 5 - 2 .5 n g /m l b a s a l l e v e l d u rin g
th e l u t e a l p h ase.
T h e re fo re , when th e co rp u s luteum i s ta k e n from th e
cow, th e t i s s u e w ould c o n ta in some unknown l e v e l o f endogenous IE .
Con­
s i d e r i n g th e i n v i t r o ex p erim en ts show a minimum e f f e c t i v e dose o f 10 n g
o f IH /g o f t i s s u e o r 2 ng o f IH /m l o f in c u b a tio n m edia (Mason and S a v a rd ,
196^ ) f o r s tim u la tio n o f p ro g e s te r o n e s y n th e s is o v e r c o n t r o l in c u b a tio n ,
I
i t seems v e ry p o s s ib le t h a t an !endogenous LH s tim u la tio n i s o c c u r rin g i n
t h e in c u b a te d c o n t r o l s .
;
A no th er o b s e r v a tio n i n d i c a t i v e o f a p o s s i b l e endogenous g o n a d o tro p in
e f f e c t may b e se e n i n t h e r e s u l t s o f a d d in g purom ycin t o in c u b a tin g l u t e a l
tis s u e .
Purom ycin e f f e c t i v e l y b lo c k s IH s tim u la tio n o f p ro g e s te ro n e sy n ­
t h e s i s b y i t s i n h i b i t o r y a c ti o n o f p r o t e i n s y n th e s is .
VZhen S avard e t a l . ,
( 1965) w ere i n v e s t i g a t i n g t h i s purom ycin e f f e c t , th e y a l s o o b serv ed a
s l i g h t d e c re a s e i n p r o g e s te r o n e s y n th e s is when purom ycin was added t o '
56
•I n c u b a tin g t i s s u e i n th e a b se n c e o f e x o g e n o u s'LH.
T h is e f f e c t i s very
s i m i l a r t o th e s l i g h t d e c re a s e i n p ro g e s te r o n e s y n th e s is o b serv ed when
ECO was added t o in c u b a tin g t i s s u e .
"When i n v e s t i g a t i n g th e e f f e c t o f a s i n g l e i n j e c t i o n o f ECO on th e
p r o g e s tin c o n te n t o f r a t o v a r i e s , L in d n er an d S h elesn y ak ( 1967) fo u n d t h a t
ECO c au sed a s h a rp r i s e i n o v a ria n 2 0 « -d ih y d ro p ro g e ste ro n e w h ile th e p r o ­
g e s te ro n e l e v e l d e c re a s e s r e s u l t i n g i n re d u c e d p h s i o l o g i c a l a c t i v i t y .
T h is
o b s e r v a tio n n e c e s s i t a t e d t h e d e te r m in a tio n o f 20 g -d ih y d ro p ro g e ste ro n e l e v e l s
i n th e p r e s e n t i n v i t r o system c o n ta in in g ECO i n t h e in c u b a tio n m ed ia.
The r e l a t i v e p e rc e n ta g e o f 20 B -d ih y d ro p ro g e ste ro n e o f t o t a l p r o g e s tin s
i n th e b o v in e c o rp u s lu teu m t i s s u e in c u b a tio n s a g re e w ith th e n o rm al 5 t o
20^ ra n g e o b s e rv e d by o th e r i n v e s t i g a t o r s (V eehuizen e t a l . , 1972; G o rsk i
e t a l . , 1958; Gomes and E rb , 1965) .
In T ab le I I a r e e x p re s s e d th e a v e ra g e p e rc e n ta g e s o f p ro g e s te ro n e
and 2 0 S-d ih y d ro p ro g e s te ro n e f o r th e f o u r in c u b a tio n c o n d itio n s .
The
p re s e n c e o f ECO i n th e in c u b a tio n m edia d id n o t seem t o in f lu e n c e th e
T ab le I I . The e f f e c t o f ECO and IH on th e r a t i o o f
203-d ih y d ro p ro g e s te ro n e t o p r o g e s te r o n e .
In c u b a tio n
C o n d itio n
In c u b a te d
C o n tro l
ECO
LH
LH + ECO ..
$ p ro g e s te r o n e
8 3 .2 + 1 .2
8 3 .3 ± 1 .4
8 6 .8 + 0 .4
8 6 .3 + 0 .5
c
Jo 203-d ih y d ro ­
p ro g e s te r o n e -
1 6 .8 ± 1 .2 . 1 6 .7 ± 1 .4
. 1 3 .2 ± 0 .4
1 3 .7 ± 0 .5
p r o g e s tin r a t i o .
IM lik e th e i n v iv o e f f e c t o b se rv e d i n r a t s , th e r a t i o
o f 20 S -d ih y d ro p ro g e ste ro n e t o p ro g e s te r o n e rem ain ed th e same u n d er th e
v a rio u s i n v i t r o tr e a tm e n t c o n d itio n s o f t h i s s tu d y .
An ex p erim en t was co n d u cted t o e s t a b l i s h th e e f f e c t i v e ra n g e o f ECO
on LH s tim u la te d l u t e a l t i s s u e .
The i n h i b i t o r y a c ti o n o f ECO was fo u n d
t o b e dep en d en t upon ECO c o n c e n tr a tio n .
As can b e se e n i n H g . 6 , ECO
re a c h e s i t s m axim al s u p p re s s io n o f IH s tim u la tio n a t a c o n c e n tra tio n o f
a b o u t 100 uM.
In c u b a tio n s w ere c o n d u cted o u t t o ECO l e v e l s e q u iv a le n t t o
500 uM w ith o u t a f u r t h e r r e d u c tio n i n IH s tim u la tio n o f p ro g e s te ro n e b i o ­
s y n th e s is .
A p r e c i p i t a t e o f ECO a p p e a rs i n th e in c u b a tio n m edia b e fo re
r e a c h in g th e 100 uM l e v e l w hich may l i m i t any c o n c e n tr a tio n e f f e c t above
t h i s ECO l e v e l .
A s e r i e s o f e x p erim en ts w ere p e rfo rm ed u s in g th e in c o r p o r a tio n o f
3
.
H -a c e ta te i n t o p ro g e s te r o n e a s an i n d i c a t i o n o f de novo p ro g e s te ro n e b i o lI
s y n th e s is i n in c u b a te d l u t e a l t i s s u e . The t h r e e l e v e l s o f IH d id n o t seem
j
t o show r e a l d if f e r e n c e s i n s tim u la te d in c o r p o r a tio n (T ab le I I I ) in d i c a t i n g
th e system t o b e m axim ally s tim u la te d a t even th e lo w e st IH . l e v e l em ployed.
T h e re fo re , t h e d a ta was combined t o show th e a v e ra g e - H - a c e ta te in c o r p o r ­
a tio n s .
In, T ab le I I I t h e d a ta i n d i c a t e s t h a t in c o r p o r a tio n i n t o th e p r o ­
g e s te ro n e o f IH s tim u la te d t i s s u e i s s i g n i f i c a n t l y (p < 0 .0 l) g r e a t e r th a n
in c o r p o r a tio n i n t o in c u b a te d c o n tr o ls e When ECO was added t o th e in c u ­
b a tio n s a lo n g w ith LH9 th e in c o r p o r a tio n l e v e l d ro p s back t o t h a t o f th e
in c u b a te d c o n t r o l s .
T h e r e f o r e 9 th e in c o r p o r a tio n o f -'H -a c e ta te i n t o
p ro g e s te ro n e d u rin g t i s s u e in c u b a tio n s r e f l e c t th e r e s u l t s o f th e
58
”
60 -
TO 40 -
C oncentration of ECO (uM)
F ig u re 6 . The e f f e c t o f ECO c o n c e n t r a t i o n on LH s tim u la t e d
s te ro id o g e n e s is .
59
I
T able I I I .
The e f f e c t o f ECO on LH s tim u la te d p ro g e s te ro n e b io s y n th e s is
a s m easured by % - a c e t a t e in c o r p o r a tio n
EXPERIMENTAL CONDITIONS
LH c o n c e n tra tio n
In c u b a te d c o n tr o l
LH'+ ECO
( d p m /g .tis s u e ) x 1 0 -3
(n g /m l)
0
LH
2 9 .3 ± 2 .3 * a
20
3 9 .1 ^ 6 .6 b
2 7 .5 + 2 .3 *
100
3 7 .3 ± 6 .2 b
2 8 .9 + 2 .4 a
200
4 2 .9 + 6 .6b
2 9 .7 + 3 .4 *
Average ^ H -a c e ta te
in c o r p o r a tio n
2 9 .3 * 2 .3 °
40.'6±6.4d
2 8 .7 ± 2 .6 c
S p e c if ic a c t i v i t y
(dpm/ug p ro g e s te r o n e )
185+21
189*34
168±26
* Each e x p erim en t r e p r e s e n ts th e mean ± s ta n d a rd e r r o r o f d e te rm in a tio n s
from f o u r t i s s u e in c u b a tio n s ..
v a lu e s w ith d i f f e r e n t s u p e r s c r ip ts d i f f e r s i g n i f i c a n t l y by p < 0 .0 5
v a lu e s w ith d i f f e r e n t , s u p e r s c r ip ts d i f f e r s i g n i f i c a n t l y by p < 0.01
60
s p e c tro p h o to m e tric d e te r m in a tio n s ,
The s p e c i f i c a c t i v i t i e s o f th e
in c u b a te d c o n t r o l , IH s tim u la te d , and IH and ECO in c u b a tio n w ere a b o u t
th e same i n d i c a t i n g t h a t th e e x te n t o f th e % - a c e t a t e in c o r p o r a tio n i s
p a r a l l e l f o r th e d i f f e r e n t in c u b a tio n c o n d itio n s .
s p e c i f i c a c t i v i t y im ply t h a t
The r e s u l t s in
IH in c r e a s e d p ro g e s te ro n e s y n th e s is w ith ­
o u t changing th e p r e c u r s o r p o o ls from th o s e o b serv ed i n c o n tr o l in c u b a ­
tio n s ,
Savard and co -w o rk ers (19 6 4,1965 and I 9 6 6 ) have shown a s l i g h t
in c r e a s e i n s p e c i f i c a c t i v i t y o f th e IH s tim u la te d t i s s u e compared t o
c o n tro ls t
T h is im p lie s t h a t IH i s s tim u la tin g
o v er c o n tr o l in c u b a tio n s a s w e ll as s tim u la tin g
I n lo o k in g a t
in c o r p o r a tio n o f a c e ta te
p ro g e s te ro n e s y n th e s is .
t h e i r d a ta th e y show v a r ia b le s p e c i f i c a c t i v i t y f o r d i f f ­
e r e n t e x p erim en ts and many o f th e s e v a lu e s a re th e same a s .th o s e s p e c i f i c
a c t i v i t i e s see n i n th e c o n t r o l .
I t sh o u ld a ls o be n o te d t h a t i n t h e i r
s p e c i f i c a c t i v i t y d e te r m in a tio n s 10-20 uC i o f ^ C - a c e t a t e was used p e r
in c u b a tio n , w hereas o n ly I uC i o f % - a c e t a t e was used f o r o b ta in in g th e
d a ta i n T ab le 111,
The im p o rta n t p o in t made by th e d a ta i n T able I I I i s
t h a t EGO i n h i b i t s th e IH s tim u la tio n o f in c o r p o r a tio n o f a c e ta te in to
p ro g e s te r o n e .
T h is s u p p o rts th e same c o n c lu s io n drawn from th e sp e c tro p h o ­
to m e tr ic d e te r m in a tio n s o f t o t a l p ro g e s te ro n e from th e t i s s u e in c u b a tio n s .
E f f e c t o f ECO on P r o s ta g la n d in S tim u la tio n
P r o s ta g la n d in s ' have th e
p a ra d o x ic a l e f f e c t o f d e c r e a s in g th e c i r ­
c u la t in g p r o g e s tin l e v e l when a d m in is te re d su b c u ta n e o u sly t o l i v e a n im a ls
and in c r e a s in g th e p ro g e s te ro n e l e v e l o f i n v i t r o b ovine co rp u s lu teu m
t i s s u e in c u b a tio n s ( S p e r o f f and Ram well, 1970; Bedwani and H o rto n , 1 9 6 8 ),
61
S in c e PGE^ was r e p o r te d t o "be th e m ost a c t i v e s tim u la to r o f th e p r o s t a ­
g la n d in s , i t was u t i l i z e d t o s tim u la te p ro g e s te ro n e s y n th e s is i n th e
s tu d y o f th e e f f e c t o f ECO on s tim u la tio n o f s te r o id o g e n e s is .
When PGE2
(10 u g /m l) was added t o th e i n v i t r o in c u b a tio n sy ste m , i t s i g n i f i c a n t l y
(p<O .O l) s tim u la te d p ro g e s te r o n e s y n th e s is compared t o th e in c u b a te d
\
c o n tr o l (T able IV ). The p ro g e s te r o n e l e v e l o f th e PGE^ in c u b a tio n s was
j u s t u n d e r b u t com parable t o th e LH s tim u la te d l e v e l . ( F i g . ? ) .
When ECO
was added a lo n g w ith PGE2 t o th e in c u b a tio n m ed ia, t h e ' p ro g e s te r o n e l e v e l
dropped s i g n i f i c a n t l y (p<O .O l) below t h e ..PGE2 s tim u la te d l e v e l .
The ECO
s u p p re ss e d PGE2 s tim u la tio n o f p r o g e s te r o n e s y n th e s is t o a l e v e l t h a t
d i d n o t s i g n i f i c a n t l y d i f f e r from t h a t o f in c u b a te d c o n t r o l s .
Ih e s t a t ­
i s t i c a l r e s u l t s w ere th e same when th e d a ta was a d ju s te d f o r a tim e e f f e c t
by c o v a ria n c e a n a l y s i s .
The e f f e c t o f ECO on e i t h e r LH s tim u la tio n o r PGE2 s tim u la tio n
a p p e a rs t o b e a b o u t th e sam e.
T h is i s n o t s u r p r i s i n g s in c e th e stim u ­
l a t o r p r o p e r t i e s o f p r o s ta g la n d in s c l o s e l y p a r a l l e l th o s e o f LH s tim u la ­
tio n .
S i m i l a r i t i e s a r e o b se rv e d i n s p e c i f i c a c t i v i t y o f th e in c re a s e d
-ik
in c o r p o r a tio n o f ~ C -a c e ta te i n t o p r o g e s te r o n e , tim e re s p o n s e c u rv e s ,
an d cy clo h ex im id e i n h i b i t i o n (S p e r o ff and Barnwell, 1970)*
a n t i s th e f a c t t h a t b o th s tim u la te a d e n y la te c y c la s e .
More im p o rt­
The common
i n h i b i t o r y e f f e c t by ECO i s i n d i c a t i v e o f some common in te r m e d ia te b etw een
LH and PGE2 s tim u la tio n o f p ro g e s te r o n e s y n th e s is .
62
T a b le IV .
E f f e c t o f ECO on PGEg s t i m u l a t i o n o f s t e r o i d o g e n i s i s in
lu te a l tis s u e
I n c u b a t io n
c o n d itio n
In c u b a te d
c o n tro l
No. O b s e rv a tio n s
9
LH '
PGE2
9
15
PGE2+ECO
15'
P r o g e s te r o n e ( u g /g )
u n c o r r e c te d
1 4 8 .3 ^ 1 1 .5a
+
h
2 1 5 .3 - 1 1 .5U 2 0 3 .s i g .Ob
1 4 7 .6 i9 .0 a
P r o g e s te r o n e ( u§ / s )
c o v a r ia n c e
c o rre c te d
16 1 .7 ^8 .6 ®
2 3 4 .8 ^ 8 .6b
+
b
2 2 6 .1 - 6 .8 °
1 7 9 .9- 6 . Sa
A v erag es on t h e same l i n e w ith d i f f e r e n t s u p e r s c r i p t s d i f f e r
s ig n ific a n tly
( p c . 0 1 ). .
63
Ug progesterone/ g luteal tissue
Total Progesterone Level
Experimental Condition
F ig u re 7 . The e f f e c t o f ECO on PGE2 s tim u la tio n o f s te r o id o g e n e s is .
( I ) in c u b a te d c o n t r o l , (L) in c u b a te d w ith 200 ng LH/ml, (? ) in c u ­
b a te d w ith 10 ug PGE2 Zml, (PE) in c u b a te d w ith PGE2 and 75 uM ECO.
6k
Mien PGEg was added t o hom ogenates o f b o v in e c o rp o ra l u t e a , a d e n y la te
c y c la s e was s tim u la te d t o p ro d u ce cAMP (M arsh, 1 9 7 0 b ).
T h is im p lie s t h a t
PGEg s tim u la tio n o f s te r o id o g e n e s is i s m e d ia te d by cAMP a s i s LH s tim u la ­
tio n .
In v iew o f th e v a s t s t r u c t u r a l d i s s i m i l a r i t i e s betw een p r o s ta g la n d ­
i n s and g o n a d o tro p in s i t was n o t s u r p r i s i n g when Rao ( 1973) d e m o n strated
s e p a r a te r e c e p to r s i t e s f o r PGE, and human c h o rio n ic g o n a d o tro p in (HCG)
i n th e c e l l membranes o f b o v in e c o rp o ra l u t e a .
The g o n a d o tro p in r e c e p to r
was s p e c i f i c i n b in d in g HCG and LH, w hereas t h e p r o s ta g la n d in r e c e p to r s
were v e ry s p e c i f i c f o r PGE^ and PGE^.
T h is e v id e n c e s u g g e s ts t h a t p r o s t a ­
g la n d in s and g o n a d o tro p in s b in d t o d i f f e r e n t s i t e s on th e same r e c e p to r
o f a d e n y la te c y c la s e o r t h a t each b in d s t o d i f f e r e n t r e c e p to r m o lecu les
f o r a d e n y la te c y c la s e .
The e x p e rim e n ta l e v id en c e b u ild s a s tr o n g c ase f o r b o th th e l u t e o l y t i c
e f f e c t and th e c o n tr a r y s te r o id o g e n ic s tim u la to r y e f f e c t t o b e e x i s t i n g
p h y s io lo g ic a l phenomena.
P erh ap s th e s e p a r a d o x ic a l e f f e c t s may c o - e x i s t ,
c o n s id e rin g t h a t p r o s ta g la n d in i n j e c t e d i n t o an a n im a l may have i t s e f f e c t
on th e corpus luteum a s a whole o rg a n s u b - s t r u c tu r e o r i t s p e r ip h e r y ,
w hereas th e i n v i t r o e f f e c t s o f th e p r o s ta g la n d in s a r e c o n fin e d t o th e
tis s u e i t s e l f .
When i n t e r p r e t i n g i n v i t r o and i n v iv o e x p e rim e n ta l r e s u l t s ,
th e d if f e r e n c e i n tim e an a g e n t h as t o e x e r t i t s a c ti o n m ust b e c o n s id e re d .
Ih e i n v i t r o in c u b a tio n s d e m o n stra te a s h o r t term e f f e c t o f p r o s ta g la n d i n s ,
w hereas th e i n v iv o ex p erim en ts d e m o n stra te a lo n g te rm e f f e c t .
T h e re fo re ,
p r o s ta g la n d in may s tim u la te p ro g e s te r o n e s y n th e s is when c o n fin e d t o l u t e a l
65
t i s s u e a s se e n i n a s h o r t te rm i n v i t r o e x p e rim e n t, b u t th e lo n g te rm
e f f e c t on th e o rg a n s u b s tr u c tu r e r e s u l t s i n l u t e o l y s i s an d r e s u l t i n g
d e c re a se d p ro g e s te r o n e r e le a s e d from t h e o v a ry .
I t h a s b e en p ro p o se d by P h a r r is s (1970) t h a t PGF2 q i s l u t e o l y t i c
due t o a v a s o c o n s t r i c t i v e e f f e c t r e s u l t i n g i n a l o c a l r e d u c tio n o f
o v a ria n b lo o d flo w .
P r o s ta g la n d in FgQ h a s b een shown t o re d u c e o v a r­
i a n venous b lo o d flo w i n th e r a t and r a b b i t ( P h a r r is s e t a l . , 1968) .
The f a c t t h a t PGF0
i s th e o n ly p o te n t v e n o c o n s tr ic to r among th e p r o s t ­
a g la n d in s (Barnwell e t a l . , 1968) would e x p la in th e l u t e o l y t i c i n e f f e c ­
tiv e n e s s o f th e o th e r p r o s ta g la n d in s when g iv e n i n v iv o .
C e r ta in i n v i t r o s t u d ie s i n d i c a t e t h a t p r o s ta g la n d in s mimic t r o p i c
a g e n ts i n th e a d r e n a l g la n d (F la c k e t a l . , 1968) and t h y r o i d g la n d
( Onaya e t a l . , 1969) a s w e ll a s l u t e a l t i s s u e .
U n lik e t r o p i c a g e n ts ,
p r o s ta g la n d in s a r e endogenous i n th e t a r g e t t i s s u e (S p e ro f f and Barnwell,
1970) and may b e s y n th e s iz e d from c e r t a i n endogenous lo n g c h a in f a t t y
a c i d p r e c u r s o r s ( C la e s so n , 195^)•
I t w ould n o t b e d i f f i c u l t to
in v is io n a l o c a l i z e d t i s s u e o r c e l l u l a r e f f e c t b y a s h o r t l i v e d ,
endogenously p ro d u ced p r o s ta g la n d i n .
S in c e p r o s ta g la n d in s a r e r a p id ly
d e g ra d ed , a lo n g -te rm e f f e c t on th e co rp u s luteum w ould n o t b e e x p e c te d .
The p r o s ta g la n d in s may s e rv e a s l o c a l r e g u l a t o r s by m o d ify in g th e
e f f e c t s o f hormones a t th e t i s s u e l e v e l .
They co u ld a c t a s i n t e r ­
c e l l u l a r m essengers o f an LH re s p o n se t o th e s u rro u n d in g co rp u s luteum
c e l l s , o r th e y may p la y a r o l e i n th e c e l l u l a r mechanism by w hich
66
hormones e x e r t t h e i r e f f e c t s .
P e rh a p s th e m o b iliz a tio n o f c h o l e s t e r o l
e s te r s , and f a t t y a c id s by LH (,Armstrong e t a l . , 1969) a r e in tim a te ly
r e l a t e d t o th e b io s y n th e s is o f p r o s ta g la n d in s and t h e i r r e s u l t i n g
s tim u la to r y a c t i o n .
E f f e c t o f ECO on cAMP S tim u la tio n
S in c e IH an d PGEg b o th s tim u la te a d e n y la te c y c la s e t o p ro d u ce
cAMP, th e e f f e c t o f ECO and cAMP s tim u la tio n was d e te rm in e d .
'When
cAMP (0 .0 2 M) was added t o th e in c u b a tio n m ed ia, p ro g e s te r o n e s y n th e s is
was s tim u la te d t o a l e v e l s i g n i f i c a n t l y g r e a t e r (p<O.Ol) th a n th e
in c u b a te d c o n tr o l (F ig u re 8 ) .
The a d d itio n o f ECO to t h e in c u b a tio n
m edia a lo n g w ith cAMP r e s u l t e d i n a p ro g e s te r o n e l e v e l s t i l l s i g n i ­
f i c a n t l y (p<O .O l) g r e a t e r th a n th e c o n t r o l in c u b a tio n .
T h is was
d e f i n i t e l y i n c o n t r a s t t o th e r e s u l t s o f ECO i n h i b i t i o n o f b o th LH and
PGEg s tim u la tio n .
I n f a c t ^ o n ly a s l i g h t d e c re a s e i n p ro g e s te ro n e
I
l e v e l i s o b se rv e d when ECO i s in c lu d e d w ith cAMP i n th e in c u b a tio n
i
m edia t o th e cAMP s tim u la te d c o n d itio n .
The s tim u la te d l e v e l s o f p r o g e s te r o n e e x p re ss e d i n T ab le V, a re
com parable t o th o s e v a lu e s o b ta in e d by M arsh and S av ard ( 1966) — 207 ug
p ro g e s te r o n e / g l u t e a l t i s s u e f o r th e 0 .0 2 M cAMP c o n d itio n and 233 u g /g
f o r th e s a t u r a t e d LH s tim u la to r y c o n d itio n .
The c o n c e n tr a tio n o f
exogenous cAMP n eed ed f o r maximal s tim u la tio n o f p ro g e s te r o n e s y n th e s is
i s r e l a t i v e l y h ig h compared t o th e endogenous, LH s tim u la te d c o n c e n tra tio n
67
Ug progesterone/ g luteal tissue
Total Progesterone Level
Experimental Condition
F ig u re 8 . The e f f e c t o f ECO on cAMP s tim u la tio n o f s te r o id o g e n e s is .
( I ) in c u b a te d c o n t r o l , (L) in c u b a te d w ith 200 ng LH/ml, (C ) in c u b ­
a te d w ith 0.02M cAMP, (CE) in c u b a te d w ith cAMP and 75 uM ECO.
68
T a b le V. . E f f e c t o f ECO on cAMP s t i m u l a t i o n o f s t e r o i d o g e n i s i s in
lu te a l tis s u e
I n c u b a te d
c o n tro l
I n c u b a tio n
c o n d itio n
N o. O b s e rv a tio n s
7
1 4 1 .8 ^ 1 2 .5a
P r o g e s te r o n e ( u g /g )
N b. O b s e rv a tio n s
9
cAMP
cAMP+ECO
;
5
2 2 2 .5 -1 5 .2 ^
!
1 4 0 . 8 - 1 2 . 5a
P r o g e s te r o n e ( u g /g )
LH .
9
2 1 5 .7 -1 1 .
9
1 9 5 .7 - 1 1 .O^
13
4-
2 3 0 .3 - 1 0 .3
T-)
13
2 1 0 .9 - 1 0 .3b
A v erag es on t h e same l i n e 1w ith d i f f e r e n t s u p e r s c r i p t s d i f f e r
s i g n i f i c a n t l y ( p - c O .Q l) . j
$
"
I
I
69
o f 8 .5 ± 4 .1 n an o m o les/g (M arsh e t a l . , 1966) fo u n d i n th e corpus lu te u m .
T his re q u ire m e n t f o r h ig h c o n c e n tr a tio n s o f n u c le o tid e h a s a ls o "been
o b se rv e d i n s t u d i e s on exogenous cAMP s tim u la tio n o f i n t a c t l i v e r and
a d r e n a l c e l l s ( S u th e r la n d an d B a l l, i 9 6 0 ) .
S in c e much s m a lle r con­
c e n tr a ti o n s o f cAMP w ere n eed ed t o s tim u la te hom ogenates, th e y d e c id e d
t h a t p e rh a p s th e c y c li c n u c le o tid e does n o t e f f e c t i v e l y p e n e tr a te th e
c e l l membrane.
T his may be t r u e i n c o rp o ra l u t e a s l i c e s , b u t Marsh
and S av ard ( 1966) w ere u n a b le t o s tim u la te s te r o id o g e n e s is b y h ig h o r
low l e v e l s o f cAMP i n hom ogenates o f c o rp o ra l u t e a and th e same a tte m p ts
w ere u n s u c c e s s f u l i n o u r l a b .
A n o th er f a c t o r w hich may in f lu e n c e th e
e f f e c t i v e c o n c e n tr a tio n o f exogenous cAMP i s th e enzyme, cAMPp h o sp h o d ie s t e r a s e , w hich may b e i n a c t i v a t i n g th e cAMP t h a t does manage
t o g e t th ro u g h th e c e l l m em branes.
I f th e d ib u ty r y l d e r i v a t i v e
- 2 ' - 0 - d i b u t y r y l - 3 13 5 1-AMP i s u s e d , t h e e f f e c t i v e c o n c e n tr a tio n can
I
b e re d u c e d 100 f o l d (M arsh, 19^9 )•
The d ib u ty r y l d e r i v a t i v e i s much
more perm eab le t o c e l l membranes an d i t i s n o t h y d r o lis e d by
p h o s p h o d ie s te ra s e .
By in c u b a tin g co rp u s lu teu m t i s s u e w ith th e th r e e known s tim u la to r s
o f s te r o id o g e n e s is a lo n g w ith ECO, i t h as b een d e te rm in e d t h a t an
i n h i b i t o r y a c ti o n o f ECO on s tim u la tio n o f p ro g e s te r o n e s y n th e s is
a p p e a rs t o ta k e p la c e p r i o r t o cAMP s tim u la tio n .
C o n sid e rin g t h a t
cAMP i s th e seco n d m essen g er f o r IH and PGEg s t i m u la tio n , th e r e s u l t s
J-
in d ic a te d t h a t th e a c t i o n o f ECO may b e t o e f f e c t i v e l y p r e v e n t an
I
70
in c r e a s e i n th e i n t r a c e l l u l a r c o n c e n tr a tio n o f cAMP due t o th e f i r s t
m e ss e n g e rs .
The i n t r a c e l l u l a r l e v e l o f cAMP i s r a i s e d by LH and PGE^
th ro u g h t h e i r s tim u la to r y a c ti o n on th e enzyme a d e n y la te c y c la s e .
T his s tim u la to r y a c t i o n may "be b lo c k e d b y ECO th u s p r e v e n tin g a r i s e
i n i n t r a c e l l u l a r cAMP.
A nother p o s s i b l e way ECO may i n h i b i t th e
in c r e a s e i n i n t r a c e l l u l a r cAMP i s th ro u g h a s tim u la to r y a c ti o n on
cA M P -phosphodiesterase.
F u r th e r i n v e s t i g a t i o n o f th e e f f e c t o f ECO on
t h e s tim u la tio n o f s te r o id o g e n e s is p ro c e e d e d w ith th e d e te r m in a tio n o f
th e e f f e c t o f ECO on a d e n y la te c y c la s e and p h o s p h o d ie s te ra s e a c t i v i t y .
The E f f e c t o f ECO on P h o s p h o d ie s te ra s e
C y c lic AMP i s h y d r o liz e d t o p h y s io lo g ic a ll y i n a c t i v e 5 *-AMP u n d er
th e in f lu e n c e o f one -or more p h o s p h o d ie s te r a s e s .
Most c e l l s c o n ta in
a t l e a s t two p h o s p h o d ie s te r a s e s , one w ith a low Km f o r cAMP (on th e
o r d e r o f IO- ^ M) an d t h e o th e r w ith a h ig h e r Km (a b o u t 10 ^ M) (Thompson
and Appleman, 1971)» lo c a t e d i n th e s o lu b le as. w e ll a s i n t h e p a r t i ­
c u la t e f r a c t i o n s . . Homogenates o f co rp u s lu teu m t i s s u e c o n ta in
p h o s p h o d ie s te ra s e a c t i v i t y a s r e p o r te d b y Marsh ( 1 9 7 0 a ).
S in c e th e
i n h i b i t o r y a c t i o n o f ECO on LH and PGEg s tim u la tio n c o u ld p o s s ib ly b e
due t o a s tim u la to r y a c ti o n o f ECO on p h o s p h o d ie s te ra s e ,, t h e e f f e c t o f
ECO on p h o s p h o d ie s te ra s e a c t i v i t y was a s s e s s e d .
Homogenates o f l u t e a l t i s s u e had an enzyme a c t i v i t y o f 40 n an o m o les/
15 m in. X 20 mg l u t e a l t i s s u e f o r p h o s p h o d ie s te ra s e a s m easured by th e
,
-
c o n v e rsio n o f
Tl
cAMP t o
' -AMP.
"When IH o r PGE2 w ere added t o th e
h Dmogenates, th e p h o s p h o d ie s te ra s e a c t i v i t y was unchanged by th e s e
s te r o id o g e n ic s tim u la to r s a s was a l s o o b se rv e d by M arsh ( l 970a , b ) .
The c l a s s i c p h o s p h o d ie s te ra s e i n h i b i t o r , th e o p h y llin e , added a t n e a r
s a t u r a t e d l e v e l s (0 .0 4 M) re d u c e d th e c a ta b o lis m o f cAMP t o a n e a r
n e g lig ib le le v e l.
D ihydroergotam in e a s .w e ll a s ECO was added to
hom ogenate in c u b a tio n s a t two c o n c e n tr a tio n s , 10“^ M and 1 0 M.
N e ith e r a l k a l o i d was c o m p le te ly s o lu b le a t th e 10“ 3 M l e v e l .
The two
e r g o t a lk a l o id s h ad a s l i g h t i n h i b i t o r y e f f e c t on p h o s p h o d ie s te ra s e a s
e x p re s s e d i n T able V I.
A t th e c o n c e n tr a tio n o f ECO commonly u sed i n
th e p re v io u s e x p erim en ts (1 0 - ^ M), th e i n h i b i t i o n o f p h o s p h o d ie s te ra s e
was 1 3 . 7$ o f t h e c o n t r o l w h ic h ^ is a s m a ll e f f e c t b u t s i g n i f i c a n t
( p O .O l ) .
Some o f th e e r g o t a lk a l o id s and l y s e r g i c a c id d e r iv a tiv e s a r e
.
known p h o s p h o d ie s te ra s e i n h i b i t o r s i n v a r io u s t i s s u e s . Kulcovetz and
Poch ( 1969) d e m o n stra te d t h a t bromo-LSD re d u c e s m y o c a rd ia l phospho­
d i e s t e r a s e i n a d ip o s e t i s s u e .
Iw an g o ff and Enz (1972) r e p o r te d th e
i n h i b i t i o n o f p h o s p h o d ie s te ra s e i n c e r e b r a l g re y m a tte r by d ih y d ro e rg o ta m in e and a la r g e group o f a n alo g o u s compounds.
Iw an g o ff and Enz
( 1973) a ls o r e p o r te d th e i n h i b i t o r y e f f e c t o f d ih y d ro e rg o tam in e and
h y d e rg in e (an e q u im o la r m ix tu re o f d ih y d ro e rg o ta m in e , d ih y d r o e r g o c r is tin e ,
and d ih y d ro e r g o k r y p tin e ) on th e p h o s p h o d ie s te ra s e a c t i v i t y i n d i f f e r e n t
o rg an s ( h e a r t , k id n e y , l i v e r , and b r a i n ) o f th e c a t .
A s lig h t
72
T ab le V I.
The e f f e c t o f e r g o t a lk a l o i d s on p h o s p h o d ie s te ra se , a c t i v i t y .
M olar Cone, o f
A lk a lo id
$ In h ib itio n o f
P h o s p h o d ie s te ra s e A c tiv ity *
IO- Sd ECO
IO-3M ECO
13-7 ±. 2 .0 #
. 1 4 .7 ± 1 -3
IO- S DHE
IO-3 M DHE'
. 15 .5 ± 2 .2
1 9 .8 + 0 .8
* P h o s p h o d ie s te ra s e a c t i v i t y o f t h e c o n t r o l was 40 n m o le s /l5 m in ./2 0 mg
t i s s u e (hom ogenized).
$ V alues d i f f e r s i g n i f i c a n t l y from c o n t r o l enzyme a c t i v i t y ( p < 0 .0 l) .
1!,
*■
i
■
I n h i b i t o r y e f f e c t o f th e a lk a l o id s on p h o s p h o d ie s te ra s e was g e n e r a lly
o b se rv e d f o r a l l e a se s i n th e c a t o rg a n s tu d y com parable t o th e •
I
■■
i n h i b i t o r y e f f e c t o f ECO and d ih y d ro e rg o ta m in e o b se rv e d i n b o v in e
c o rp o ra l u t e a hom ogenates.
The c a t b r a i n had th e h ig h e s t phospho­
d i e s t e r a s e a c t i v i t y (39 u m oles/3 0 m in . X 100 mg t i s s u e ) and was
i n h i b i t e d '15.3$ o f th e c o n tr o l by IO- ^ M d ih y d ro e rg o ta m in e .
T his can
b e compared t o an a c t i v i t y o f 4 0 n m ole/1 5 m in X 20 mg t i s s u e f o r
b o v in e c o rp o ra l u t e a hom ogenates and a 1 3 . 7$ i n h i b i t i o n o f th e c o n tr o l
by IO"4 M ECO.
Marsh ( 1970a) r e p o r te d a 250 n m ole/1 5 m in . X 20 mg
t i s s u e f o r p h o s p h o d ie s te ra s e a c t i v i t y i n b o v in e c o rp o ra l u t e a a t a
h ig h e r s u b s t r a t e l e v e l .
T h is s l i g h t i n h i b i t i o n o f p h o s p h o d ie s te ra s e by ECO i n b o v in e c o rp o ra
l u t e a hom ogenates i n d i c a t e s t h a t ECO w ould te n d to in c r e a s e th e i n t r a ­
c e l l u l a r l e v e l o f cAMP.
An in c r e a s e i n cAMP r e s u l t s i n an in c r e a s e i n
p r o g e s te r o n e s y n th e s is w hich i s c o n tr a r y t o th e i n h i b i t o r y a c ti o n o f
ECO o b serv ed on s tim u la te d s te r o id o g e n e s is i n in c u b a te d l u t e a l t i s s u e .
The s l i g h t i n h i b i t i n g e f f e c t o f p h o s p h o d ie s te ra s e i s n o t l i k e l y t o have
a d o m in atin g e f f e c t on i n t r a c e l l u l a r l e v e l s o f cAMP compared t o an LH
o r PGEg s tim u la tio n o f a d e n y la te c y c la s e .
S in c e p h o s p h o d ie s te ra s e i s
i n h i b i t e d r a t h e r th a n s tim u la te d by ECO, i t can n o t b e th e s i t e o f th e
ECO i n h i b i t i o n o f s tim u la te d p ro g e s te ro n e s y n th e s is .
I t is d iffic u lt
t o a s s e s s t h i s e f f e c t o f ECO i n h i b i t e d p h o s p h o d ie s te ra s e on th e o v e rI
shadow ing ECO i n h i b i t e d l e v e l s o f p ro g e s te r o n e s y n th e s is o b se rv e d i n
rJk
t i s s u e in c u b a t io n s .
P e rh a p s th e i n h i b i t i n g e f f e c t o f ECO on s tim u la te d
s te r o id o g e n e s is w ould be even g r e a t e r i n th e ab sen ce o f in h ib ito ry e f f e c t on p h o s p h o d ie s te ra s e .
The E f f e c t o f ECO on A d e n y late C y clase A c tiv ity
The a ss e ss m e n t o f a d e n y la te c y c la s e a c t i v i t y was made i n c o rp o ra
l u t e a hom ogenates and i n l u t e a l t i s s u e s l i c e s th ro u g h th e m easurem ent
o f a ccu m u lated new ly form ed cAMP from r a d io a c ti v e p r e c u r s o r s added to
th e two s y ste m s .
T iss u e and c e l l u l a r i n t e g r i t y a r e p re s e rv e d i n th e
t i s s u e s l i c e system p ro v id in g th e i n t a c t c e l l u l a r sy stem som etim es
r e q u ir e d f o r f u l l e x p re s s io n o f horm onal a c t i v a t i o n .
The t i s s u e
i n t e g r i t y may b e an im p o rta n t f a c t o r c o n s id e rin g th e n a tu r e o f a d e n y la te
c y c la s e and i t s a c t i v a t i o n by more th a n one a g e n t.
One d i f f i c u l t y o f
th e t i s s u e a d e n y la te c y c la s e a s s a y i s l a b e l i n g th e ATP s u b s t r a t e p o o l.
P h o s p h o ry la te d n u c le o tid e s a r e r e l a t i v e l y im perm eable t o th e c e l l .
Homogenates p ro v id e a c e l l f r e e sy stem t h a t w i l l r e a d i l y a c c e p t r a d i o ­
a c ti v e ATP i n t o i t s s u b s t r a t e p o o l.
Of c o u rs e , c e l l u l a r i n t e g r i t y i s
l o s t a lo n g w ith some a d e n y la te c y c la s e a c t i v i t y .
The homogenate does
p ro v id e a system w here any d e g ra d a tio n o f a cc u m u la tin g cAMP by
p h o s p h o d ie s te ra s e can be r u le d o u t t y co m p lete i n a c t i v a t i o n o f th e
enzyme by an i n h i b i t o r su ch a s th e o p h y llin e .
The e f f e c t o f ECO on IH s tim u la tio n o f a d e n y la te c y c la s e was
d e te rm in e d by m easu rin g th e a c c u m u la tio n o f 1^C-CAMP1 i n t i s s u e s l i c e s .
75
The I n t r a c e l l u l a r ATP s u b s t r a t e p o o l was la b e l e d by th e c o n v e rsio n o f
e x tra c e llu la r
-a d e n in e t o i n t r a c e l l u l a r 1^C-ATP.
A denine was r e p o r te d
t o p e n e t r a t e l ip o c y te s w ith r e l a t i v e e a s e a n d , i n t h e p re s e n c e o f
g lu c o se a s an en erg y s o u rc e , was fo u n d t o be a lm o st e x c lu s iv e ly
c o n v e rte d t o i n t r a c e l l u l a r ^C-A TP (Humes e t a l . , 1969)*
T his
1^C-a d e n in e ---------- >■ 1^C-ATP ---------- >- 1^C-CAMP
a d e n y la te
c y c la s e
c o n v e rsio n was d e m o n stra te d t o ta k e p la c e i n b o v in e c o rp u s luteum s l i c e s .
IrJhen la b e le d a d e n in e was added t o in c u b a tin g t i s s u e s l i c e s , an in c r e a s e
in
C-ATP and th e c o n c u rre n t d e p le tio n o f
w ith tim e .
As can be se e n i n F ig u re
-a d e n in e was m easured
m axim al c o n v e rs io n t o ^C-ATP
p la te a u e d a f t e r 60 m in u te s on in c u b a tio n .
. A f te r a 60 m in u te p r e - in c u b a tio n , EH and ECO w ere added t o th e
a p p r o p r ia te in c u b a tio n s t o d e te rm in e th e e f f e c t o f ECO on ES, s tim u la t i o n o f a d e n y la te c y c la s e a c t i v i t y .
The m easured am ounts o f
14
C-cAMP
accu m u lated u n d e r th e c o n t r o l in c u b a tio n c o n d itio n , EH s tim u la te d
c o n d itio n , and EH p lu s ECO c o n d itio n in c u b a te d f o r d i f f e r e n t tim e
i n t e r v a l s a r e com pared i n F ig u re 1 0 .
The r e s u l t s a r e e x p re s s e d a s th e
mean ± th e s ta n d a r d e r r o r o f t h r e e e x p e rim e n ts .
The a d d itio n o f EH
( l.O u g /m l) r e s u l t e d i n a m arked in c r e a s e i n cAMP a c c u m u la tio n i n th e
l u t e a l t i s s u e com pared t o in c u b a te d c o n t r o l t i s s u e . * Mien ECO (100 uM)
/
70-
c pm / 100 mg l u t e a l t i s s u e X 10
60 -
50-
40 -
T
30 ON
20-
10 -
10
20
40
In c u b a tio n
F ig u re 9.
60
Time
14
14
C onversion o f C-a d e n in e t o
C-ATP in l u t e a l t i s s u e .
( O ) *14C-ATP, ( A ) 14C -a d en in e .
90 min
/ 100 mg l u t e a l t i s s u e X 10
C pm CA MP
120 min
In c u b a tio n
F ig u re 10.
Time
C y c lic AMP
accu m u latio n in l u t e a l t i s s u e . ( o ) in cu b ated
c o n t r o l , ( p ) LH p lu s ECO, ( - ^ ) LH s tim u la te d .
78
was added to the in c u b a tio n m edia a lo n g w ith LH, th e a c c u m u la tio n o f
_cAMP was s u b s t a n t i a l l y d e c re a s e d from th e LH s tim u la te d c o n d itio n .
These r e s u l t s s u g g e s t t h a t ECO h a s an i n h i b i t o r y e f f e c t on th e LH
s tim u la tio n o f a d e n y la te c y c la s e a c t i v i t y i n i n t a c t c o rp u s luteum
tis s u e .
L u te a l t i s s u e hom ogenates w ere a l s o u s e d t o a s s e s s th e e f f e c t o f
ECO on th e s tim u la tio n o f a d e n y la te c y c la s e i n a c e l l f r e e sy stem .
The
ATP s u b s t r a t e p o o l was ta g g e d w ith th e a d d itio n o f 10 uC i o f ^H-ATP t o
each in c u b a tio n .
A d e n y late c y c la s e a c t i v i t y was d e te rm in e d from th e
amount o f “’H-ATP c o n v e rte d t o %-cAMP d u rin g th e in c u b a tio n .
The
in c lu s io n o f a h ig h c o n c e n tr a tio n o f th e o p h y llin e "(0.04 M) i n th e
a s s a y in c u b a tio n and th e r e l a t i v e l y s h o r t in c u b a tio n tim e o f 15 m in u tes
a s s u r e s a n i n e f f e c t u a l p h o s p h o d ie s te ra s e a c t i v i t y , i n t h e hom ogenate.
The a d d itio n o f LH (20 u g /m l) t o t h e homogenate in c u b a tio n , con­
t a i n i n g th e e q u iv a le n t o f 40 mg o f l u t e a l i s s u e , r e s u l t s i n s tim u la te d
a d e n y la te c y c la s e a c t i v i t y a s p r e v io u s ly d e m o n stra te d by M arsh ( 1 9 7 0 a).
The mean a c t i v i t i e s + stan d a rd , e r r o r a s d e term in e d b y M arsh i n b o v in e
c o rp o ra l u t e a l hom ogenates w ere 116 + 25 P m o les/2 0 m ih . X 40 mg f o r th e
c o n t r o l , 469 ± 95 f o r ..LH s tim u la te d , and 4 l4 0 + 720 f o r NaF s tim u la te d
a d e n y la te c y c la s e .
As can be se e n i n F ig u re 11, th e enzyme a c t i v i t i e s
d e term in e d u n d e r t h e same c o n d itio n s a r e i n th e same ra n g e a s th o s e
r e p o r te d by M arsh.
The a d d itio n o f ECO (100 uM), once a g a in , had. th e
e f f e c t o f i n h i b i t i n g LH s tim u la tio n o f a d e n y la te c y c la s e .
The r e s u l t s
79
1700 -
p moles cAMP / 15 min / 40 mg o f ti s s u e
1500
C o n tro l
LH
NaF+
ECO
EXPERIMENTAL CONDITIONS
F ig u re 1 1 .
A d en y late c y c la s e a c t i v i t y in l u t e a l t i s s u e hom ogenates
8o
e x p re ss e d i n F ig u re 11 a r e th e
means + th e s ta n d a rd e r r o r o f th r e e
e x p erim en ts f o r IH and LH p lu s ECO w h ile th e re m a in in g v a lu e s a r e th e
means + th e ra n g e f o r two e x p e rim e n ta l g ro u p s .
S t a t i s t i c a l a n a ly s is
d e m o n stra te th e enzyme a c t i v i t i e s f o r th e LH c o n d itio n and th e LH p lu s
ECO c o n d itio n t o h e s i g n i f i c a n t l y ( p O .O l) d i f f e r e n t . '
The a d d itio n o f PGEg (10 u g /m l) t o th e homogenate a ls o r e s u l t s i n
s tim u la te d a d e n y la te c y c la s e a c t i v i t y h u t n o t t o th e same e x te n t as
does LH (F ig u re l l ) .
T his same o b s e r v a tio n was made h y Marsh ( IgYOh) .
The a d d itio n o f ECO a p p e a rs t o re d u c e th e s tim u la to r y e f f e c t o f PGEg
on a d e n y la te c y c la s e i n th e same manner, a s i t re d u c ed LH s tim u la tio n .
An even g r e a t e r s tim u la to r y e f f e c t o f a d e n y la te c y c la s e was
o b serv ed w ith th e a d d itio n o f NaF (0 .0 1 M) t o t i s s u e hom ogenate.
The
s tim u la tio n o f a d e n y la te c y c la s e h y NaF h as b een o b se rv e d c o n s is te n tly
i n th e s tu d y o f th e e f f e c t o f o t h e r hormones on t h i s m e d ia to ry enzyme.
i n v a r io u s t a r g e t t i s s u e s (S u th e rla n d e t a l . 3 1962) .
The m agnitude o f
th e NaF e f f e c t alw ays seems t o s i g n i f i c a n t l y exceed th e e f f e c t o f a
s tim u la to r y horm one.
The a d d itio n o f ECO d id n o t e f f e c t th e s tim u la ­
t o r y a c ti o n o f NaF a s i t h as s u p p re ss e d LH and PGEg s tim u la tio n o f
a d e n y la te c y c la s e .
In a s i m i l a r f a s h io n , a n o th e r e r g o t a lk a l o i d ,
d ih y d ro e rg o ta m in e , i n h i b i t s th e n o re p in e p h rin e s tim u la tio n o f a d e n y la te
c y c la s e b u t does n o t e f f e c t NaF s tim u la tio n o f th e enzyme (Ward and
F a in , lg Y l) .
I t seems l i k e l y t h a t NaF i n t e r a c t s w ith some component
o f th e a d e n y la te c y c la s e system w hich i s common to a l l t i s s u e s and t h a t
81
t h i s i n t e r a c t i o n i s d i f f e r e n t from th e e f f e c t o f th e hormone, w hich i s
v e ry s p e c i f i c i n m ost t i s s u e s s tu d ie d .
The n a tu r e o f th e e f f e c t o f ECO on a d e n y la te c y c la s e i n b o v in e 1
corpora l u t e a t i s s u e i s d i f f i c u l t t o a s s e s s .
The e r g o t a lk a l o id
i n h i b i t s th e s tim u la to r y a c ti o n o f b o th IH and PGE^ on a d e n y la te c y c la s e .
I n i t i a l l y i t seems l o g i c a l t o s p e c u la te t h a t th e a l k a l o i d p re v e n ts th e
b in d in g o f IH and PGEg t o an a d e n y la te c y c la s e r e c e p to r s i t e .
However,
one m ust keep i n m ind th e s p e c i f i c i t y th e s e s i t e s have d e m o n stra te d .
S e p a ra te r e c e p to r s i t e s f o r g o n a d o tro p in and p r o s ta g la n d in were shown
t o e x i s t i n b o v in e co rp u s luteum t i s s u e (Bao, 1973)•
C o n sid e rin g th e
s t r u c t u r a l d i s s i m i l a r i t i e s betw een IH , PGEg, and ECO i t i s d i f f i c u l t t o
i n v i s i o n th e a l k a l o i d t o b e com peting w ith IH and PGE^ f o r t h e i r r e s p e c ­
t i v e b in d in g s i t e s .
Hormones su ch a s a d r e n o c o r tic o t r o p i n , g lu cag o n ,
n o re p in e p h rin e and e p in e p h rin e w hich s tim u la te a d e n y la te c y c la s e In
t h e i r r e s p e c tiv e t a r g e t tis s u e ,! have no e f f e c t on a d e n y la te c y c la s e in
b o v in e l u t e a l t i s s u e (M arsh, IjjJO a).
Y et e r g o t a lk a l o id s have demon­
s t r a t e d i n h i b i t o r y a c tio n s on n o re p in e p h rin e s tim u la tio n o f a d e n y la te
c y c la s e i n l ip o c y te s (Ward and F a in , 1 9 7 1 ), c a te c h o la m in e a c t i v a t i o n o f
a d e n y la te c y c la s e i n t h e h e a r t and l i v e r o f dogs (Murad e t a l . , 1962)
and r a t l i v e r ( B e r th e t e t a l . , 1957) ; and glu cag o n and c ate c h o lam in e
in c r e a s e o f cAMP i n th e r a t l i v e r (Yeung and O liv e r , 1968) .
The g e n e r a l
i n h i b i t o r y e f f e c t o f e r g o t a lk a l o id s on s tim u la tio n o f a d e n y la te c y c la s e
m ust r e s u l t from some common a c ti o n on th e enzyme t o p r e v e n t i t s
82
a c tiv a tio n .
B ecent e x p e rim e n ta l e v id e n c e (S ch m id t, e t a l . , 197^-) i n d i ­
c a te s l i v e r and p l a t e l e t a d e n y la te c y c la s e h a s a c t i v a t e d dephospho—
and i n h i b i t e d phospho—form s c o n tr o lle d b y an a d jo in in g p h o sp h o p ro te in
p h o s p h a ta s e w hich a c t i v a t e s phospho—a d e n y la te c y c la s e b y d e p h o sp h o ry la ­
tio n .
P e rh a p s th e e r g o t a l k a l o i d s a r e p r e v e n tin g hormone s tim u la tio n
th ro u g h a d i r e c t i n h i b i t o r y a c t i o n on th e a c t i v a t i o n o f a d e n y la te c y c la s e
by a common a c t i v a t i o n p ro c e s s su ch a s th e one j u s t m en tio n ed above.
;
i
SUMMARY
The E f f e c t o f ECO on th e S tim u la tio n o f S te ro id o g e n e s is
The i n i t i a l e x p e rim e n ta l e v id en c e d e m o n strated s, d i r e c t e f f e c t o f
e rg o c o m in e on th e p ro d u c tio n o f p ro g e s te r o n e i n th e co rp u s lu te u m .
The
p h y s io lo g ic a l im p o rtan c e o f th e ECO i n h i b i t e d p ro g e s te ro n e would b e h a rd
t o d e te rm in e i n l i g h t o f th e a c ti o n of.ECO on p i t u i t a r y hormone o u tp u t.
However, t h i s d i r e c t e f f e c t i n t i s s u e p ro g e s te ro n e b io s y n th e s is m ust b e
ta k e n i n t o a c c o u n t when d is c u s s in g th e e f f e c t s o f ECO on mammal r e p r o ­
d u c tiv e d y s f u n c tio n s .
A lthough th e d e c re a s e d p r o g e s te r o n e l e v e l s i n r a t s fo llo w in g ECO
i n j e c t i o n s h a s b een t r a c e d t o i n h i b i t i o n o f p i t u i t a r y r e l e a s e o f p r o l a c t i n
and LH (W uttke, e t a l . , 1971), t h e r e may b e o th e r s i g n i f i c a n t e f f e c t s
r e s u l t i n g from a d i r e c t e f f e c t o f ECO on l u t e a l t i s s u e .
K ra ic e r and
S tr a u s s (1970) p o in te d o u t t h a t ECO may i n t e r u p t a p r e o v u la to r y su rg e o f
p r o g e s tin s r e s p o n s ib le f o r s e t t i n g o f f th e g o n a d o tro p in o v u la to ry p u l s e .
E rg o c o m in e was a ls o fo u n d to d e c re a s e p ro g e s te ro n e l e v e l s when a d m in ste re d
t o th e mouse ( C a rls e n e t a l . , 1961) , th e f e r r e t (B ia tc h le y and Donovan,
1967) , and man ( S h e le sn y a k e t a l . , 1963) a lth o u g h t h i s l a t e r o b s e r v a tio n
' came i n t o q u e s tio n fo llo w in g su b se q u e n t i n v e s t i g a t i o n (L in d n e r e t a l . ,
1 9 6 7 ).
84
Hormone l e v e l s o f c a t t l e t r e a t e d w ith e r g o t a lk o id s have n o t b een
m easu red .
However, c a t t l e consuming t o x i c l e v e l s o f e r g o t have shown
r e p ro d u c tiv e <3ys f u n c tio n in g .
S in c e th e h o v in e c o rp u s lu teu m i s a w e ll
c h a r a c te r iz e d , s p e c i a l i z e d t i s s u e and s in c e i t i s r e l a t i v e l y la r g e i n
m ass, i t seemed t o b e th e i d e a l t i s s u e to u s e t o i n v e s t i g a t e th e e f f e c t
o f ECO on s t e r o i d o g e n e s i s .
The corpus lu teu m i s c h a r a c te r iz e d a s a s p e c i a l i z e d t i s s u e w h ic h ■
f u n c tio n s t o p ro d u c e p r o g e s te r o n e .
T h is p ro g e s te ro n e i s r e le a s e d i n t o
th e b lo o d s tre a m w here i s a c t i v a t e s o r m a in ta in s th e fe m a le r e p ro d u c tiv e
sy stem .
The p ro d u c tio n o f p ro g e s te r o n e i n th e co rp u s lu teu m i s c o n tr o lle d
by th e serum l e v e l o f IH r e le a s e d from th e p i t u i t a r y .
T h e re fo re , th e
p i t u i t a r y , u n d e r th e in f lu e n c e o f th e h y p o th allm u s and r e p ro d u c tiv e hormone
fe e d b a c k , c o n tr o ls th e c y c li c fem ale re p ro d u c tiv e rhythm .
I n v e s t i g a t o r s have shown t h a t LH s tim u la te s p ro g e s te r o n e s y n th e s is
i n th e co rp u s lu teu m v i a th e "seco n d m essen g er" cAMP.
The p r o s ta g la n d in s
a l s o s tim u la te s te r o id o g e n e s is i n l u t e a l t i s s u e v i a cAMP.
However, n e i t h e r
th e r e l a t i o n s h i p betw een IH and p r o s ta g la n d in s n o r th e p h y s io lo g ic a l s i g n i ­
f ic a n c e o f p r o s ta g la n d in s tim u la tio n h a s b een d e te m in e d .
m essenger system " (F ig . 3 , p . 31) p ro v id e s a means f o r :
The "second
( l ) th e r e c o g n i­
t i o n o f a hormone by s p e c i f i c t a r g e t t i s s u e s , (2 ) th e tr a n s m is s io n o f th e
hormone m essage a c r o s s th e c e l l membrane, and ( 3 ) th e a m p lif ic a tio n o f
th e hormone m essage i n t o th e s tim u la te d c e l l u l a r p r o c e s s e s .
I n i t i a l e x p erim en ts d e m o n stra te d t h a t ECO i n h i b i t e d IH s tim u la tio n
and PGEg s tim u la tio n o f s t e r o i d o g e n e s i s . ( F ig . 1 2 ) .
However, th e a l k a l o i d
d id n o t r e s u l t , i n t h e l a r g e r e d u c tio n o f s te r o id o g e n e s is when th e t i s s u e
in c u b a tio n s w ere s tim u la te d by exogenous cAMP.
These r e s u l t s p la c e th e
e f f e c t o f t h e e r g o t a l k a l o i d a t t h e c e n te r o f th e " se c o n d a ry m essenger
sy ste m " .
The. i n h i b i t o r y e f f e c t o f ECO i s p r i o r t o cAMP m e d ia tio n o f LH
and PGEg s tim u la tio n o f p ro g e s te r o n e s y n th e s is ( F ig . 1 3 ) .
The i n t r a c e l l u l a r l e v e l o f cAMP i s c o n tr o lle d by t h e a c t i v i t y o f
two enzyme sy stem s ( F ig . 1 3 ) .
E rg o c o m in e c o u ld i n h i b i t IH and PGE^
s tim u la tio n o f s te r o id o g e n e s is by i n h i b i t i n g a d e n y la te c y c la s e o r
in c r e a s in g p h o s p h o d ie s te ra s e b o th r e s u l t i n g i n th e break-dow n o f th e
m e d ia tio n o f IH o r PGEg s tim u la tio n .
E rg o c o m in e was fo u n d t o have a
s l i g h t i n h i b i t o r y a c t i o n on p h o s p h o d ie s te ra s e w hich w ould r e s u l t i n
in c r e a s e d i n t r a c e l l u l a r cAMP l e v e l s .
However, e rg o c o m in e was fo u n d t o
b lo c k LH and PGEg s tim u la tio n o f a d e n y la te c y c la s e , w hich w ould r e s u l t
i n d e c re a s e d i n t r a c e l l u l a r cAMP and s u b se q u e n t break-dow n o f m e d ia tio n
o f IH and PGEg s tim u la tio n o f s t e r o id o g e n e s is .
The m ag n itu d e o f ECO
i n h i b i t i o n o f a d e n y la te c y c la s e sh o u ld over-shadow t h e s m a ll i n h i b i t o r y
e f f e c t o f ECO on p h o s p h o d ie s te r a s e .
As a r e s u l t o f h o m o g e n iz atio n ,
p h o s p h o d ie s te ra s e does have a much h ig h e r en zy m atic a c t i v i t y th a n
a d e n y la te c y c la s e i n hom ogenates.
However, Marsh (1970a) p o in te d o u t
100
■
CAMP
cAMP +
ECO
EXPERIMENTAL CONDITIONS
F ig u re 1 2. The e f f e c t o f ECO on th e s tim u la to r s o f p ro g e ste ro n e
s y n th e s is i n l u t e a l t i s s u e . In c u b a tio n c o n d itio n s as
d e s c rib e d i n p re v io u s f i g u r e s .
8?
F ig u re 1 3 .
The e f f e c t o f ECO
on s te r o id o g e n e s is
FDase
LH
BGE2
cAMP
p r o te in k in a s e
s id e - c h a in c le a v a g e
enzyme h y d ro x y la se
la n o s te ro l
s q u a le ne
a c e ta te
p reg n en o lo n e
\
88
t h a t cAMP acc u m u la te s i n t h e c e l l i n th e p re s e n c e o f t h i s la r g e p o t e n t i a l
p io s p h o d ie s t e r a s e a c t i v i t y i n d i c a t i n g t h a t e i t h e r v ezy l i t t l e o f th e
p h o s p h o d ie s te ra s e a c t i v i t y i s e x p re s s e d o r t h a t th e r e i s some com partm e n ta liz a tio n w hich s e p a r a te s cAMP from t h i s d e s t r u c t i v e enzyme.
Of
c o u rs e hom o g en izatio n r e l e a s e s th e p h o s p h o d ie s te ra s e a c t i v i t y w hich m ust
h e i n h i b i t e d by th e o p h y llin e when a s s e s s in g a d e n y la te c y c la s e a c t i v i t y .
The p rim a ry e f f e c t o f erg o c o m in e i n h i b i t i o n o f s tim u la te d p r o g e s t e r ­
o n e , t h e r e f o r e h a s b e en d e m o n stra te d t o ta k e p la c e by an i n h i b i t o r y a c ti o n
o f t h e a l k a l o i d on th e a c t i v a t i o n o f a d e n y la te c y c la s e . ' Zrom th e e x p e r­
im e n ta l d a ta p r e s e n te d , one may o n ly s p e c u la te on th e n a tu r e o f t h i s ECO
i n h i b i t o r y a c ti o n on a d e n y la te c y c la s e .
S in c e th e s tim u la to r y a c ti o n o f
b o th LH an d PGEg a r e i n t e r r u p t e d i t a p p e a rs t h a t EQO i n t e r f e r s w ith an
a c t i v a t i o n p ro c e s s o f a d e n y la te c y c la s e betw een th e r e c e p to r s i t e s and
th e enzyme.
A g e n e r a l i n h i b i t o r y a c ti o n o f e r g o t a lk a l o id s on a d e n y la te
c y c la s e a c t i v a t i o n w ould a ls o e x p la in e r g o t a l k a l o i d d is r u p tio n o f hormone
s tim u la tio n i n th e o th e r t i s s u e s m e n tio n e d — a d ip o se (Ward and F a in , 1971) j
h e a r t (Murad e t a l . , 1962) , and l i v e r (B e r th e t e t a l . , 1957, Yeung and
O liv e r , 1968) .
As a r e s u l t o f th e ECO i n h i b i t o r y a c t i o n , LH o r PGEg f a i l to s tim u la te
an in c r e a s e i n th e i n t r a c e l l u l a r l e v e l o f ■cAMP i n b o v in e l u t e a l t i s s u e .
N orm ally, LH o r PGEg s tim u la te d a d e n y la te c y c la s e w ould in c r e a s e th e
i n t r a c e l l u l a r l e v e l o f cAMP- w hich a c t i v a t e s c e r t a i n p r o t e i n k in a s e s
b o v in e l u t e a l t i s s u e a s shown by Menon (1973) (F ig - 1 3 )•
in
The a c t i v a t e d
p r o t e in k in a s e s
in d u c e
in c r e a s e d p r o g e s te r o n e s y n t h e s is
p h o s p h o ry la tio n r e a c t i o n s .
th r o u g h c e r t a in
B oth LH and cAMP have b een sh o rn t o ■d i r e c t l y
a c t i v a t e a p r o t e i n k in a s e t h a t p h o s p h o ry la te s h i s t o n e s , w h ereas, o n ly
cAMP a c t i v a t e s a d i f f e r e n t p r o t e i n k in a s e t o ph o sp h o ry l a t e rlb o so m a l
p r o t e i n s (A zhar an d Menon, 197*0*
These p h o s p h o ry la tio n s c o u ld p o s s ib ly
in c r e a s e th e p r o t e i n s y n th e s is o f enzymes i n th e s te r o id o g e n ic pathw ay.
A nother p h o s p h o ry la tio n r e a c t i o n has b een shown by Caron e t a l . , (197*0
t o s tim u la te t h e a c t i v i t y o f th e r a t e - l i m i t i n g enzyme o f th e s te r o id o g e n ic
path w ay .
The s id e - c h a in c le a v a g e enzyme system i n b o v in e l u t e a l t i s s u e
i s s tim u la te d by a cAMP-dependent p r o t e i n k in a s e p h o s p h o ry la tio n o f some
p r o t e i n i n th e cytochrom e P-450 f r a c t i o n o f th e enzyme com plex.
From t h i s d i s c r i p t i o n i t can b e se e n how -the e r g o t a l k a l o i d in d u c e d
re p r o d u c tiv e d y s fu n c tio n s o b se rv e d i n c a t t l e c o u ld p o s s ib ly r e s u l t from
a d i r e c t e f f e c t o f th e a lk o id on th e p ro g e s te ro n e s y n th e s is i n th e c o rp u s
lu te u m .
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*
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