The oestrogenic and anti-oestrogenic activity of some synthetic steroids and non-steroids

897

THE 0ESTROGENIC AND ANTI-0ESTROGENIC ACTIVITY OF S0~E SYNTHETIC STEROIDS ANDNON-STEROIDS by IRINAPOLLABD~ and L. K k R T I ~ Department of V e t e r i n a r y Physiology~ U n i v e r s i t y of Sydney, Sydney, N.S.W. A u s t r a l i a t

Recleved February 21, 1968

ABSTRACT A number of s t e r o i d s i n h i b i t e d o e s t r a d i . l - 1 7 ~ s t i m u l a t e d t e t r a z o l i u m r e d u c t i o n when g i v e n i n t r a v a g i n a l l y a t t h e same time as t h e o e s t r o g e n ~ b u t none of them was as p o t e n t as d i m e t h y l s t i l b o e s t r o l (D~S). The most e f f e c t i v e compounds p o s s e s s f r e e or e s t e r i f i e d h y d r o x y l s a t 3 and 17 and t o t h i s e x t e n t resemble o e s t r a d i o l - 3 : l T ~ . A l l of t h e s t e r o i d s which were a n t i - o e s t r o g e n i c were a l s o to some e x t e n t o e s t r o g e n i c in t h a t t h e y i n c r e a s e d v a g i n a l t e t r a z o l i u m r e d u c t i o n , u t e r i n e weight or u t e r i n e e p i t h e l i a l m i t o s i s , when g i v e n i n h i g h doses subcutaneously. Although t h e r e were e x c e p t i o n s t h e more p o t e n t a n t i o e s t r o g e n s a l s o t e n d e d to be t h e more p o t e n t o e s t r o g e n s . Of t h e n o n - s t e r o i d s , ~RL-~I was o e s t r o g e n i c b u t n o t a n t i - o e s t r o g e n i c , SC7801 was a r e l a t i v e l y p o t e n t a n t i - o e s t r o g e n but was o n l y weakly oestrogenic. ~-37 was weakly a n t i - o e s t r o g e n i c l o c a l l y . It s i g n i f i c a n t l y i n c r e a s e d t e t r a z o l i u m r e d u c t i o n u t e r i n e weight and u t e r i n e e p i t h e l i a l m i t o s i s a t 1000 pg s u b c u t a n e o u s l y , b u t h i g h e r doses increasingly depressed these responses. INTRODUCTION Emmens~ Cox & M a r t i n 6 found t h a t a number of n o n - s t e r o i d a l anti-oestrogens

inhibited all

s t a g e s s t u d i e d of the v a g i n a l r e s p o n s e

t o o e s t r o g e n , whereas a small s e r i e s of s t e r o i d a l inhibited only vaginal cornification

anti-oestrogens

and were w i t h o u t e f f e c t upon

t h e e a r l y r e s p o n s e s of i n c r e a s e d m i t o s i s and t e t r a z o l i u m r e d u c t i o n . Department of P h y s i o l o g y , Monash U n i v e r s i t y , C l a y t o n , V i c t o r i a , Australia. ~Tresent address: I m p e r i a l Cancer R e s e a r c h Fund, LincolnWs Inn F i e l d s , London, G r e a t B r i t a i n . t Address f o r r e p r i n t s .

898

ST ER O ID S

Recently~m, ens 5 has investigated oestrogenic

activities

the vaginal

smear assay.

o f t h e s e and o t h e r of their

effects

of further

the oestrogenic

steroids

The p r e s e n t

11:6

and a n t i -

and n o n - s t e r o i d s

paper describes

compounds i n t h e t e t r a z o l i u m

using

the activity

a s s a y 9 and a l s o

some

upon t h e u t e r u s . ~ T E R I . k I ~ AND METHODS

Randomly b r e d o v a r i e c t o m i z e d Q.S. m i c e were u s e d 6 - 1 0 d a y s a f t e r p r i m i n g w i t h a s i n g l e s u b c u t a n e o u s d o s e o f 1 ~g o f o e s t r a d i o l in oil. E x p e r i m e n t a l s o l u t i o n s were p r e p a r e d f r o m a l c o h o l i c s t o c k s or from the solids immediately before use. In local (intravaginal) t e s t s , d o s e s o f o e s t r a d i o l and o f t h e compounds t e s t e d w e r e a d m i n i s t e r e d t o g e t h e r i n s i n g l e i n t r a v a g i n a l i n j e c t i o n s o f 0 . 0 0 5 ml o f d i s t i l l e d w a t e r and t h e m i c e w e r e k i l l e d 2~ h o u r s l a t e r . In the solutions containing higher concentrations o f t h e compounds, p r o p y l e n e g l y c o l was a d d e d in amounts up t o 25% i n order to increase solubility. The e s t i m a t i o n o~ v a g i n a l t e t r a z o l i u m r e d u c t i o n was c a r r i e d o u t a s d e s c r i b e d b y M a r t i n . The t r a n s f o r m a t i o n 100 l o n g (1000 x o p t i c a l d e n s i t y ) was u s e d f o r t h e a n a l y s e s . C o n t r o l a n i m a l s w e r e i n j e c t e d w i t h 0 . 0 0 5 ml o f t h e a p p r o p r i a t e v e h i c l e of injection. I n s y s t e m i c t e s t s t h e compounds w e r e a d m i n i s t e r e d i n s i n g l e subcutaneous injections o f 0.1 ml o f o i l . The a n i m a l s were k i l l e d 2~ h o u r s a f t e r i n j e c t i o n , and e s t i m a t e s o f v a g i n a l t e t r a z o l i u m r e d u c t i o n , u t e r i n e w e t w e i g h t and u t e r i n e e p i t h e l i a l m i t o s i s were made. The u t e r i w e r e d i s s e c t e d o u t , w e i g h e d on a t o r s i o n b a l a n c e , f i x e d i n B o u / n l s f i x a t i v e and p l a c e d i n an a u t o m a t i c t i s s u e p r o c e s s o r w h i c h c a r r i e d them t h r o u g h t h e a l c o h o l s and x y o l t o wax. T r a n s v e r s e s e c t i o n s were c u t a t 5~, s t a i n e d i n H e i d e n h a i n ' s haematoxylin, differentiated and c o u n t e r s t a i n e d i n Van G i e s o n ' s picro-fuchsin. E s t i m a t e s o f m i t o t i c a c t i v i t y were made b y c o u n t i n g t h e t o t a l number o f m i t o s e s p r e s e n t i n t h e e p i t h e l i u m i n each section. The compounds w e r e o b t a i n e d f r o m G.D. S e a r l e & Co; ( U . S . A . ) and f r o m W i l l i a m s S. M e r r i l Co; ( U . S . A . ) and w e r e : SC12222 1 7 ~ - e t h y n y 1 - 1 9 - n o r - a n d r o s t _ ~ _ e n _ 3 ~ 9 1 7 ~ _ d i o 1 , 1 7 _ a c e t a t e SCl1800 1 7 a - e t h y n y l - 1 9 - n o r - a n d r o s t - 4 - e n - 3 ~ , 1 7 ~ - d i o l , 3 , 1 7 - d i a c e t a t e SC692~ 3 - m e t h o x y - 1 6 ~ - m e t h y l - l ~ 3 9 5 ( l O ) - e s t r a t r i e n - 1 6 ~ , 1 7 ~ _ d i o l SC9263 1 6 ~ - c h l o r o - l , 3 9 5 ( 1 0 ) - e s t r a t r i e n - 1 7 - o n e SC8916 3 - m e t h o x y - 1 6 ~ - m e t h y l - l , 3 , 5 ( l O ) - e s t r a t r i e n - 1 6 ~ - o l - 1 7 - o n e SCl1195 1 7 a - m e t h y l - S a - a n d r o s t - l - e n - 1 7 ~ - o l - 3 - o n e SCl1~6~ 1 7 a - m e t h y l - S a - a n d r o s t - l - e n - 3 ~ , 1 7 ~ - d i o l , 3 - a c e t a t e

June 1968

s T ER O ID S

899

SC10017 SC5888 SC7801 SC8~70 SC6091

21-fluoropregn-~-en-17~-o1-3,20-dione,17-acetate 17a-oxa-D-homo-5~-androstan-3,17-dione 3-(~-methoxyphenyl)-4-ethyl-7-hydroxycourmarin 17a-ethynyl-androst-~-en-17~-ol-3-one,17 acetate 17~-ethynyl-19-nor-androsta-3,5-diene-3,17~-diol,3,17diacetate SC43~1 1 9 - n o r - a n d r o s t - ~ - e n - 1 7 ~ - o l - 3 - o n e ( 1 9 - n o r t e s t o s t e r o n e ) MRL37 1 - [ ~ - ( 2 - d i e t h y l a m i n o e t h o x y ) p h e n y l ] - 2 - ( ~ - m e t h o x y p h e n y l ) 1-phenylethane MRL~I 1-chl o r o - 2 - [~- ( 2 - d i e t h y l aminoethoxy)pheny~ 1,2-diphenylethylene ~ER25 l-[~-(2-diethylaminoethoxy)phenylJ-2-(~-methoxyphenyl)l-phenylethanol RESULTS Table 1 snmmarizes t h e r e s u l t s of a s e r i e s of t e s t s of t h e s e compounds f o r a n t i - o e s t r o g e n i c a c t i v i t y u s i n g the i n t r a v a g i n a l tetrazolium assay.

D i m e t h y l s t i l b o e s t r o l ( I ) ~ ) was i n c l u d e d f o r

comparison i n most of t h e experiments and c o n s i s t e n t l y i n h i b i t e d o e s t r a d i o l - 1 7 ~ (5Opg) a t a dose of 0.015 ~g. e x h i b i t e d a c t i v i t y camparable to t h i s .

However, SC7801,

SC12222, SC8~70 and SCl1800 s i g n i f i c a n t l y a t doses of 0.16 ~g.

No o t h e r compound

inhibited oestradiol

The remaining compounds of Table 1 were

i n a c t i v e a t t h i s dose l e v e l , and were t h e r e f o r e t e s t e d a g a i n a t h i g h e r doses ( 0 . 5 , 1.5 and ~.5 ~g).

SC6091 was n o t f u r t h e r

tested due to exhaustion of stocks.

SCll~6~, SC692~ and ~ L 3 7

significantly inhibited oestradiol at doses of 0.5 ~g~ ~.5 ~g and 4.5 ~g respectively.

The highest doses of SCII195 and

SCI0017 decreased tetrazolium reduction but these effects were only barely significant (P(O.05).

SC58888, SC8916, SC9263, SC43~I

and progesterone were ineffective at all doses tested.

The

highest dose of ~ L ~ I depressed tetrazolium reduction but not significantly.

Subsequent tests (Table 4) showed that MRL~I was

900

ST ER O ID S

oestrogenic

locally

11:6

a t a d o s e o f 10 pg.

A l t h o u g h SCl1800 and SC8470 i n h i b i t e d tetrazolium

reduction

at relatively

compounds were i n a c t i v e thought that

oestrogen

stimulated

low d o s e l e v e l s ,

in the vaginal

these

s m e a r a s s a y 5.

in the longer term vaginal

smear tests

I t was the

oestrogenicity

o f SCl1800 (F~mens 1965, and T a b l e 3) m i g h t o b s c u r e

any inhibition

of oestradiol,

tetrazolium until

assay,

well after

oestrogenic

24 h o u r s .

SCl1800 were a d m i n i s t e r e d

a r e shown i n T a b l e 2.

that

m i g h t n o t become o b v i o u s

Doses o f 0 . 0 4 ,

0 . 1 6 and 0 . 6 4 ~g o f

24, 30 and 36 h o u r s l a t e r . Separate

analyses

The r e s u l t s

w e r e made o f t h e

o f SCl1800 i n t h e p r e s e n c e and a b s e n c e of o e s t r a d i o l .

It significantly effects

responses

term

a l o n e o r w i t h 50pg o f o e s t r a d i o l

and t h e m i c e w e r e k i l l e d

effects

whereas in the shorter

inhibited

oestradiol

in the absence of oestradiol

the response to the highest

from control

values

at any time,

d o s e was s i g n i f i c a n t l y high dose treatments reason for this

higher

with self-inhibition

times,

but its

a r e somewhat p u z z l i n g

dose did not differ

in

significantly

whereas the response to the lowest

than responses

a t 24 h o u r s ,

is obscure,

at all

to either

control

b u t n o t a t 36 h o u r s .

b u t may r e f l e c t

at the high dose level.

or

The

an o e s t r o g e n i c

effect

However, the effects

a r e s m a l l compared w i t h t h o s e o f o e s t r a d i o l . T a b l e s 3 and 4 l i s t alone,

subcutaneously

w e i g h t and u t e r i n e

on v a g i n a l

epithelial

With t h e e x c e p t i o n tetrazolium

the effects

reduction

o f t h e compounds a d m i n i s t e r e d

tetrazolium

reduction,

uterine

mitosis.

o f SC10017 w h i c h d i d n o t i n c r e a s e

b u t which d i d i n c r e a s e

uterine

w e i g h t and

J u n e 1968

mitosis,

STEROIDS

901

t h e r e was good agreement between t h e t h r e e t e s t methods;

any compound which i n c r e a s e d t e t r a z o l i u m r e d u c t i o n a l s o i n c r e a s e d u t e r i n e weight and m i t o s i s , w hi l e compounds i n a c t i v e i n t h e t e t r a z o l i u m t e s t were a l s o i n a c t i v e i n t he u t e r i n e w ei ght and m i t o s i s tests. The s t e r o i d s ,

SC12222, SC8~70 and SCl1800 were o e s t r o g e n i c

a t i 0 ~g~ SC692~ a t I00 ~g and SC8916, SC9265, SCII195 and SCII~6~ a t 500 ~g. criterion

SC5888 showed no s i g n of o e s t r o g e n i c a c t i v i t y a t 500 ~g.

by any

Indeed t h e h i g h e r doses a p p e a r e d t o d e c r e a s e

u t e r i n e weight. Of the n o n - s t e r o i d s , ~/RL~I was o e s t r o g e n i c a t 50 ~g, SC7801 a t 100-500 ~g, and MItL37 a n d S 5

a t 1000 ~g.

However, i n t h e

case of ~fltL37 h i g h e r doses t h a n t h i s s i g n i f i c a n t l y

reduced a l l

three responses, although not to control levels. Some of t h e compounds were a l s o t e s t e d f o r o e s t r o g e n i c a c t i v i t y by t h e l o c a l r o u t e ( f i n a l

coll,mns Table 3 & ~).

Although

e x t e n s i v e t e s t s were n o t c a r r i e d out i t i s c l e a r t h a t t h e doses o f SC12222, SC8470, SCl1800, SCl1~6~, SC7801, ~ L ~ I and MP~37 r e q u i r e d l o c a l l y to e l i c i t

significant

r e s p o n s e s approached and i n

some c a s e s exceeded t h o s e r e q u i r e d s u b c u t a n e o u s l y . compounds have s u b c u t a n e o u s - l o c a l r a t i o s a p p e a r to be p r o - o e s t r o g e n s 1 ' 2 ' 3 ' ~

Thus t h e s e

of u n i t y or l e s s and would

There i s i n s u f f i c i e n t

data

to make any comment in t h i s r e g a r d on SC692~. DISCUSSION Many of t h e s t e r o i d s

i n v e s t i g a t e d not only i n h i b i t the

t e t r a z o l i u m r e s p o n s e b u t a l s o r e d uce t h e l e v e l s of o e s t r a d i o l

9o2

ST ER O ID S

retained

b y t h e v a g i n a 9.

11:6

Moreover the most potent

of these

compounds, SC8470, SC12222, SCl1800 and SCl146~ a l l - 3 and - 1 7 h y d r o x y l r a d i c a l s this

extent

either

free

resemble oestradiol-3:17~.

they are acting

as competitive

oestrogens

in tetrazolium

in vaginal

s m e a r t e s t s 5.

or esterified Thus i t

the

and t o

seems l i k e l y

that

antagonists.

SC9263 w h i c h

SC5888, SC8916 and

possess

tests

were i n a c t i v e

as a n t i -

h a v e a l s o b e e n f o u n d t o be i n a c t i v e

T h e r e was a l s o good a g r e e m e n t b e t w e e n

t h e two a s s a y m e t h o d s f o r m o s t o f t h e compounds which showed oestrogenic

activity.

oestrogenically vaginal

active

s m e a r t e s t s 5.

high dose level results

However, SCl1195 and SCl1464, w h i c h were in the tetrazolium 0estrogenic

in sensitivity

Emmens e t a l . 6 f o u n d t h a t oestrogens

they studied

was some c o r r e l a t i o n extent here.

this

activity

were n o t a c t i v e

between the present

s m e a r a s s a y may w e l l be due t o

o f t h e two m e t h o d s . all

of the non-steroid

were a l s o p r o - o e s t r o g e n s

b e t w e e n t h e two a c t i v i t i e s .

also appears to hold for the steroids

SC8~70, SC12222, SCl1800,

the most potent

were also the most potent oestrogens;

anti-

and t h a t

there

To a l i m i t e d investigated anti-oestrogens,

SC692~, SCII195, SC9263,

and SC8916, considerably less active as oestrogen antagonists, were also considerably less active as pro-oestrogens, while SC5888 devoid of anti-oestrogenic activity was also devoid of oestrogenic activity.

An exception was SCII~6~, one of the

more potent antagonists, which was only weakly active as an oestrogen.

in

was o n l y shown a t t h e

o f 500 ~ g , so t h e d i s c r e p a n c y

and t h o s e o f t h e v a g i n a l

the difference

test,

J u n e 1968

STEROIDS

903

TABLE 1 A n t i - o e s t r o g e n i c a c t i v i t y of a s e r i e s of p u t a t i v e o e s t r o g e n a n t a g o n i s t s in the i n t r a - v a g i n a l t e t r a z o l i u m a s s a y This t a b l e s,,mmarizes the r e s u l t s of a number of t e s t s c a r r i e d out a t d i f f e r e n t times. To save space the r e s u l t s l i s t e d are r e s t r i c t e d to the c o n t r o l group f o r each compound (50 pg o e s t r a d i o l a l o n e ) , the lowest dosage group to show s i g n i f i c a n t i n h i b i t i o n or where a compound showed no a c t i v i t y , the h i g h e s t dose t e s t e d . The r e s u l t s are expressed as mean o p t i c a l d e n s i t i e s w i t h 10 or w i t h 5 ( t ) a n i m a l s / g r o u p . Compound D~ SC7801 m:ff,37 MRL41 SC12222 SC8470 SCll80O sc6091 ( , ) sc11464 SC6924 SCl1195 SC10017 SC5888 SC8916 SC9263 SC4341 PROGmTm0NE

Dose (u~)

Control

Treated

0.015 0.160 4.50 4.50

0.681 0.681 0.476 0.365

0.486 ~ 0.205 0.256

0.160 0.160 0.160 o.160 0.50 4.50 4.30 4.50 4.30 4.30 4.50 4.50 4.50

0.368 0.368 0.330 0.681 0.622 0.639 0.551 0.545 0.483 0.394 0.478 0.659 0.523

0.248 XXX 0.216"** o.2o5 ~ * 0.409 o.274 ~X~ o.308 xxx 0.449* 0.294* 0.609 O.324 0.385 0.609 0.513

*o. Ol ~P < o. o3 xx x

P~O. 001

0.258xxx

90~

ST ER O ID S

11:6

TABLE 2 The e f f e c % o f l o c a l l y

applied

response to oestradiol Results group.

a r e e x p r e s s e d a s mean o p t i c a l

Time ( h r )

S C l l 8 0 0 on t h e % e t r a z o l i u m a t 241 ~0 and ~6 h o u r s densities,

Dose o f SCl1800

wi%h f i v e

animals per

Dose o~ o e s t r a d i o l

(~g)

o

5o pg

24

0.0 o.16 0.64

0.119 0.207 0.080

0.536 0.454 0.388

30

0.0 0.16 O.64

0.133 o.168 0.093

0.595 0.428 0.313

36

0.0 o.16 0.64

0.115 0.119 0.122

0.312 o.371 0.251

Analyses of variance SCll800 alone Source of variation

scn8oo

DF

High dose; control low d o s e

(T)

Linear Quadratic S x T Linear Residual Error

Mean s q u a r e s

(s)

High dose v scontrol

Time

Mean s q u a r e s

SCll800 + oes%radiol

1

986

1

2 , 5 8 1 ~-~

2

89

1,703"

vs 150 ....

1 1

m__

29253 ~-~ 3~4

4

----

255

1 3

19520* 121

35

** o.ool(v_.(o. Ol;

342

* o. o1¢£(o. 05

.... .... 280

June 1968

STEROIDS

905

TABLE The e f f e c t s of p u t a t i v e o e s t r o g e n a n t a g o n i s t s g i v e n a l o n e on v a g i n a l %etrazolium r e d u c t i o n ~ u t e r i n e weight and u t e r i n e m i t o s i s R e s u l t s are expressed as mean o p t i c a l d e n s i t i e s , mean u t e r i n e weights (mg) or as mean no. m i t o s e s / u t e r i n e s e c t i o n (Mi%), w i t h 10 or 5 (*) mice/group. Compounds were given s u b c u t a n e o u s l y i n o i l . S o m e of them were a l s o g i v e n i n t r a v a g i n a l l y , when o n l y %etrazolium r e d u c t i o n was measured. Subcu%aneous Compound

Dose

0estradiol

0.01 0.10

o.117 0.159 0.656 ~

27.7 31.g ~6.8"*

0.5 6.9 29.5 ~

50 pg -

SC12222

1 10

0.1g0 37.3 0.525"** 50.0"*

6.0 15.9"

1 10

0.13~ 0.18~*

SC8~70

1 10

0.37~ 0.~18"*

~2.2 1~.1 g7.g ~-~ 11.9"

1 10

0.151 0.163"

SCl1800

1 10

0.117 35.3 0.673*** ~8.5"

1 10

0.193 0.203*

(I/g)

0.D.

Intravaginally Mi%.

Ut.'t

Dose(I/g

0.7 9.3 ~x~

)

0.D. 0.330 -

sc11~6~

10o 500(,)

0.175 ~2.9 3.6 0.205 *~* 5g.8 *~a 28.~ ~ ' ~

SC692g

1 10 100(,)

0.101 35.5 0.28~ 38.3 0.580*** 58.3**

0.g 12.9 27.6***

100{, / 500(*)

O.lgl 38.8 0.390*** 38.7*

19.8 ~

-

-

lOO{, / 500(,)

0.106 0.186

~3.8 37.9**

3.0 16.2"

-

-

-

-

SC5888

500(,)

0.16g

23.9

0.8

-

-

SC8916

100{, / 500(,)

0.067 0.602**

30.5 52.~***

1.~ 5.~**

-

-

-

-

100{, / 500[*)

0.110 3~.3 3.0 0.6~7 ~x-~ 67.5 **~* 31.~ xx~

scn195 SClOOl7

SC9263

*0.01 ('P('O.05

~O.O01-(P(O.O1

10{, / 0.171 1 0 0 [ , ) O.lg6 -

10{,) 100(,)

~.2

*** (P(O.O01

-

0.~32 0.735***

-

-

-

-

906

ST ER O ID S

11:6

TABLE 4 The e f f e c t s

of putative

on v a g i n ~ t e t r a z o l i u m

oestrogen antagonists

reduction,

uterine

given alone

w e i g h t and u t e r i n e

mitosis

R e s u l t s a r e e x p r e s s e d as mean o p t i c a l d e n s i t i e s , mean u t e r i n e w e i g h t s (mg) o r as mean n o . m i t o s e s / u t e r i n e s e c t i o n ( M i t ) , w i t h 10 o r 5 ( t ) mice/group. The compounds were g i v e n s u b c u t a n e o u s l y i n o i l . Some o f them were a l s o g i v e n i n t r a v a g i n a l l y , when o n l y t e t r a z o l i u m r e d u c t i o n was m e a s u r e d . Subcutaneous Compound

Intravaginally

Dose ( p g )

O.D.

Ut.Wt

Mit.

Oestradiol

0.01 0.10

0.117 0.159 0.656 ~

27.7 31.g 46.8 ~

0.5

-

6.9

50 pg

29.5 ~

-

-

SC7801

10 100 500(,)

0.121 0.217 0 . 3 5 0 "~

33.5 10.4 36.8 13.3 5 1 . 0 *~'x" 2 9 . 0 ~'v*

10(t) 100 -

0.199 0.4~0 -

0.045 0.166 0.568 ~

29.6 29.5 39.6**

0.6 8.5 33.0*'**

0.1 1 10

0.174 0.256 0.371

mm~l

1(,) 10 50(,)

Vose(gg)

O.D. -

0.736 ~ *

MILL37

500 1000 2000 4000

0.091 0 . 5 4 0 "~v* 0.470 0.310

41.4 43.6** 40.5 39.9

0.6 4 7 . 2 *~* 29.4 27.6

10(,) 100 -

0.194 0.225 -

~25

1000 2000 4000

0 . 3 0 0 ~* 0.280 0.280

30.5 33.4 33.2

2 7 . 8 *~ 29.8 27.0

-

-

~*O.O01(P(O.01

××~ P ( 0 . O01

We w i s h t o t h a n k P r o f e s s o r C.W. Emmens f o r much h e l p f u l a d v i c e and c r i t i c i s m . This work was a i d e d b y a g r a n t f r o m t h e P o p u l a t i o n C o u n c i l ( U . S . A . ) and t h e Wool R e s e a r c h T r u s t Fund.

REFEBENCES

l,

T~,,mens, C.W., J. ENDOCRIN. 2; ~

(1941).

2.

k'~,,ens, C.W., J. ENDOCRIN. ~; 168 (19~2).

June 1968

STEROIDS

3.

Emmens, C.W., J. ENDOCRIN. 3; 174 (1%2).

/~.

k'hnmens, C.W., J. ENDOCRIN. ~; 316 (19t~2).

5.

R,-,ens, C.W., J. REPROD. FERPIL. 9; 277 (1965).

6.

]~mmens, C.W., Cox, R.I. and Mar%in, L., RECI~Pr PROGR. HORMONE 1.._88;415 (1962).

RES.

7.

Mar%in, L., J. ENDOCRIN. 30; 21 (196~).

8.

Mar%in, L., J. ENDOCRIN. 30; 337 (196~).

9.

Pollard, I.

Unpublished resul%s.

907