Further studies on implantation inhibitors

657

FURTHER STUDIES ON IMPLANTATION INHIBITORS Gregory Pincus,

Upendra K. Banik and Jean Jacques

The Worcester Foundation for Experimental Shrewsbury, Massachusetts and the College de France, Paris

Biology

ABSTRACT Received

August

14,

1964

Twenty-three compounds injected on day 1 or days 1 through 3 of pregnancy in rats have been tested as possible inhibitors of implantation. Among them eight have proven active at total doses of 1. 5 mg per rat or less. Administration of some of the active compounds by gavage has also led to implantation inhibition. The group of compounds found to be active were also the most potent in uterotrophic assay in immature mice. Among them a highly active compound, A-nor-androstane-2a, 17cr-diethynyl-2fl, 178 diol (V) has been examined in detail. It appears to act primarily by causing expulsion from Fallopian tubes and uterus of the free, pre-implantation ova, and was ineffective in the usual sterilizing dose in terminating the implanted embryos. Nonetheless, at the sterilizing pre-implantation dose compound V proved to be antideciduomagenic in pseudopregnant rats. Thus a dual action on oJum progression and uterine development is suggested, A direct ovicidal effect appears to be excluded since ova transplanted from treated females to pseudopregnant recipients implanted and developed into normal young in the same manner as ova transplanted from untreated control females. Compound V acted as an estrogen upon the uterus and vagina in spayed mice, but its implantationpreventing effect was not counteracted by progesterone or a highly active synthetic progestin. Following administration of a sterilizing dose of V on day 1 of pregnancy a two-week period of pseudopregnancy ensues during which mating is refused, Subsequently normal mating behavior and normal pregnancies occur. Administration of V on days 18, 19 and 20 of pregnancy does not affect the gonad development of the offspring. In previous inhibiting

papers

(1,2,3)

action of certain

coitum to rats and mice. to be effective

we have described antiprogestins

administered

Some of the compounds

in preventing

implantation

the fertilitypost

that we found

in these species

have

STEROIDS

658

45

been found by Chang (4) and Chang and Yanagimachi

(5) to be

similarly

found by

active in the rabbit.

Among the compounds

Chang (5) to be at least partially two orally active

gestagens,

diacetate

(7).

Norethynodrel

pregnancy

in rats (8,9).

effective

norethynodrel

in the rabbit were (6) and ethynodiol

has also been found to prevent

Accordingly

this paper we have included several

in the studies reported in active

progestins

as well

as antiprogestins. MATERIALSAND METHODS The method is essentially the same as in our previous communications (1,lO) unless, in certain instances, as otherwise stated. Most of the experiments were performed on female Sprague Dawley rats weighing 170-250g. The compound was administered on different dates before or after mating with known fertile males. All animals were killed between days 9 and 10 after mating. The number of implanted embryos were counted and observed. Any animal having at least one or more implanted embryos was considered as pregnant. Statistical analysis of the number of pregnant and nonpregnant animals was made using Mainland and Murray’s (11) four-fold contingency test tables. The average number of fetuses in pregnant animals in the experimental series were compared with those of the controls according to the procedure laid down by Dunnett (12). All animals, before and after mating, were maintained on uniform husbandry conditions and fed a standard Purina laboratory diet with drinking water ad libitum. Uterotrophic assays of the compounds studied were made in immature mice by the method of Rubin et al. (13). Methods used in special studies with compound V are detailed in the text. RESULTS The prevention compounds

of implantation.

tested are presented

the number of pregnancies presented

in Table 1.

The structural formulae of the in Fig.

occurring

At the dosages

1.

Their effects

upon

and on mean litter size are used,

statistically

sig-

STEROIDS

Nov. 1964

nificant

reductions

compounds

in both of these parameters are found for

V, IX8 XVI XVII, XVI& XX and XXII,

ing XVI had significantly reduction

659

reduced

in numbers pregnant.

litter sizes,

Animals receivbut a nunsignificant

Among these are five A-nor steroids

STEROIDS

660

4:5

Table I Effects

of CORIDOundS Dose

Compound Control 0,2 mi, oil I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI

XVIII XIX XX XXI XXII XXIII

0 0 1. 5 1. s** 1. 5 1. 5 1. 5 1.5 0. I 0. 5 0. 5 0. 5 0. 1 0, 5 1. 0 1. 0 1. 0 1. 0 1.0 0.5 1.5 0. 5 1. 0 0.01 0.01** 0. 1 1.0 0. 5 0. s** 1.5 1.5** 0. 1 0. 5 0. 1 0. 5 0. 1 1. 0 0. 1

of Fiaure 1 on Imolantation Number of rats

Number preqnant

8 6 7 6 6 8 9 10 6 6 6 5 4 5 5 5 6 6 4 5 6 5 7 5 5 5 5 4 5 6 5 5 5 5 6 6 5

7 4 5 4 6 7 1 1 4 5 6 5 1 4 5 5 6 6 3 2 3 3 7 3 1 0 5 0 5 5 4 2 5 3 0 0 4

of Fertilized

P+ Pr

N, S. #

N, S. N, S. N. S.

$;i 0

N. S. N. S. N. S.

Esus of Rats

Litter size (mea nl

10. 6 12.7 12, 2 13. 2 11. 2 12. 8 3. 0 11.0 7. 2 10.4 10.7 9.0 6. 0 12.7 9.4 10. 8 11. 5 10, 6 2,6 4. 5 6,O 2. 3 11. 8 10, 8 3.0 0 11.0 0 8. 5 11.0 10. 5 3. 5 8, 6 8. 6 0 0 13.2




* Injected on Day 1 of pregnancy only. ** Injected from Day 1 through Day 3. + Compared with control (Pr - Number of pregnant rats and l-litter # Not significantly different from control at 5% level.

P+ 1

N. N. N. N.

S. Se Se S.

c,“i N. S. N, S.

N. S. N. S.


N. S. N.S. N, S.


$K . < N, S. N. S. 0.01 (0.01
size.)

Nov. 1964

STEROIDS

661

fv, IX, XV, XX and XXII), structural relatives viously found active (I, 3).

as antiprogestins

&VI and XVII) previously

Rorig (14) to be antifertility contrast

several

analogues

to prevent pregnancy

stanes with various failed

agents and one progestin

to exhibit

The potency

significant of estrogens

groups

(15,16),

used, 4,

inhibiting each.

as implantation

inhibitors

reexamined

assay,

has been

by Emmens and his may be used

We are thus able to

“estrogenicity”

of our test

implantation-

on the basis of minimum effective

doses

This we have done in Table 2, setting the activity

estrone

in each test at 100,

Table 1.

It is notable

compounds (listed)

XII, XIII, XIV, Xxi) also

and to compare that with relative activity

(VI, VII, XI)

and five A-nor andro-

and we have found that estrone

in a uterotrophic

compounds

or implantation.

activity.

as a standard for the test used here (17). express,

In

(II, IV, XIX, XXIII) or

at the doses

long known and has been recently colleagues

of

(XVIII).

of active A-nor compounds

substituent

inhibitors

found by Saunders and

(I, III, VIII) failed to prevent pregnancy analogues

pre-

are two analogues

highly potent progestins

Three androstane failed

and implantation

The only other active compounds

stilbestrol

of compounds

and listing

of of

the active compounds

that the uterotrophic

potency

of

of the inactive

of Table 1 were all less than those found for the

active

compounds.

the next @XI) 1/7000th

The most potent (XIII) was 1/5500th,

and the progestins

ranging from l/lOOOOth

662

STEROIDS

45

Table II B ComwciSpgqf_Utemic

n Inhua

awlantatio

Activa

Relative Potencv Implantation lnhibitina m 100 SO 4 2 4 20 5 2 100

100 20 0.4 0. 25 0. 2 0.02 0. 25 0. 14 2.50

Estrone V Ix xv XVI XVII XVIII xx XXII

Ratio

1 0.40 0. 10 0. 13 0.05 0.001 0. 05 0. 07 0.025

(XIX) to less th an l/50 OOOth&XIII) as active as estrone. would thus seem that a degree of “estrogenicity” with implantation

prevention.

However,

It

is associated

as indicated

by the last

column of Table 2, considerable differences are had in the ratio of one activity uterotrophic

to the other,

potency

tation inhibition. implantation

compound XVII having l/SOOOth the

of e&one

and l/S*

its potency

Compound V, in contrast,

inhibitor but also considerably

in implan-

is more active as an more uterotrophic

than

XVII. A number of the compounds activity

of Table 1 have been tested for

upon oral administration.

3, indicate

substantial

compounds

tested.

higher doses

activity

The data, presented

in Table

by this route for five of the seven

Since there is a suggestion

are needed by this route (cf. -

that somewhat

data in Table 1 for

663

STEROIDS

Nov.1964

TableIII Effect of Administratian bv Gavase of Different Compounds on Imnlantation of Blastocvsts of Rats* Duse

ComRuu~d jma/rat), Control WI V

Ix

Number

of rats

0

P+ Pr

_L-

48.01 <0. of,

9*0

N. S.

N, S, N. S.

11.0 6.3

N. Se < 0, OS

N. S, 0.01 < (0.01

11.5 6.0 0

N. S, < 0. 05 (0. OL

6.0

(0.05

2.0

(0.01

6

3 1

0.5

s

3

N. S, **

4 4

2

3

0. 1 0.5

4 5 5

4 x 0

0.5

5

< 0.05 (0.01

1.0

5

4 1

XXI

0.1 0. s

4 6

4 5

N. S, N. S,

XXII

0.02

5

5

0. 1 0. s

4 6

0 0

N. S, < 0.01 (0.01

xx

P+

[mean)

3*6 3. 0

7

0.01

Litter size

10.7

10

0,s 1.5

xv XVII

x3

Number preanant

9,s

N. S, N. S,

9.6 0 0

N. S. <0. 01

11.4

(0.

oi

* Fed by feeding tube attached tu a glass syrfnge on Day f of pregnancy in a volume of 0.4 ml oil, Control animals received same quantity of vehicle only by the same procedure. ** Not significantly different from control,

* Comparedwith control, #

Fed on Day 3 of pregnancy, compounds V and XX), the compounds fai&ng tu exhibit significant activity

might do so at higher oral doses

than those used thus far.

STEROIDS

664

Activities

of compound V.

4:5

We have conducted

a series

of studies

on the action of compound V as a most potent A-nor steroid. data of Table 4 demonstrate: down to 100 pg on single

(a) a consistent

injection

by 30 pg given over three days,

3,4,5

and 8 (Table 5).

of the single

inhibition

100 pg dose on

injection

Failure to prevent pregnancy

on days

or to affect the

is seen only for the rats injected

has taken place.

that the pre-implantation

ranging

but not by 6 pg so administered.

the same dose in single

number of embryos surviving day 8 when implantation

at doses

on day 1, (b) an effective

Ln view of the clear effectiveness day 1, we have employed

activity

The

stages

Therefore,

on

it would appear

are primarily susceptible

to the action

of this compound. The action upon tubal and uterine ova may be direct upon the ova themselves

(i.e.,

by preventing

ovicidal)

or indirect

by causing

the uterus from developing

have attempted to study these possibilities effect

of compound V on deciduomagenesis

ovum expulsion

receptivity by:

to the ova.

(a) determining

in pseudopregnant

(Table 6) and (b) by tracing the movement of the fertilized control

and V-injected

rats into pseudopregnant

We the

rats

ova in

recipients

The data of Table 6 are taken from pseudopregnant

(Table 8).

rats.

The

left uterine horn of each animal was stimulated on day 5 by inserting oviduct

a barbed needle through the tubal sphincter down to the cervix,

thus effecting

metrial aspect for the entire horn length.

or

beneath the

trauma to the antimesoIn the experimental

Nov. 1964

665

STEROIDS

Table IV

yprvino pnsaaes

of Comoound V Administered Post-Coitallv

Dose

Litter size

Number preanant

Number gf rats

iiLibmL 10.57

0.2 ml. oil

8

7

20**

6

4

loo**

10

1

< 0.01

soo**

5

1

< 0.05

1500**

5

0

lo/day

for 3 days***

11

0

2/&y

for 3 days ***

6

5

*

** *** + /

to Female RaU

7.75

N.S.+

N. S.

11.0

N. 8.

t

3. 0

0.01

0.01

0

H. s.+

< 0.01

0

H. 8.

9.0

N. S.

<

N. 5.

Compared with control. Single injection on day 1. Starting from day & of pregnancy. Not significantly different from control at 5% level. Highly significant. group 100 pg of compound V was injected immediately received

after the traumatization

the oil vehicle.

weight

It is obvious

increase

horn whereas difference

the control

at 72

that in the control group significant

in the experimental

in weight between

group

and the uterine horns dissected

due to deciduoma

may have affected

whereas

The animals were sacrificed

hours after the traumatization and weighed.

subcutaneously

formation took place in the left group there is no significant

the two horns.

That compound V

the normal growth of the pseudopregnant

uterus

666

STEROIDS

45

Table V

Infection (DaYf

Number or rats

Number preanant

8

7

1

10

1

3

7

0

4

7

1

5

6

0

6

6

6

Control: 1

*

** ;

Litter size (Mean)

P** pr

P+* 1

12.85 < 0.01 <

7‘ 0

0.01

0

< 0.01 <

7.0 0

0.01 N.S.#

10.83

< 0. OS IG s.+ < 0.05 Ii. 5. N, S.

lOOpg/rat was injected on different dates after mating. Control animals received vehicle only on Day 1. Compared with control. Highly significant. Not significantly different from control at S% level.

Table VI

Treatment

No. mts

Left horn*

(rnol oer 100 u bodv we&& Right horn &lean i&,&J

Oil control

8

350.73 l54. 23

111.66 *lo. 86

0.001

Compound V 100 pg/rat

8

82.77 f 6.32

76.27 f 5.38

N.S.+

~

*

$ +

Stimulated by scratching with a barbed needle. Comparlson between left and right horn. Not significantly dlfferent.

P)

Nov. 1964

667

STEROIDS

is suggested

by the considerable

horns in the experimental

group compared to the control

That compound V also causes Fallopian

lower mean weight of the right

expulsion

animals sacrificed

junction

eggs is

They demonstrate

on day 4 of pregnancy

found at the utero-tubal 1 and 2).

of ova from the

tubes and uterus of rats carrying fertilized

evident from the data of Table 7.

group.

that in control

almost all of the ova are

and a few in the uterus (expts.

But in animals receiving

100 pg of V on day 1 no ova

are found in the tubes or the uterus on day 4(expt.

3).

search made one day earlier also fails to disclose

any ova

(expt.

4).

From 17 animals injected

on day 3 and examined

hours later only 4 ova were obtained, and three in the uterus (expt. of ova produced been present. injection Expulsion

(1).

24

one at the tubal junction

5); on the basis

of the mean number

in this strain of rats over 100 eggs should have Examination

on day 3 disclosed

of the oviducts (expt.

at 12 hours after V-

6) most of the ova in the uterus.

of free ova is thus obvious.

We have previously blastocysts

Indeed,

reported morphological

of rats receiving

normality of

an implantation-inhibiting

steroid

The data of Table 8 on ova taken from rats of the type

described

for experiment

plantation

to untreated pseudopregnant

42% implanting as PUPS.

6 of Table 7 indicate

(as observed

that their trans-

recipients

results

in

on day 10 or 11) end 36% emerging

This is not significantly

different

from the 47% of ova

STEROIDS

668

4:5

Table VII

No. of Blastocvsts Fallopian Tubal tubelunction-

Bxpefiment # 1 2 3 4 5

6

Control: 0.2 ml oil on Day 1. Bggs on Day 4

6

0

37

0

Control: 0.2 ml oil on Day 3. Eggs on Day 4

6

0

54

6

10

0

0

0

6

0

0

0

17

0

1

3

9

0

9

46

Compound V on BY Bggs on Day 4

1.

Compound V on my Bggs on Day 3.

1.

Compound V on -Y 24 hours before collection

3,

Compound V on Day 3, 12 hours before collection

* Bach rat in experimental implanting

series received

100 pg by a single injection.

and the 39% born alive in the control

Since compound V is significantly

animals.

uterotrophic

in immature

mice (Table 2) we have examined its “estrogenicity” other tests:

(a) uterotrophic

activity

on administration

(20 to 30 g) C 57 mice for three days beginning ovariectomy activity injection

with sacrifice

by two to spayed

three weeks

on day 4, and (b) vaginal

after

cornifying

in adult spayed mice injected

subcutaneously

in a single

at 15 days after ovariectomy.

The potency

of V relative

Nov. 1964

STEROIDS

669

Table VIII Imp:lantation and DeveloBment

No,

of Eros fn3m Comoound V-Treated Rats* of eggs

Treatment

Number )mdanied

No, pups &rn

Oil Control

Sl

24

20

100 &rat on Ray 3: 12 hours before collection

64

27

23

Remarks AI1 pups were found to be in normal shape and good health All pups were found to be in normal shape and good heaith

* All eggs were collected on Day 4 of pregnancy of treated animals and transferred to uterus at ths tubal end on Day 4 of pseudopregnancy.

to estrone was in (a) 7.1% antiprogestin

and in (b) 7.0%.

(18) we would expect

by progestational

its activity

steroids * As indicated

neither progesterone,

nor progesterone

highly potent synthetic

progestin

Since it is also an

have,

to be antagonized

by the data of Table 9

plus estradiol,

upon subcutaneous

injection,

been able significantly

to antagonize

preventing

effect

This is entirely

previously

reported data on attempts to reverse

inhibiting

of compound V.

activity

acetoxy-17a-ethinyl-1 progesterone

the implantation-

experiment

in rats and mice of the enol acetate

of 178-

g-nor androsta-3,5-diene-3,17-diol

and three synthetic

progestins

1 administration

at proestrus

by

(1).

in rats housed with males under various

with the proportion

similar to our

the implantation

In Table 10 we list the mating performance occurring

nor a

and the implantations conditions.

In

did not appear to interfere

mating (compared with the oil-injected

controls)

670

STEROIDS

4:5

&tempts to Reverse the ImPlantation-Inhibitina Dose/ratfbay

Comnound

for 3 days

Number of rats

Activitv of CornPound V*

Number presrnant

Pf Pr

Litter size fmmsanf

Contrclf

0, 2 ml. 011

6

0

Progesterone

IO

mg.

7

3

N. 5, #

6.6

S mg, **

5

0

N*S.

0

N, 5.

5

0

N. S.

0

N. Se

6

0

N. 5.

0

N” se

4

0

N.S.

0

N,S.

Progesterone Progesterone

IO mg, **

Progelsrerone + Estradiol

+ 0.5 iip.

Synthetic Progestf n*

S

mg,

LO

mg,

0

Compound V wag administered (100 W/rat) on Day 1 of pregnancy tc all rats. * Progesterone injected from D8y 1 through Day 5. Compound V (100 pg/rat) was given on Day 3. / Control animals, tn addition to compoun4 received vehicle only for 3 days. * 3&l%-Macetoxy-k-methyl pregn-4-en-20-one (This compound has been found to have about 20 times the potency of progesterone, Pincus and Merrill, 1964,) # Not significantly different from control at 5% level. l

l

but a reduced number exhibited implantations,

This implies either

interference with fertilization or perhaps an ovum expelling effect in most of the injected animals.

In experiment 2 apparently normal

mating and pregnancy occurred after the “pseudopregnancy” consequent on compound V administration.

That the sterile period

induced by compound V is indeed a pseudopregnancy is attested by the lack of mating to males housed continuously with day 1 Vinjected females (expt, 31, There thus appears to be no carryover of the sterilizing action of compound V. Recently Kletchel and Pincus (19) have recorded the gonadal abn~rma~i~es in rabbit fetuses after in vitro treatments of fertilized

Nov. 1964

STEROIDS

671

TableX

When mating No. of snimslroccurred

To 11 within

12

Control - at proestnis

7 days** Experlmental

- at

9

10. 5

10.0

12

To 10 within 7 days**

3

15

To 13 within

12

10.3

13

12.0

Pl-OWtNS

Day 1 of pregnancy

Mean no. of mtlons

No. with im~lantatlons_

2 weeks of end of induced sterile period l ** 3

Day 1 of pregnancy

To 15 within

16

one week of end of induced sterile period**

*

Administered subcutaneously in a single 100 wg dose. Housed with males throughout. l ** Housed with males at end of the two week sterile period,

l*

eggs with estradiol the effects

The compound

on days 18,19

by Cesarean

section

distance

et al.

to study

and anogenital

(100 pg/rat/day

and 20 of pregnancy.

distance

for 3 days) was Fetuses were removed

on day 21 and the anogenital

measured by vernier calipers. Revesz

Thus it was of interest

of compound V on sex abnormality

of rat fetuses. injected

and stilbestrol.

distance

It is now well established

was by

(20) and Kincl and Dorfman (21) that the anogenital

of the male fetuses

Sex of each individual

is greater than that of female fetuses.

fetus was confirmed

by opening the abdomen.

Results in Table 11 show that the compound has no adverse either on sex reversal

or on anogenital

distance.

effect

672

STEROIDS

4:5

Table Xl

Anogenital (mean)

Treatmentr* oil control

5

27

8

1

3.4 27

Compound V

didance

1.6

46

3.5 38

1.8

* Each animal received low per day for 3 dayr, 1. e. on Days 18, 19 and Poetuserr were removed by Cerarean section on 20 of pregnancy. Day 21 and anogenital distance of each foetus was measured by Vernier Caliper.

DISCUSSION All of the eight compounds exhibit

found to be inhibitors

in immature mice a degree of uterotrophic

that of any of the 15 having no significant effect.

It may therefore

estrogens, tests clearly

that the activity

activity

implantation-inhibiting

its estrogen-like

activity.

of V cannot be reversed

gestagens.

cannot be reversed He observed,

Also our observation

by progestins

ratios employed, inhibited.

by progesterone

moreover,

other characteristic

Therefore,

pregnancy

act as

of compound V in two additional

is paralleled

by the finding of Martin (22) that in mice the interruption by estrogens

exceeding

be said that the potent compounds

and indeed the behavior indicates

of implantation

of pregnancy

or several

19-nor

that at the progestin:estrogen estrogen

prevention

effects

could be

may involve

a special

Nov. 1964

673

STEROIDS

qualitative

effect

of steroidal

effecters.

We have shown that the primary effect appear to be ovum expulsion. rabbits

This is an effect

(23), rats (24) and guinea pigs (25).

deciduomagenic

as is XVIII and norethynodrel

pointed out that norethynodrel given post coitally

of compound V would of other estrogens

However, (26).

it is also anti-

Saunders (27) has

which also is an antifertility

agent

to rats (9) may act as an antigonadotrophin

pituitary and thus depress

ovarian function

in

and consequent

to the

sustain-

ment of pregnancy. It is obvious implantation

that the exact sequence

prevention

by the types of compounds Is the locus

require further clarification. effect

receiving

may inhibit implantation

pregnancy-maintaining

For that compound’s

effect

It would seem that a careful

studied here

of the ovum expulsion

in the tubal and uterine musculature?

that norethynodrel

of events leading to

Davis (28) has shown in ovariectomized

rats

doses

of estrogen

and progesterone.

the presence

of ovaries

is not obligatory.

study of effects

on myometrium would

be rewarding. Perhaps the most surprising the observation

of estrogen-like

A-nor androstanes.

bolically

estrogens

convertible

activity

To our knowledge

has thus far been associated non-steroidal

outcome

of several

to phenols.

of the saturated

a degree of ring A unsaturation

with steroidal

have phenolic

of our studies has been

estrogens,

rings or benzene

and even the rings meta-

To what extent these A-nor

STEROIDS

674

androstanes

are estrogen-like

to be seen.

Their metabolic

course,

a possibility,

4:5

in other measurable conversion

functions

to phenolic

remains

steroid is,

of

but if so it would be an unprecedented

process. The use of the mouse for uterotrophic for assay

of anti-implantation

disadvantage necessarily

since estroqenic

species

(1,3).

implantation

inhibitors

would seem to have some

potency

by standard assay

pounds may involve

We have shown,

in the A-nor series are active

The divergence inhibiting

and of the rat

activity

parallel in the two species,

that implantation

assay

between uterotrophic

potency

differences

but it cannot be regarded as a qualitative

however, in both

and

seen in some of the active

quantitative

is not

com-

in “estroqenicity” species

difference.

ACKNOWLEDGMENTS The investigations described in this paper were aided by research grants from Merck, Sharp and Dohme, the Population Council and G, D. Searle and Co. We are indebted to the following donors for generous supplies of the indicated compounds: Dr. B. L&ken: II, III, IV and XXIII; Dr. M. Tishler: XV and XXII; Dr. F. Colton: I and XVIII; Dr. J. Brown: VIII; Dr. K, Rorig: XVI and XVII; Dr. H. Gibian: XIX. A number of the A-nor steroids have been prepared by one of us (J*3; ) in collaboration with M. Minssen, M. J. Brienne, D. Varech and J. J. Basselier; they are the subject of a series of notes that will appear in the Bul1eti.n de la Societe Chimiuue de France. REFERENCES 1.

Banik, U. K., 111,595(1962).

and Pincus.

G.,

PROC. SOC. EXP. BIOL, MED.,

2. Jacques, J., and Pincus, G., PRCC. 1st INT. CONG. ON HORMONAL STEROIDS, Excerpta Medica Press, Amsterdam, 1962, p. 3.

Nov. 1964

3.

STEROIDS

K., PROC. 7th INT. PLANNED Excerpta Medica, Series No.

and Banik, U. Pincus, G., PARENTHOOD FED. CONF., 494 (1963).

4.

Chang,

M.

C.,

FERT.

5.

Chang,

M.

C.,

and Yanagimachi,

6.

Pincus, G., and Merrill,

7.

Elton, R. L. $ and Nutting, 107, 991(1961).

8.

Saunders,

AND STERIL.,

9.

Davis,

B. K.,

10.

Banik,

U.

11.

Mainland,

12.

Dunnett,

13.

Rubin, B. L., ENDOCRINOL.,

14.

Saunders, (1964).

15.

Martin, L., 20,307(1960).

16.

Emmens,C.W., (1962).

17.

Banik, U.K., and Pincus, (in press) (1964).

18.

Pincus,

19.

Ketchel, M., 115,419(1964).

20.

Revesz, C., 66,140(1960).

Chappel,

21.

Kincl, (1962).

and Dorfman,

15, R.

J.,

ANN.

D.,

(in manuscript)

(1964).

EXPTL.

I.

M.,

STAT.

EXP.

RES.,

C.,

BIOL.

MED.

20,395(1964).

K.,

C. W.,

and Finn,

and Merrill,

L.,

FERTIL.

C.A.,

J.

G.,

PROC.

and Pincus,

C.L.,

and Dorfman,

R. I.,

J.

REPROD.

SOC.

(unpublished

G. I PROC.

SOC.

and Goudry,

R. I.,

116,591(1952).

AND STERIL.,

and Cox,

A.

22,57(1962).

50,1096(1955).

Black,

and Rorig,

MED.,

SCIENCE,

ASS.,

Dorfman, A.S., 49,429(1951).

Emmens,

SOC.

HORMONE

BIOCHEM.

J. AM.

F. J.,

PROC.

M.

157,308(1963).

and Murray,

C.W.,

F.,

PROGR.

NATURE,

K.,

G.,

REC.

E.

72,

97 (1964).

Chang, M. C., Hafez, E.S.E., Zarrow, A., ENDOCRINOL., 59, 695 (1956).

F.

F.A.,

675

15,202

ENDOCRINOL.,

FERTIL.,

EXP.

data)

BIOL.

3,239

MED.,

(1964).

EXP.

R.,

R. I.,

BIOL.

MED.,

ENDOCRINOL.,

ACTA ENDOCRINOL.,

41,274

STEROIDS

676

22.

Martin,

L.,

J.

ENDOCRINOL.,

23.

Greenwald,

G.S.,

J.

24.

Greenwald,

G.S.,

ENDOCRINOL.,

25.

Deansley,

26.

Miyake,

27.

Saunders,

28.

Davis,

R., T.,

J.

F. J., B.K.,

J.

26,31

(1963).

ENDOCRINOL.,

REPROD.

Kakieshi,

45

H.,

26,133(1963). 69,1068(1961).

FERTIL., and Hara,

ACTA ENDOCRINOL., ENDOCRINOL.,

5,49(1963). K.,

STEROIDS,

46,157(1964B).

27,99(1963).

2,749(1963).