Macromolecular binding and metabolism of progesterone in the decidual and pseudopregnant rat and rabbit uterus

441

MACROMOLECULAR BINDING

AND METABOLISM OF PROGESTERONE IN THE DEC I DUAL

AND PSEUDOPREGNANT RAT AND RABBIT UTERUS.’

J,

R.

Reel,

Department

of

S.

D.

Pharmacology,

Parke,

Received:

Van Dewark,

Davis

Y.

Division & Co,,

Shih,

of

and M. R.

Medical

Ann Arbor,

Ca 1 lant ine2

and Scientific

Affairs

48106

Michigan

7720771 ABSTRACT

The present study was undertaken to determine whether progesterone and/or its early metabol ite(s) associate wfth macromolecules in the decidual (traumatized) and pseudopregnant rat and rabbit uterus. Under both --in vivo and --in vitro conditions progesterone-7&H-derived radioactivity binds to 4-5s protein(s) in the uterine cytosol fraction of both species. I n addi t ion, it was shown that the decidual rat uterus rapidly converts progesterone to 3X-OH-3%pregnan-20-one and that this is the major early metaboli te of progesterone in the cytoplasm of this tissue. Both progesterone and 3C%-OH-!3-pregnan-20-one bind to 4-9 cytoplasmic protein(s), suggesting that the early metabolite, in addition to progesterone, may play some role in regulating the progestational response of the uterus.

Presently,

the

nism

of

action

with

target

action

hormone

and,

organ

demonstration

ing

proteins

ing

progestin

oviduct

1

rests

as such,

the

in

(24-26)

generally on

receptors

Support

response,

most

is for

of

thls

in

have

also

for

that

the

initial

the

the

organs

m~alian

become

of

been

a unitary

hormone

of

gained

mecha-

interaction

event

expression

has

androgen-,

target

concept

premise

hypothesis

estrogen-,

binding

the

represents

essential

respective

accepted

in

hormone

the

hormone

primarily

from

and corticoid-specific (l-17).

uterus

Recently, (18-23)

and

binddata

the

concern-

chick

available.

The research described in this report involved animal care facilities fully accredited by the for Accreditation of Laboratory Animal Care. 2 Mead Johnson Research Center, Present address :

animals maintained American Association Evansville,

Ind.

in

47721.

STEROIDS

442

The early derived

observation

radioactivity

led

us to examine

and

rabbit

described one,

target

selectively this

tissue

a “progestin

here

an early

protein(s) is

for

demonstrate major

metabol

these

uterine

in organ

by Wiest

(27)

the

receptor”.

ite

showing

accumulated and

that

18:4

in

The --in

the

viva

decidual

rat

uterus

of

and --in

vitro

uterus

the

rat

studies

and 3a-OH-5a-pregnan-20-

progesterone,

preparations

progesterone-

pseudopregnant

progesterone of

that

associate

and show that

with this

4-5s

binding

specific.

MATERIALS AND METHODS Induction of Pseudopregnancy and Decidual i zation: The method of Yochim and DeFeo (28) was employed to initiate pseudopregnancy and decidual ization of uteri of sexually mature Ho1 tzman rats weighing 200-250 gms. Decidualization was induced in the right cornu while the contralateral cornu served as the pseudopregnant control D On the 9th day of pseudopregnancy (the day of maximum decidualization) the rats were sacrificed with ether or by the injection of air into the heart and the decidual and pseudopregnant cornua were excised, sl i t long i tudi nal 1y, blotted, and weighed on a torsion balance. The wet weights of decidual cornua ranged from 1500 to 2500 mg, whereas the pseudopregnant cornua ranged from 130 to 180 mg. Estrous rabbits of the Dutch-belted strain weighing 2 to 3 kg were mated on day 0 with vasectomized bucks to initiate pseudopregnancy. The “thread loop method” of Courrier and Kehl (29) was employed to induce uterine trauma. On day 7 (the day of ova imp1 antation in normal pregnancy) the right cornu was traumatized by inserting a threaded needle at the utero-tubal junction and drawing it through the lumen, exiting near the cervix. The two exteriorized ends of surgical thread were knotted forming a loop. The left cornu was left undisturbed in the pseudopregnant condition. On day 14 of pseudopregnancy the rabbits were sacrificed by cervical dislocation and the uteri were handled as described for rats, except that 500 mg segments from the central portion of cornua were taken for study. It was noted that the traumatized and pseudopregnant cornua did not differ significantly in weight which is in accord with the 1 imi ted decidual response often observed in this species (30,31). For this reason wa conservatively refer to this preparation as a traumatized rather than a decidual cornu. In Vivo Administration and In Vitro Incubation with Progesterone-7a or 1, 2-5H : On the 9th day of pseudopregnancy two rats per group (under ether athesia) were injected with progesterone-7c+3H (New England Nuclear, 20 C/mmole) via the tail vein using either 60 )rC in 0.5 ml or 500 PC

Oct. 1971

STEROIDS

443

Five and 15 minutes later the in 1.0 ml of 0.151 NaCl - 5% ethanol. rats were thoroughly bled via cardiac puncture and then perfused with Because of the quantity of isotope required only one 0.15J NaCl. On the 14th day of pseudorabbit per --in vivo experiment was used. pregnancy 1 hour prior to autopsy, 1.0 mC of progesterone-7&3H in 0.5 ml of propylene glycol was injected into the marginal ear vein. In the --in vivo rat experiments the decidual cornua were preincubated for 1 hour at 4OC, whereas in the rabbit the uterine --in vivo experiment tissues were preincubated for 2 hours at 370C before preparation of the cytosol fraction. For _in vitro rat and rabbit experiments, uterine and other tissues were preincubated for 1 hour at 37OC in Eagle’s HeLa (Difco) or KrebsRinger-Henseleit-glucose medium (32), respectively, prior to incjbation of the tissues or cytosol fractions with progesterone-7a or 1,2H. Preincubation was found to wash out considerable quantities of extracellular serum proteins. Detai 1 s of each incubation with progesterone3H are given in the figure legends. Preparation of Cytosol Fractions and Sucrose Gradient Analysis: All tissues were homogenized in the cold in O.OlM tris-0.0015fi EDTA, pH 7.4 using a motor-driven al 1 -glass Kontes apparatus. The homogenates were centrifuged at 105,OOUXg for 90 minutes to obtain a supernatant cytosol Two-tenths ml aliquots of the cytosol fraction were layered fraction. on 6-21% linear sucrose gradients containing O.OlM tris-0.0015! EDTA, was carried out in a ~~-56 rotor with a pH 7.4. ul tracentrifugation Beckman ~2-65~ operating at 55,000 RPM (average centrifugal force = 297,OOOXg) for 16 hours at 4’C, except where indicated in figure legends. The ultraviolet absorbance (1 = 276 np) and radioactivity profiles of the gradients were determined by placing the tubes in a Beckman fractionating system and pumping the gradients upward with 88% glycerol through a LKB flow cell equipped with an ultraviolet detector and collecting 0.2 ml fractions by means of an LKB fraction collector.. The 0.2 ml fractions were counted in 3.0 ml of ethanol and 15 ml of scintillation solvent’ using a Packard 3375 spectrometer. Quench correction was made by external standardization. Steroid Extraction: In the --in vivo rat experiment pooled 0.2 ml gradient fractions from the 4-5s region, the whole cytosol fraction, and serum were adjusted to 0.5% NaOH’wi th 2.5% NaOH and extracted 3 times by partitioning against 2 volumes of diethyl ether:ethyl acetate For the --in vitro rat experiments, in which either the (1:’ v/v). decidual cornua r the cytosol fraction was incubated directly with ‘j H, pooled progesterone-7o+ 0.2 ml gradient fractions from the 4-5s reg’on and the whole cytosol fraction were extracted as above, except the addition of NaOH was omitted. The steroid extractions for both exper.iments yielded >,99% of the radioactivity in --In vivo and --in vitro the organic phase. The organic phase was washed twice with 0.1 volume

1

0.1 gm POPOP, 8.0 dioxane.

gm PPO,

and

110.0

gm of

naphthalene

per

1 iter

of

STEROIDS

444

18:4

Fol lowof H20 and the residual H20 was removed with anhydrous Na2S04. the organic extract was taken to dryness in a flash ing filtration, The residue was dissolved in a small volume of evaporator at 45OC. CHC13:CH30H (1 :1 v/v) and subjected to paper chromatography. Paper Chromatoqraphy: Steroid extracts or eluates were sequentially paper strips (45 x 2 cm) in the chromatographed on Whatman No. 1 filter toluene/propylene glycol (33), 1 igroine/propylene following systems: steroids were detected gl ycol (34), and hexane/formami de (27) o Labeled by scanning the paper chromatograms in a Packard Model 7201 strip The major radioactive components were eluted with methanol counter. and counted in a Packard 3375 scintillation spectrometer. (a) Acetyl ation: The Steroid Identification and Characterization: steroid was dissolved in 0.2 ml of ovridine and 0.1 ml of acetic anhydride and allowed to stand at room’temperature overnight. One-half ml of methanol was added and the pyridine and excess acetic anhydride The steroid residue was were evaporated with a stream of N2 at 60OC. (b) Digi tonide then rechromatographed in the hexane/formami de sys tern. Precipitation: One mg of 3@-OH-w-pregnan-20-one in 0.5 ml of 95% ethanol at 55OC was added to duplicate samples consisting of 5000 DPM of presumptive progesterone of 3a-OH-5a-pregnan-20-one, respectively, previously separated by sequential chromatography in the systems Five-tenths ml of 1% digitonin in 50% ethanol was described above. the mixture stirred and al lowed to stand at room temperature added, The digi tonide precipitates were co1 lected by centifugaovernight. The precipitate was tion and washed with cold 95% ethanol and ether. and the steroid extracted by dissolved in 0.1 ml of pyridine (45’C) (c) Chromic Acid Oxidapartitioning against 2.0 ml of diethyl ether. Two-tenths ml of freshly prepared 0.4% Cr03 in 90% acetic acid tion: was added to the presumptive progesterone and 3a-OH-5a-pregnan-20-one The vessel was stoppered and allowed to stand at room residues. After the addition of 3 ml of H20, the sample temperature for 1 hour. was extracted 3 times with 3.0 ml of hexane. The extract was dried and subjected to silica gel thin layer chromatography in a saturated In this system the mobi. ity of the benzene:acetone (8: 2 v/v) system. presumptive progesterone was unchanged, whereas the presumptive ~CXOH-w-pregnan-20-one was oxidized to a derivative having the same mobi 1 i ty as 5cr+pregnan-3,20-dione. (d) Recrystall ization to Constant Recrystall ization of the steroid residues, resultSpecific Activity: ing from the elution of radioactive zones from the H/F chromatograms, was carried out using the methods of Axelrod et al. (35).

RESULTS Proqestin terone

Binding binds

was administered

to

in

the

uterine to

rats

Rat

Uterus:

macromolecule(s) which

had one

In order in

to

vivo,

decidual

determine

if

proges-

progesterone-7aand one

pseudopregnant

STEROIDS

Oct. 1971

uterine

cornu.

activity

in

sucrose cornua

the

density were

not

After

a 5 or

cytosol

of

gradient

15 minute

two pooled

in

this

circulation decidual

the

analysis;

studied

445

time cornua

contra1

ateral As seen

experiment.

the

binding

was assessed

by

pseudopregnant in

Figure

1,

1600 51s 5 min. ....... 15min.

0

4

TOP

8 12 16 FRACTION NO. Bottom

Sucrose gradient analysis of _in vivo progestin binding Fiqure 1. and 15 minutes after i.v. in the cytosol of decidual rat cornua 5 H. injection of 60 PC of progesterone-7a-

progesterone-derived fraction to

of

the

radioactivity cytosol

5.1 S when compared

Martin

and Ames

4 to 5S complex

(36). is

which to yeast

It

is

considerably

binds has

--in

vivo

to

a sedimentation

alcohol interesting reduced

coefficient

dehydrogenase to

note

a macromolecular

that

in comparing

of

by the the the

4.7

method

amount

15 to

of

of

the

5

18:4

STEROIDS

446

minute

This

group.

other

subcei

gressively

luiar

may indicate

compartment

converted

4-5s

deciduai

containing

complex

that

i tes

which

--in

complex

H it

vitro

(Fig.

2).

FRACTION

TOP

is moving

progesterone do not

and pseudopregnant

progesterone-7ol-3

forms

the

and/or

to metabol

By incubating medium

that

is

in

establ

Interestingly,

NO.

being

some pro-

bind.

cornua

was also

into

tissue

ished the

culture

that

a

binding

Bottom

Sucrose gradient analysis Fiqure 2. in the cytosol following incubation rat cornua for 30 min. at 37OC with

of --in vitro progestin binding of decidual and pseudopre nant 3 H. 5x10-9 i progesterone-7Ck’-

activity

decidual

ment

was considerably

with

Wiest’s

experiments to

4-5s

cytosoi.

have

(27) further

macromolecules

higher earlier

progestin

shown when

in

that

incubated

cornua

retention

which data.

progesterone-7cI-‘H directly

with

is

agree-

Additional readily

the

in

uterine

binds

STEROIDS

Oct. 1971

In order terone

to examine

binding,

intestine,

cytosol

in

the

were

Figure uterine

2

4

In order macromolecule(s), gesterone-7c+3H

the

prepared

3 shows

TOP

of

of

cytosol,

’ 0

Fiqure 3. Sucrose progestin binding. tine, and diaphragm p roges terone-7c+3H.

question

fractions

and diaphragm

progesterone-7cu-3H. occurred

the

447

tissue decidual

and

that

specificity

incubated

target

directly

6 8 10 12 14 FRACTION NO.

the

information decidual

and various

uterine enzymes.

as

to

only

specificity.

16 18

Bottom

gradient analysis of the t issue specificity Cytosols from the decldua 1 cornu small were incubated for 30 min . at 37&C with

to obtain

with

binding

tissue

proges-

smal I

uterus,

progesterone

implying

of

the

cytosol As shown

nature was in

of

the

incubated Figure

of intes5x10-9

1

binding with

4 only

propronase,

18:4

STEROIDS

448

a proteolytic

enzyme,

suggesting

that

the

abolished binding

progesterone

macromolecules

binding are

at

in

least

the in

4-5s part

region, protein.

_

DNAaSe 1 RNAaSe .----*NEURAMINIDASE *.-.* PRONASE

9000r

I

4

0

TOP

0 12 16 20 FRACTION NO. Bottom

Figure 4. Sucrose gradient analysis of the effects of various enzymes on progestin binding in decidual cornua. Two cornua were homogenized in 0.01 i Tris-0.0015 4 EDTA, pH 7.4 containing 8 mM MgC12 and 1 .O ml al iquots of the cytosol were incubated at 37OC for30 min. with 200 pg DNAase, 200 pg RNAase, or 400 pg neuraminidase. 200 pg pronase,

Proqestin

Bindinq

terone-7a-3H-derived molecules to

rabbits

in

other

bearing

in

the

Rabbit

radioactivity species, one

Uterus: also

To ascertain interacts

progesterone-W

traumatized

and one

whether

proges-

uterine

macro-

with

‘H was given pseudopregnant

intravenously cornu

and

STEROIDS

Oct. 1971

the

animal

small

1 hour

autopsied

degree

of

binding

in

so0 A. IN VW0

later. the

,200

4-5s

449

Figure

5A shows

region

of

8. IN VITRO

the

that

cytosol

there

was a

from

the

6om,, C. CYTOSOL

:

I :

TRAUMATIZED ....‘I..’ PSfUDOPRfGNANT ---- FACES

0

5

10

IS 20 Bottom

TOP

0

5

TUBE

10 15 20

FRACTION

NUMGfR

Fiqure 5. Sucrose gradient analysis of progestin binding fn traumat i zed and pseudopregnant rabbi t cornua. (A) In vivo binding in the cytosol 1 hr. following i .v, injection of 1.0 mc of progesterone-7c+ (6) --In vitro binding in the cytosol following incubation of 3H. whole tissues for 30 min. at 370C with 5x10-9 1 progesterone-7cr-3H. (C) --In vitro binding following incubation of the cytosol for 30 min. at 370C with 5x10-9 E progesterone-7@3H. In (B) and (C) ultracentrifugatlon (297,000 x g) of the sucrose density gradients was for only 10 hrs.

traumatized cytosol

of

experiment cl rculation or

cornu; the

hours

have

time

following

little

pseudopregnant

might

dispensing

however,

of

with

better

the the

removal

no binding

cornu. been

injected incubation from

or

the

In

occurred

retrospect,

performed

of

uterine

rabbit.

howaver,

by shortening

progesterone-7a-%i

the

the this in

viva*

and by shortening

tissues Thus,

in

the

at low

37’C degree

for of

2

binding the

observed

injected

poor (3)

could

distribution

of

to one or

factors:

by endogenous

injected --in

more

(1)

progestins,

progesterone-7Cu-3H

vivo

and --in

vitro

(2)

to

(2 hour

dilution

the

of

relatively

uterus,

or

preincubation)

progesterone-7W3H.

it

was decided

--in

vitro

to circumvent

to

study

conditions.

(1)

500 mg central

and

fallopian

H and

(2)

the

Figures

region

as

occurred of

this

--in

vitro

the little

those the

in

There

traumatized but

the

did

the

tube

would

tend

appear

bound

regarding

Metabolism

of

found

the

this

3H

wi th

experiments

under

two ways: cornua

progesterone-7adirectly are

that

with

presented

sediment

No such the

assoc

was no difference

support

our

traumatized

cornua

in

was

too

in

in

rabbit

small

vivo to

in

the re was these

experi-

between

the

experiment

allow

nature

(unl i ke

that

cornu

same

binding

cornua

however,

the --in

fit in

contention

the

at ion

uterine-spec

and pseudopregnant

radioactivity

in

incubated

to be some difference,

any

(Fig.

definitive

point.

Proqesterone-7athat

were

uterus.

there

uterus

progesterone-j’cX-3H-derived

indicating

to

and pseudopregnant the

rat

that

in

incubated

these that

rabbit

performed

macromolecules

traumatized

ization

conclusion

was

in

The fact

rats)

decidual

with

fallopian

between

be seen

the

influences

and pseudopregnant

tissues of

complicating in

were

were

these

can

observed

binding.

ments.

It

binding

traumatized

results

associates

in

case

s of The

5B and 5C.

radioactivity

of

respectively,

cytosol

these

experiments

portions

tubes,

some of

progesterone These

proges terone-7CX-3H.

it

the

metabolism

In an attempt

5A),

be due

progesterone-70+3H

extensive

of

3

18:4

STEROIDS

450

radioactivity

3H

in

the

Decidual

associates

with

Rat

Uterus:

4-5s

Since

cytoplasmic

STEROIDS

Oct. 1971

macromolecules injection learn

in

of the

the

of

progesterone-7CY-3H

fraction

were from

whole

then

cytosol

broad

i n each rerun

are

in

region the

the

in of

in the

sucrose

fraction.

In

acetylated

the

Figure

of gave

while

the that

zone

for 6.

the

system,

zones system.

whole

insufficient

however,

to

cytosol

were

the

same mob11 1ty corresponding

chromatographed of

the

to

ligroine/propylene components together

fraction amount

and

and of

the

of

from

the

observed the

serum

3H radio4-5s

rechromatography

and serum

progesterone

yielded

corresponded

in

whole

in the

L/PG

cytosol

components

chromato-

respectively.

as 3a-OH-Sa-pregnan-20-one to

three

radiochromatograms

two zones

and 3a-OH-SC+pregnan-20-one,

the

system

eluted

originating

those

gradient

steroid.

1y separated

for

the

in mob11 1ty

the

The

y,

zones

the

in

cytosol

Unfortunate1

was

(T/PG)

which

two partial

(H/F)

closely

as progesterone having

this

0.74

cytosol

gradients,

were

glycol

separated

system;

H/F

extracts

cornua

density of

of

500 JJC of

the

sucrose

region

to

decidual

of

species

i.v.

interest

Chromatography

an Rf of

however,

gradients

corresponded

4-5s

and the

two chromatographic

system

The zone

with

system

hexane/formamide

graphed

the

bound

after

this,

and the

to

toluene/propylene

peak

H/F

subjected

and Methods.

hexane/formamide

illustrated

activity

the

was of

Al iquots

the

from

it

rats

later.

isolate

The partially

the

obtained

to

and serum

sys tern,

case.

in

were

Rechromatography

(L/PG)

into

5 minutes uteri

radioactive

1),

5 minutes

TO determine

i .v.

Materials in

progesterone. gl ycol

injected

fraction,

extracts

within

Fig.

(cf.

extracted

under

uterus

radioactivity.

in order

as described

one

was

decidual

were

steroid

this

collected

centrifugation Steroids

rat

progesterone-7CX-3H

nature

and blood

decidual

451

could

could not.

be

Thus,

452

STEROIDS

18:4

Chr~atography in the hexane/formamide system of 3HFigure 6, labeled progestins from the cytosol fraction of decidual cornua Cytosol and serum extracts had previously been and the serum. chromatographed in two other systems (cf. Materials and Methods). Decidual cornua and blood were taken 5 minutes after i.v. injection of 500 PC of progesterone-70+3H.

progesterone

and 3~-Ofl-5~-pregnan-ZO-one

as

labeled

the

major

progesterone tration

predominated

of

early

molecules

in

metabol

the

To confirm

incubated Aliquots

in

in

the

progesterone-iW3H,

f ts major

cornua

steroids

and

the

the

decidual

cytosol with cytosol

the

uterine

serum In

5 minutes

with

i fied

whereas

after

both 4-5s

--in

vivo

adm i nis-

i .v.

progesterone

cytoplasmic

from

observations,

decldual

progesterone-7a-3H fraction

ident

a nd macro-

uterus.

these

fraction

tentatively cytoplasm,

addition,

associated rat

and extend

directly of

i te

were

from

cornua for

each

of

the of

30 minutes the

two

decidual

rats at

were 37°C.

incubations

STEROIDS

Oct. 1971

were

then

pooled

4-5s

s terol

to

fractions

sucrose and

ds chromatographed

glycol The

subjected

system

results

those

the

obtained

in

whole

in

as an the

were

in

cytosol

the

with

decidual

4-55

direct not

cytoplasmic

incubation result

cytosol

in

9)

appear

the

to

principal tion

aid

in

(2)

by digi

tonin,

was

and

cytosol

in

cytosol

decidual in

by chromic

(4)

the

acid

recrystallized

acetylatable,

3,20-dione,

(3)

not

to constant

specific

(2)

precipitated activity

unmetabol

ized

sucrose

any

in

the

other

the

zone

oxidized

(Table

1).

gradients

decidual

progesterone

cornu extent. were

of

was

(1)

not

not

the

two

incuba-

specificity

by chromic and

acid (4)

results

acetyla-

precipitated

as separated

These

whole

following

(3)

by digitonin,

did

The presumptive

system

to constant

hand,

procedures

fraction

oxidation,

found

significant

cornua. H/F

and

cytosol

dens! ty

of

to

other

uterine i tes

to

associated

and characterization

the

with

the

methods,

identification

steroids

On the

fraction

similar

components

metabol of

3@OH-5a-pregnan-20-one

(1)

that

region

as separated

unchanged

The presumptive

evidence

the

with

progesterone

observed

zone

table,

system

isolated

progesterone-7a-‘H

progesterone

4-55

to chromatographic

steroids

of

the

8).

step.

progesterone

and both

progesterone

to metabolize

In addition employed

in

is,

the

1 i groi ne/propylene

were

labeled

(Fig.

progesterone-7CY-3H

or

Thus,

10). not

of

that

7)

(Fig.

the

and

chromatographic

principal

macromolecules

any detectable

(Fig.

(Fig. does

uterine

the

the

extracted

incubations

experiment,

3~-OH-5~-pregnan-2O-one

that

intermediate tissue

centrifugation; were

except

whole

the --in vivo

gradient

cytosols

as before,

was omItted

obtained

density

453

(Table

1).

in

the

H/F

to

5c+pregnan-

recrystallized provide

and 3C+OH-5Wpregnan-20-one

strong

l&4

STEROIDS

454

DISTANCEFROM ORIGIN(Cfi)

Figure 7. Chromatogr$phy in the toluene/propylene glycol and hexane/ formamide systems of H-labeled progestins extracted from the cytosol fraction of qdecidual cornua. The cytosol extract was chromatographed initially in the TfPG system and the partially separated zones were eluted individually as shown by the dashed 1 ines and rerun in the H/F Decidual cornua ware incubated at 37’C for 30 min. in Eagle’s sys tern. Heta medium containing 2.5x10-8 !j progesterone-1,2-3H (Amersham-Searle, specific activity = 41 C/mmole) l

PROGESTERONE I 3cc-OH,-Sa;P-20-t

”

0

+TL‘+-J-i

-r--

10

20

w 30

40

DISTANCE FROM ORIGIN (CM)

Fiqure 8. Chromatography in the T/PG and H/F system of 3H-labeled progestins extracted from the 4-5s region of 6-21% sucrose density gradients. The 4-5s extract was chromatographed as in Fig. 7. Aliquots of the cytosol fraction from the decidual cornua of Fig. 7 were subjected to sucrose density centrifugation to obtain the bound 3H-labeled progestins.

Oct. 1971

455

STEROI.DS

DISTANCEFROMORIGIN(CM)

in the T/PG and H/F systems of 3H-labeled Fiqure 9. Chromatography progestins extracted from the cytosol fraction of decidual cornua. The extracts ware chromatographed as in Fig. 7. The cytosol fraction was incubated directly with 2.5x10-8 L of progesterone-l, 2-3H for 30 min, at 37OC,

too-

A. T/PG

80.

0

10

20 30 DISTANCEFROMORIGIN(CM)

40

Fiqure 10. Chromatography in the T/PG and H/F systems of 3H-labeled progestins extracted from the 4-55 region of 6-21% sucrose density The 4-5s extract was chromatographed as in Fig. 7. Aligradients. quots of the cytosol fraction from Fig. 9 were subjected to sucrose density centrlfugation to obtain the bound 3H-labeled progestins.

18:4

STEROIDS

456

TABLE 1 RECRYSTALLIZATION Crystal1 ization Number

OF STEROIDS

Sol vent

TO CONSTANT SPECIFIC

WOH-5a-Pregnan20-one

Sol vent

Proges teronf

ACTIVITY

DPM/mg

DPM/mg

1346

864

Initial 1

Hexane

780

2

Ethanol / Pet. Ether

830

Methanol

3

Hexane

777

Dioxane/H20

Final Mother Liquor

1372

Ike tone/Hexanc

1439

/H20

1339

1305

842

The radioactive steroid was eluted mixed with S-10 mg of unlabeled carrier from the designated solvent.

are

the

major

(30

min.)

of

steroids the

present

decidual

in

uterus

from the steroid,

.the cytosol to

H/F chromatogram, and crystal 1 ized

ng short

follow

exposure

progesterone.

DISCUSS ION The

foregoing

results

progesterone-derived protein(s) rabbit

decidual

major

more

interesting

4-5s

cytosol

progestational

it

that

associates

(traumatized)

In addition,

converts

the

demonstrated

radioactivity)

the

uterus.

rapidly is

in

have

with

that

the

progesterone

to

3a-OH-5a-pregnan-20-one

metabolite

of

progesterone

early

was proteins, (or

the

finding

suggesting

anti-progestational)

that that

4-5s

cytopl

and pseudopregnant

was shown

in

decidual

this

and

effect

metabolite on the

asmic

rat

and

rat

uterus

that

tissue.

3a-OH-5a-pregnan-20-one this

(or

progesterone

Perhaps binds

may exert uterus.

this

to

some In

Oct. 1971

STEROIDS

relati‘on

to

(371,

this

Liao

reductase

(381, of

turn

(41)

work from

Wilson

and Tveter

rat

androgenical in

possibility,

ly

binds

recently

(391,

ventral

active to

prostate metabol

specific

progesterone-treated traumatization

rats and

113%.

In

rats

device

in one

similarly uterine

contralateral

cornu

one, in

converted

which

led might

have

stimulated

the

to

and

propose

that

a physiological

11% of

decidual from

estrone

to castrated,

growth

decidual

was not

5a-reductase

nuclear

in

the

inhibited. was

3C+OH-5a-pregnan-20of

progesterone These

progesterone

in mediating

response

activity

preparations.

uterine

by

an intrauterine

response

i te

following

activity

observed

metabol

slice

Armstrong

the

to

highly

which

that

enzyme

major

role

the

~CX-

(3-7).

of

inhibited to

the

tissue

the

to

SC+reductase

placement

completely

to be the

Baulieu

shown that

decldual

nuclear

by an extra-nuclear

homogenates

Armstrong

of

resulting

was shown

uterine

administration

activity

The 5c+pregnan-3,20-dione

of

testosterone

tissue

uterine

in which

has

this

treated,

Sa-reductase

(40)

in

markedly

cornu

laboratories

17~-OH-5~-androstan-3-one,

the

increased

and depressed

rapidly

i te,

that

the

converts

receptors

reported

457

the

both results

5c&reductase

biological

actions

of

progesterone, fn the respect

study

to progesterone

pregnant derived

rabbit

similar

It binds

macromolecules

to of

no experiments

metabolism

uterus.

radioactivity

cytoplasmic

those

present

that

observed

Rao and Wiest

(42)

in

the

was shown, both having

in

have

the

--in

that

and _in vitro

a sedimentation rat.

These

who reported

performed

traumatized

however,

vivo

been

results

a specific

with

and pseudoprogesteroneto uterine

coefficient are

in

(4-5s) accord

5s progesterone

with

STEROIDS

458

binding

protein

and McGui re in

the

in

binding

mature

is

siderable

available

other

protein

(CBG,

transcortin)

exists

within binding

the

via

are the

synthesized

shown by their

high

absence

fallopian

present, f icance.

in

the

however,

mediating

the

physiological

for

target

questions

can only

the

the

protein.

very

little

uterine

progestin-

specific

This

rat

and

cell

in small

or action

be resolved

con-

progesterone globulin

not

apparently

readily

accessible

the

progestin-binding

or is

delivered

clearly

uterus

intestine,

speculate

hormone

presented

protein

is

rabbit

localization

only

have

(21).

uterine

proteins,

organ

receptor”

data the

rabbit

corticosteroid-binding

as transport

serum

of

(20)

and as such,

tube,

as do certain

other

is

concentration

They may serve

togen

these

nature

that

corticoids

one can

a “proges

and Baul ieu

cell,

their

pseudopregnant

species.

a CBG-like

by the

ci rcul ation,

the

uterus

whether

the

Beyond

indicates

uterine

of

this

rat

adrenal

Regardless teins

the or

uterus.

Mi lgrom

which in

of

in

of

who observed

concerning

hand,

evidence

binding

cytosol

rabbit

macromolecules

On the

for

uterine

DeDel 1a (23)

and

sexually

information

the

18:4

they of

as

tissue

uterus

specific

to

their

may play

a more

progestins

At

functional

act

signi-

as a reservoir, active

as has

(1,2,3,4,10).

as

virtual

and diaphragm.

or

by further

the

and .by their

proteins

receptors

to

pro-

been

role

envisioned

Obviously

experimentation.

ACKNOWLEDGMENT We would like Lee whose talents

to dedicate and friendship

this paper to the late are deeply missed.

Mr.

in

Shiao-lung

these

Oct. 1971

459

STEROIDS

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