Testosterone metabolism by rat placenta

427

TESTOSTERONE

M E T A B O L I S M BY RAT P L A C E N T A S. Sybulski

Hellenic R e s e a r c h L a b o r a t o r y D e p a r t m e n t of O b s t e t r i c s and Gynaecology Royal V i c t o r i a H o s p i t a l Montreal, Canada

Received July 7, 1969 ABSTRACT T e s t o s t e r o n e - 4 - C 1-~4 was i n c u b a t e d with h o m o g e n a t e s of 15- and 21- day rat p l a c e n t a l tissue and the r e s u l t i n g r a d i o a c t i v e m e t a b o l i t e s more polar than t e s t o s t e r o n e were studied. The most a b u n d a n t l y o c c u r r i n g of these metabolites was shown to be identical to 5~-androstane-3~, 17~-diOlo The p r e s e n c e of exogenous N A D P H 2 in incubations of 21-day p l a c e n t a s a p p e a r e d to be n e c e s s a r y for the elabo r a t i o n of this and other u n i d e n t i f i e d metabolites. N e i t h e r estrone nor e s t r a d i o l were b i o s y n t h e s i z e d in d e t e c t a b l e amounts from testosterone. The r e s u l t s suggest that both 15- and 21- day rat p l a c e n t a l tissue h o m o g e n a t e s

have ~4-reductase activity. In contrast to the human, rat placental tissue appears to be relatively deficient in aromatizing enzyme activity and perhaps in NADPH 2 content.

A variety

dehydrogenase

activities

have

been detected by means of h i s t o c h e m i c a l

techniques

in rat

stages of gestation,

and it

placental

of s t e r o i d

tissue at different

has been s u g g e s t e d

that the rat placenta may be capable

of b i o s y n t h e s i z i n g

c e r t a i n steroids

of these enzyme activities, hydroxysteroid

dehydrogenase

as a result of some

i n c l u d i n g that of A 5 - ~ (1-3).

However,

-

rat placental

o,28

STEROI tissue

does not appear

steroids

(4)(5).

of p r o g e s t e r o n e quantities

Evidence

of estrogens,

was o b t a i n e d

(3) suggest

rats.

synthesis

but not

to m a i n t a i n p r e g n a n c y

(6) using parabiotic

of Dearie et al

adrenal-like

for some p l a c e n t a l

and possibly

sufficient

14:4

to elaborate

f o l l o w i n g ovariectomy, Segal

DS

to term

by M u n e m i t s u

Histochemical

placenta may be active

giant t r o p h o b l a s t s in this respect

13 to 15 days of gestation, ~5-3~-hydroxysteroid In the present placental

experiments

more polar than testosterone formed)

placenta,

its capacity

in vitro

at a p p r o x i m a t e l y

activity

indicates

incubated w i t h 4-C 14

enzyme activity

(which w o u l d

(7-11).

include any

to readily convert certain

androgens,

to estrogens

it is a tissue rich in a r o m a t i z i n g Recently

Smith and A x e l r o d

have reported that a hrei p r e p a r a t i o n is also able to convert

products

In the case of human

and androstenedione,

that

at this time°

both 21- and 15- day rat

were

was studied.

such as testosterone

steroids,

of the fetal

and the nature of the m e t a b o l i c

estrogens

studies

since they show a peak of

dehydrogenase

tissue homogenates

testosterone

and

that term rat placenta may

not be capable of s y n t h e s i z i n g p r o g e s t a t i o n a l but that the p e r i p h e r a l

in

(12)

of human placenta

dehydroepiandrosterone

to 5o(-

Oct. 1969

ST E R O I D S

androstane presence

products of

some

to ring

a minor A

reductase

MATERIALS Incubation

extent

AND

429 suggesting

activity

as

the well.

METHODS

procedure

Pregnant Wistar rats were killed by concussion on day 15 or 21 of gestation. The placental tissue was immediately removed and homogenized with Krebs-Ringer phosphate buffer (at pH 7.2 with the usual NaCI and KCI concentrations reversed) in a chilled Potter-Elvehjem homogenizer. The buffer was fortified with the following substances (mg%): glucose (200), Na pyruvate (88), nicotinamide (488), NAD (18) and ATP (25). The homogenate was centrifuged for ten minutes at 800 X g and the supernatant was then added to beakers containing 1 mg. of nonradioactive testosterone plus 1.3 million counts/min. of the 4-C 14 isotope (obtained from New England Nuclear Corp.) dissolved in 0.2 ml. of propylene glycol. The cofactor NADPH 2 (nicotinamide adenine dinucleotide phosphate, reduced form) was added to some of the samples. Incubation was carried out in a Dubnoff shaking water bath under 100% oxygen at 37°C. Extraction

and

isolation

of

steroids

At the end of two hours of incubation, homogenates were treated with five volumes of a methanol/methylal (1/4) solution to precipitate the proteins and extract the steroids. The mixture was mechanically shaken and allowed to stand overnight. The protein was then filtered off and the supernatant evaporated down to an aqueous residue from which the steroids were extracted twice with five volumes of chloroform. The extracts were applied separately on washed Whatman No.l paper strips and chromatographed in the hexane/propylene glycol system of Savard (13) to separate the substrate, testosterone, from metabolites elaborated during incubation. Concomitant chromatography of authentic estrone and estradiol revealed that these compounds would be in a position on papergrams between the area of application and that of testosterone.

430

STE

ROI

DS

14:4

Paper strips were dried at room t e m p e r a t u r e after c h r o m a t o g r a p h y and kept against N o - S c r e e n m e d i c a l X-ray film for at least two days. Dark areas of v a r y i n g intensity on the d e v e l o p e d film r e v e a l e d the exact p o s i t i o n of r a d i o a c t i v e material on the chromato~rams. Also, t e s t o s t e r o n e and any m e t a b o l i t e with t h e A ~ - 3 ketone grouping in ring A still intact could be detected on p a p e r g r a m s with the aid of a m e r c u r y hand lamp by virtue of their strong a b s o r p t i o n of u l t r a v i o l e t light at 240 m~o E s t i m a t i o n of r a d i o a c t i v i t y The radioactive zones were eluted from chromatograms with chloroform/methanol (3/1) o Eluates were evaporated to dryness under nitrogen and the residue re-dissolved in ethanol. Aliquots were pipetted in duplicate on aluminum planchets and air dried. The preparations were essentially infinitely thin and counted in a windowless proportional flow counter. The determined radioactivity in counts/mino related to the protein N content of the incubated genate preparation. The latter was estimated by modification of the biuret reaction.

was homoa

Controls for the experiments were prepared and treated through all procedures exactly as other samples, except that they were not incubated. All values for radioactive metabolites formed were corrected for any radioactivity present in the corresponding area of the control papergrams. Further

chromatography

F o l l o w i n g the s e p a r a t i o n of m e t a b o l i t e s on paper c h r o m a t o g r a m s in the Savard system and e s t i m a t i o n of their radioactive content they were s u b j e c t e d to further c h r o m a t o g r a p h i c analysis in some experiments. The thinlayer system (85% benzene, 15% methanol) of Lisboa and D i c z f a l u s y (14) was used and also instant thin-layer c h r o m a t o g r a p h y media (ITLC type, Gelman Instrument Co.) with the solvent system 95% benzene, 5% ethyl acetate. R a d i o a u t o g r a p h y was used to detect r a d i o a c t i v e m e t a b o l i t e s as d e s c r i b e d above.

Oct. 1969

$T ER O I D S The

results

rat

placental

are

shown

tissue in

without

added

activity

in

in

the

obtained with

Table

Io

Savard

fraction

controls.

experiments,

homogenates

definite

of

material

on

paper

content

ranged

from

106

incubated

strips

contributed as

to

compounds

radioactive

there

was

(362

and

three 838

975

at

in

the

six

of

seven

out NADPH

to

content six

in was

times

counts/min.)

2 showed

more its

slowly radioactive

counts/min./mg,

least

X 2 and

to

present

chromatograms; to

radio-

testosterone

with

two

of

They

measured

than

the

were

X 2

paper

six

designated and

seven

separately;

radioactivity

in

(99

and

in 137

X 1

counts/mino,

respectively). In

an

additional tissue

effort

to

experiment from

six

rats

identify was (at

compounds

performed day

21

X 1 and in

of

N

compounds

experiments

more

protein

distinct

radioactivity.

X 1 and

their

in

incubated in

Radioautography

that this

amount

testosterone

homogenate.

indicated

than

incubated

conversion

samples

increase

polar

2l-day

testosterone

of

no

However,

moving

of

was

the

of

labelled

cases

more

over

non-incubated

a

six

there

system

incubations

4-C 14

In

NADPH2, the

with

431

which

pregnancy)

X2,

an

placental was

pooled

b,32

STE

R Ol DS

14:4

and h o m o g e n i z e d with an equal volume of K r e b s - R i n g e r buffer,

incubated with 4 - c l 4 l a b e l l e d

plus NADPH2,

extracted,

testosterone

and c h r o m a t o g r a p h e d

in the

usual way. Radioautography

of paper c h r o m a t o g r a m s

the presence of four distinct material

more slow m o v i n g

Savard system,

revealed

bands of r a d i o a c t i v e

than t e s t o s t e r o n e

as well as r a d i o a c t i v e

at the s t a r t i n g

line

on the paper).

These four bands

in the

material

(area of a p p l i c a t i o n

remaining

of the extract

included the compounds

X 1 and X 2 and two other more slow m o v i n g m e t a b o l i t e s designated graphy on on

ITLC

as X 3 and X 4 (Table 2)° paper,

compounds

graphic

mobilities

estradiol

in

metabolites when

silica

chromatography

unknown

or

on

media was

at

X 2 and

The abundantly testosterone

to

or

one

X 3 each on

remain

latter

estrone

least

thin-layer

revealed

differed

chromatographed

appeared

gel

Successive

that

of

these

media

of

while

of

since

two

these chromato-

standard

systems into

and

none

estradiol,

separated

ITLC

plates that

from

chromato-

estrone

used.

The

compounds

compound

X 1

homogeneous.

compound

occurring

of

in

paper

the

(XI) the

was

consistently

metabolites

chromatographic

more

the polar

system

most than used.

Oct. 1969 An

ST ER O I D S

ethanolic

material

solution

X2)

failed

absorption

at

therefore,

considered

testosterone aliquot

to

either

with

of

of

compound

show

240

compound

or

X 1 was,

3~,

17~-diol,

and

specific with the

activity.

X 1 was

tissue

testosterone too,

produced

properties stane-3o~ placenta. quantitatively C 14

labelled

substrate

i0

of

metabolites as

the

17~-diol) As

to

with

with

compounds and the

estrone

or

testosterone.

X1

latter

tested

estradiol

of

for

possible constant

obtained

indicates

that

compound.

2

(Table

the

same

of

15-day

labelled

4).

These,

chromatographic as

by

near

compound

detectable were

each

to

3)

(identified

No

of

(all

4-C 14

tissue,

was,

5d-androstane-

this

X 2 elaborated

predominant.

to

data

with

NADPH

it

An

added

preparations

incubated mg

and

saturated.

and

the

a derivative

(Table

homogenate

plus

2)

recrystallization

identical

were

of

light

17~-diol

17~-diol

different

placental

be

crystallization

5~-androstane-3~

Two

might

Co.) by

The

UV

17~-diol,

Chemical

homogeneity

also

(Table

5~-androstane-3~

Sigma

metabolite

my

therefore,

5~-androstane-3K,

radiochemical

(and of

A completely

compounds

from

280 it

the

obtained

X1

a maximum

that ring

433

5~-androterm

X 1 was

amounts

derived

rat

from

of the

14:4

STEROIDS

434

DISCUSSION The p r e s e n t they

suggest

experiments

that

both

have~4-reductase 3~

17~-diol

more polar

metabolites with

tissue

of

the

of

this

of

endogenous

21-day

to

during

oxidation

placentas

of

from testosterone

observe

that

elaborated

in

that

this

endogenous

metabolism

in

NADPH2.

the

be d u e t o

excessive

catabolic

enzymes or

o f NADPH2 d u e t o

this

NADPH2 was a d d e d

suggests in

the

during

The f a c t

were

placentas

NADPH2 d u e t o

that

occurring

when e x o g e n o u s

also

in

c o m p o u n d 5o(- a n d r o s t a n e -

deficient

coenzyme could

excessive

absence

destruction

competing

to

systems

incubation.

The f i n d i n g tissue

in the

others

(15)(16)

being

only

failure

rat

homogenates.

may be r e l a t i v e l y

However,

interest

abundantly

metabolites

quantity

incubation

the

formed

tissue

compound and other

to

most

of

and 21-day

activity~

was t h e

incubations

detectable

15-

~re

present

ofA4-reductase

present as

to

study the

contrasts

possibility

i n human p l a c e n t a ;

products

were

elaborated

mid-term

placentas

with

activity

during several

in

with of

the

such

apparently in situ

rat

reports

of

activity

no ~ 4 - r e d u c e d

perfusions

androgens.

placental

of

However,

Smith

Oct. 1969 and

Axelrod

(12)

5ok-androstane of

labelled

recently

brei

was

The

of

of

near

(at

by

the

present

techniques

in

the

rat

is

probably

biosynthetic

This

tend

would suggest

thesizing

rat

to

of

of

not

of

such

tissue either

findings

observations

in

of

21-day

three

with

tritium-

evidence

of

5~-

rat

et

to

when

2 was

added

the

human

that

of

as

(3)

it

present

respect

is

in

the

placentas

Even

though that

may

experiments

(2)(3) of

syn-

giant be

active

homogenate

did

not

behave

to

the

possible from

differently biosyn-

testosterone.

with the

human.

the

the

placentas

by

this

studies incapable

contrast

placenta

via

is

estradiol

tissue

the

estrogen

suggest

rat

placentas

rat

detectable

suggests

placenta

al

or

NADPH

histochemical

15-day

estrone

with

tissue

steroids.

with

with

placental

a source

with

term

approximately

preparations

of

following

quantities

pathway

Deane

respect,

in

used)

agree

that

even not

progestational

observations

These

term

least

particular

thesis

placentas No

4-C I-~4 testosterone

incubations

from

amounts

premature

to

this

in

small

formation

observed.

failure

aromatize

cells

the

dehydroepiandrosteroneo

reduction

which

observed

metabolites

incubation

in

435

STEROIDS

previous

present

author;

436

STE homogenate

preparations

biosynthesized incubated

of

estrone

under

experiments

the

from

similar

either

R OI DS

with

14:4

latter

tissue

testosterone

readily

in vitro

conditions

as

in the

or without

exogenous

when

present NADPH2o

ACKNOWLEDGEMENT This work was supported by Foundation, Inco The author thanks Mr° AoHo assistance°

the

John

Burney

A. for

Hartford technical

R E F E R E N C E S

Botte, Vo, Materazzi, Jo ENDOCRINo 34, 179

i°

.

.

.

.

°

Ferguson, 38, 291

M.Mo and (1967).

Deane, HOW., and Leipsner,

Christie,

Chieffi,

G.Ao,

Rubin, BoL., Briks, Go, ENDOCRINOLOGY

Greep, RoOo, Trans. 3rd Conf. Foundation. 1956o po 229. Sybulski, PHYSIOL.

So 39,

and 203

Venning (1961).

Munemitsu, So and Segal, MORPHOL. EXPTL° 48B, 173

7.

Ryan,

8°

Baggett, Bo, ENDOCRINOLOGY

o

G. and (1966)o

KoJ.,

Jo BIOL.

CHEMo

Jo ENDOCRIN.

E.Co, Lobel, BoLo 70, 407 (1962).

The

E.Ho,

Josiah

CAN~

SoJo, ARCH. (1959). 234,

268

Engel, LoLo, Balderas, 64, 600 (1959)o

Gual, C., Morato, To, Hayano, Dorfman, RoI., ENDOCRINOLOGY

Go,

Macy,

Jr°

J. BIOCHEMo

ANAT.

MICROSCOPo

(1959). L and

Lanman

M., Gut, M. and 71, 920 (1962) o

A.,

Oct. 1969

STE R O I DS

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k

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0 0

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> .r'l

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0

mll~

0

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0

0 0

O • "~ "1~

0

0

0

0

0

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0

% >

O r~

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b~

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0

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0

0 O 0

0

0 LO

0 ~D

0,1 b~

¢0 0

Oq

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0 ;~1

0 ~-I

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r-.-I O

0

~ O ~r..)

r~ 4-~

0

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o

0

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0,1

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O ~ 4.~ m

e~:

m

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•

O

0 r~ +

N

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q-38

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DS

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0 .r-I 4~

0

0

CXl

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0

0

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@

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0

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0

+

+

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¢o qo o

o

--

o

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0 ~

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0

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b~ ~

0

~: 0 0 o

~ 0

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0

"t~

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o

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b-

o

0

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Oct. 1969

STE

R O I D S

TABLE RECRYSTALLIZATION RAT PLACENTAL

Crystallization

439

3

OF METABOLITE X 1 (DERIVED FROM INCUBATION OF TISSUE WITH 4-C 14 LABELLED TESTOSTERONE) WITH 5~ANDROSTANE - 3~, 17~-DIOL*

no

Specific (dpm/mg) mother liquor

activity crystals

1

2330

2300

2

2310

2250

3

2255

2140

Following successive chromatography on paper, silica gel thin-layer plates, and ITLC media, an aliquot of compound X 1 (dissolved in ethanol) was added to 80 mg of 5~-androstane -3~, 17~-diol - the specific activity of this original solution was 2305 dpm/mg. Solvents used for crystallizations were mixtures of ethanol, methanol, and chloroform°

440

ST E R O I DS

..~ 0

xfl

0

~

o o ~

o o (53

0

&

&

O,1 r/l

r..)

Z

I

O,l

o .,-I

o,1 "~

~ c,l "0 ~D

~ m M . ~ '

c~

o o 0b

o o u~

0 h0

0 0

o ,--4

0 I,-,-t

x~ 0

Z

0

~

m o

E~

~oo

~D

°;

o,I

o,I

&

&

LO

0,I

c~ o Z

~ Z

m ~ ~

N

"il ~)

o

co co

o~ o,1

Cxl o,1

o~

cq

h0 0 0

r-~

o,1

14:4