Endocrinol.

Japon.1975,22(4),361∼366

NOTE Steroid

Hormone

Formation

in Bovine

TAKAHIDE

Ovarian

Follicles

MORI

Department of Obstetrics and Gynecology, Kyoto University School of Medicine, Sakyo-ku, Kyoto 606

Synopsis

ment

In

an

of

the

chorionic

from

acetate

the

gesterone,

in

the

dissected

ovaries absence

on

incubation

by

30%.

luteinization.

formed

from

atretic

follicles.

radioactive

The

acetate was

not

major

was addition

into

the

in

by

of

vitro

the

Corpora lutea have been classified into two types according to their content or types of steroids formed from radioactive precursor on incubation in an in vitro system (Savard et al., 1965). One type is bovine corpus luteum which produces only progestins (Mason et al., 1962; Savard and Cassy, 1964; Savard and Telegdy, 1965). The other type, e.g., human corpus luteum, produces an array of steroids including progestins and estrogens (Hammerstein et al., 1964). A complete profile of steroids formed by the human follicle has been determined following in vitro incubation with radioactive acetate (Ryan and Smith, 1961; Mori and Savard, 1975). However, sufficient infor-

by

steroids

under

mations

are

in vitro

May

8, 1975.

among

the

steroids

profile

of

bovine

incorporation the

exprimental

lacking

The

on

terone, one,

steroid

following

pregnenolone,

of range

of

conditions.

at

the

present

time

for

trivial

biosynthesis. names

are

used

the

pregn-4-ene-3,

20-dione;

17-hydroxy20-dione;

androstenedione,

androst-4-ene-3,

epiandrosterone,

3ƒÀ-hydroxyandrost-5-en-17-one;

17-dione;

3,

5

(10)-triene-3,

(10)-trien-17-one 17,ƒÀ-diol.

dehydrotes-

17 ƒÀ-hydroxyandrost-4-en-3-one 5

:

17-hydroxypregnenol-

17ƒ¿-hydroxypregn-4-ene-3,

3,

text

proges-

3ƒÀ, 17ƒ¿-dihydroxypregn-5-en-20-one;

progesterone,

estra-1,

in

3ƒÀ-hydroxypregn-5-en-20-one;

hydroxyestra-1,

for publication

undergoing

proper estimation of the follicle as a functional compartment participating in steroid biosynthesis of the bovine ovary. The present study was attempted to assess histophysiological significance of the follicle in steroid hormone formation of the bovine ovary and to investigate the effect of hCG

tosterone,

Received

pro-

proliferation

although

the

of Histology

cells

overall

50%,

dehydro-

neither

steroidogenic

increased

from

of

theca

radioactivity

the

formed

formed.

showed

and

decreased

amounts

were

dominant

represent

estrone

amounts small

which

approximately

individual

lesser

human

follicular

testosterone of

; hCG

addition,

cells,

to

radioactive

estradiol-17ƒÀ

distribution

hCG

steroids in

and

of of

products

with

compart-

effect

amounts

radioactive

characterized

considered of

the

uniform

of

with

testosterone

granulosa

follicular

the

testosterone-4-14C

In

was

the

minces

Significant

with The

of

pattern

acetate-14C The

incubated

hCG.

estrone

amount

and

steroidogenesis,

were of

and

specimens

small

of

formation

follicular

17-hydroxyprogesterone.

follicle with

on

and

17-hydroxypregnenolone,

implication

hormone

vitro

androstenedione and

luteinization

increase

formed

were

epiandrosterone

steroid

bovine

presence

approximately

acetate-1-14C

histophysiological in

(hCG)

were

conversion

nor

assess

ovary

non-gravid in

estradiol-1713

of

to

bovine

gonadotropin

tissues or

attempt

; estrone, ;

estradiol-17ƒÀ,

3-

362

Endocrinol. JaponAugust 1975

MORI Incubations scribed

Materials

and

Methods

were

(Mason

contained

in

(pH

2ƒÊCi

7.4),

sodium

Chemicals and gonadotropin All solvents were reagent grade and redistilled before use. Radioactive materials were purchased from New England Nuclear Corporation, Boston, Mass. Sodium acetate-1-14C (61.8mCi/mmole) was used without further purification. The purity of testosterone-4-14C (58.8 mCi/mmole) was checked by paper chromatography. Tritiated steroids of high specific activity, which were used as internal standards for the measurement of analytical losses, were purified by thin layer chromatography within a month before use. Human chorionic gonadotropin (hCG) was a gift of Ayerst Laboratories (Ayerst-APL), New York, N. Y. Acetylation and saponification Acetylation was performed by incubating the dried samples in 0.5ml of pyridine and 0.5ml of acetic anhydride overnight at room temperature. Saponification of testosterone acetate was carried out by treating the dried samples with 20ml of 5% sodium hydroxide in 50% ethanol for 24 hours at room temperature in the dark.

for

Table

1.

Sources

4ml

an

3

of

concentration tion

a

media

most

of

since

the

effective

human

in

corpus

oxygen

100

5%

of per

concentration

at

incubator

and

IU

carbon

hCG.

ml

The

of

was

incuba-

found

to

steroidogenesis

slices

(Rice

of

incubated

absence

stimulating

luteum

100ƒÊCi

metabolic

and was

buffer

or was

95%

hCG

desystem

bicarbonate

Dubnoff

presence of

previously

incubation

testosterone-4-14C in

the

as

The

The flask

hours

in

out

1962).

Krebs-Ringer

of

atmosphere

dioxide,

et

be

by

al.,

the

1964).

The

reaction wasterminated byfreezing quickly thecontents until

of the vessels. commencement

Extraction

and

The tion

5)

in

et

were

a

the

glass

and

To a

tritium

steroids

as

an

of each

steroid

mixture

The

was

and residue

between

of

lipid

phenolic

and

distribution ene

(Bagget

et

al.,

chromatographed 1

paper

for

system

and

was

The

17 the

glycol

The

hours

in

resulting

in

activity

one

and

(containing zone

steroid

III

72

and

tolu-

fraction

on

was

Whatman

No. glycol

containing hours

re-

countercurrent

phenolic

in

estrone

ligroin/propy-

chromatographed glycol

located carrier

by

and

scanning.

testosterone,

carrier

A

gesterone)

were Zone

and substrates

of

polar

a

the

carrier

pregnenolone

run-off

(countaining

located I

and was

used.

17

radiozone

central

zone

dehydroepiandrosterone),

(containing and

for zones

I

17-hydroxyprogester-

17-hydroxypregnenolone),

stenedione)

separation.

by

to separated

touene/propylene

was

ligroin/propylene were

(containing

then

hydroxide

run-off for

partitioned

system.

neutral

hours

was

estradiol-17ƒÀ

rechromatographed

lene

of materials

1956).

to water.

methanol

fraction sodium

cor-

added.

and

and

1 N

for

were

aqueous

neutral

of

loss

evaporated

benzene

components,

between

for

ether,

of

0.1ƒÊCi

indicator

fraction

90%

and

400ƒÊg

and

,determined

benzene

and

media

sample

between

the

four No.2

homogenized

carrier

with

partitioned

non-polar

into

be

extracted

ligroin

move

to

of

The were

each

as

The

contents

extracted.

steroid

dilu-

laboratory

(Incubation

samples

homogenizer.

carrier

this

The

non-radioactive

rection

in

frozen

steroidsby

acetate-14C

incubation

labelled

radioactive

analyzed

1964).

with

combined

of

were stored

used

al.,

incubated

tissues

were

generally

(Hammerstein to

of

formed

technique

vessels

The samples of analysis.

separation

steroids

dryness

Tissues and incubation (Table 1) Fresh ovaries of non-gravid cows obtained from a local slaughter house were immediately chilled in cold physiologic saline and transported to the laboratory. Intact follicles of 5 to 30mm in diameter were dissected from the ovary in the cold ; five to thirteen ovaries were used at a time. The combined follicles were minced and divided in to two roughly equal portion. The tissues were weighed and placed in an appropriate incubation vessel as described below. One part was used for control incubation, and the other part was used for incubation with addition of hCG in vitro. Small pieces of tissue were taken randomly for histology before mincing. Under the microscope, follocular tissue showed predominantly cells of the theca interna and externa together with a small amount of granulosa and stromal cells. The theca cells were undergoing epitheloid change but were not completely luteinized. The granulosa cells showed degenerative change with neither mitotic figures nor luteinization.

al.,

acetate-1-14C.

37•Ž in

carried

et

collected

rechromatographed

II

a mobile and carrier for

androprofurther for

6

Vol.22,

No.4

STEROIDOGENESIS

IN BOVINE

steroid in the above procedures, as well as by radiochemical purity of each isolated steroid. Radiochemical purity was determined by repeated crystallization to constant specific activity (Savard et al., 1960). Radiochemical purity of radioactive testosterone was further established by demonstrating constancy of the specific activity before and after saponification (Rice et al., 1964). Total radioactivity incorporated into each steroid was corrected for recovery and expressed as dpm per g of tissue.

hours in toluene/propylene glycol to separate the area containing 17-hydroxypregnenolone from the area containing testosterone and 17-hydroxyprogesterone. The former steroid was eluted and rechromatographed for 16 hours in toluene/propylene glycol, while the latter two steroids were treated for acetylation of the hydroxy steroids. Zone II, III and the run-off zone were separately eluted and subjected to acetylation procedure. The resulting steroid acetates and free steroids from all four zones were separated on a thin layer plate of silica gel using benzene/ethyl acetate (3: 1) system followed by scanning for radioactivity. Detection

and

measurement

Δ4-3-Ketosteroids with

a

were

shortwave

ultraviolet

spectrophotometrically absorption ards

on

out et

by

at

240mƒÊ.

paper

strip

1952)

phomolybdic 1952) termined

thin

layer

Turnbull and

reagent

at

281 by

reagent

mƒÊ.

steroids

and

quantified

their

ultraviolet

of

steroid

plate

was

blue a

and

and

Eik-Nes,

tissue

a

Conversion

a:

Bovine bicarbonate

b:

Combined

follicle

follicle

accumulation

pH

of

acid-ethyl

in

vitro

incubated for

of

by

precursor.

experiment of

the

aromatize

using

As

was minced androgen

testosterone-14C

shown

was and

amouts

testosterone-4-14C

were 7.4

radio-

in

Table

as

converted

to

estradiol-17ƒÀ

by

control

2, radioactive

in

and

test-

significant

hCG-added

tissues.

1959).

minces

minces buffer

the first capability to

estrogen

estrone

3

hours

radioactivity

the

with at

in

and

and

control

and

0.37%

for

and

only

Stitch

Graafian

follicles

active

testosterone

to

μμmoles

the

amount

per

g

hCG-added data

indicated

under

radio-

and 197

of that

converted

from

tissue

of

,upmoles

in

estrone

be

yield 611

available

(1968)

bovine

in

to

hCG-added

The were

430ƒÊƒÊmoles The

Oakey

17ƒÀ

calculated

estradiol-17ƒÀ

incubations.

estradiolup

identical

to

871

experi-

mental conditions. Hence, it was able to judge that the follicular tissue minces were enough active in aromatizing androgen into estrogen.

2.0ƒÊ

into

control

were

respectively.

estrone

radioactive

effect

37•Ž

for

rates

incubations

into

and

conversion

0.49%

Quantitative measurement of the radioactive steroids formed Identification of radioactive steroids was made by coincidence of radioactivity with the authentic carrier 2.

of the

osterone-14C

dewere

Scanning and measurement of radioactivity Areas of radioactivity on paper chromatograms were located by scanning in a Vanguard Model 800 autoscanner. Scanning of radioactivity on thin layer chromatograms was carried out in a Varian Aerograph Thin Layer Chromatography Radio Scanner, Type LB 2007H. Radioactive zones were eluted and duplicate aliquots of each sample were counted in Packard Tri-carb liquid scinitillation counter, Model 3375, with an efficiency of 44.5% for 14C and 43.5% for 3H. Radioactivities were recorded as disintegrations per minute (dpm).

bovine

to

estradiol-17ƒÀ

preparation

into

Kirk,

were

sulfuric

purpose

examine

The

Table

testosterone-14C and

phos-

3ƒÀ-Hydroxy-J5-steroids

(Oertel

The

carried

of

Estrogens

using

of estrone

to

(Barton

spray

(Kritchensky

colorimetry

Conversion active

stand-

reagent

by

3ƒÀ-hydroxy-ƒ¢5-steroids.

determined alcohol

measuring

estrogens

acid

for

or

Results

chromatograms

Visualization

with for

on lamp

by

spraying

al.,

of carrier

located

363

FOLLICLES

Ci under

estrone

of

of

human

estrone

and

chorionic

testosterone-4-14C 95% and

oxygen estradiol-17ƒÀ.

estradiol-l7ƒÀ

in and

by

gonadotropin.a

5%

4.0ml carbon

of

Krebs-Ringer

dioxide.

364

Endocrinol. Japon. August 1975

MORI

Distribution of radioactivity among steroids formed from acetate-14C A complete profile of steroidal products from radioactive acetate was obtained in the second experiment. The results were shown in Table 3 and illustrated in Fig. 1. The major steroidal products formed from acetate-1-14C were androstenedione and

testosterone,

progesterone. 14C into lone,

estrone

major

and

added

effect

of

Bovine of

ence

chorionic minces

acetate-1-14C

bonate 95%

human

follicle

pH

oxygen and

and

5%

absence

gonadotropin.

Four

Table

3.

of

for

3

100

Bovine

at

dioxide, IU

of

follicle

minces

bicarbonate

buffer,

incubations

were

pH collected.

bicar-

in

steroids effect

were 7.4,

3

pres-

collected.

hours

with at

and out

into

total

same

progesterone

rone

in

84.3%

in

hCG-

radioactivity

steroids.

Combined

was

as

combined and

control

to

17-hydroxypregnenolone

dehydroepiandrosterone the

two

androstene-

86.6%

of

the

into

into

approxi-

incorporation

17-hydroxyprogeste

incubation.

Effect of hCG in vitro The addition of hCG to the incubation media in vitro increased approximately by 50% the overall incorporation of acetate-14C into the steroids (Table 3). However, response of the individual steroids to the addition of hCG was not uniform. Incorporation of radioactivity into any of the androgenic and estrogenic steroids increased significantly, while increase of the accumulated radioactivity in 17-hydroxyprogesterone or progesterone did not occur in the presence of hCG in vitro. Combined incorporation of radioactive

100ƒÊCi 37•Ž

i.e.

less. the

amounted

incubation

actate-

was into

steroids,

formed from acetate-14C by follicle of human chorionic gonadotropin.a

incubated for

under

the

chorionic

were

Radioactive

vitro.

100ƒÊCi

37•Ž

human

incubations

the

a

with

Krebs-Ringer hours

carbon

of

in

incubated

4.0ml 7.4

acetate-1and the

gonadotropin

were

in

buffer

from follicles

estradiol-17ƒÀ

incubation

mately

formed bovine

of

17-hydroxypregneno

testosterone

control

and

steroids tissues of

,

were

17-hydroxy

incorporation

androgenic

dione

products

imcorporation

incorporation

1. Radioactive 14C by minced

minor and

and

incorporated

Fig.

the

The progesterone

Combined

in ACETATE-1-14C INCORPORATION INTO STEROIDS IN BOVINE OVARIAN TISSUE EFFECT OF hCG-IN VITRO

and

dehydroepiandrosterone

under

of

acetate-1-14C

95%

oxygen

minces

in and

5%

in vitro and

4.0ml carbon

of

Krebs-Ringer dioxide.

Four

Vol.22,

No.4

acetate

STEROIDOGENESIS

into

17-hydroxypregnenolone

dehydroepiandrosterone following

of

incorporation

is

that

the

was

following

reduced the

com-

clear

and

unchanged.

into

of and

approximately of

by

hCG

in

the

firstexperiment(Table 2).

365

FOLLICLES

steroids

covering

than

androgens.

different

formation the

estrone

addition

ple other

incorporation

testosterone

BOVINE

66%

progesterone

remained

estradiol-17ƒÀ 30%

whereas

into

interesting

radioactive

by

hCG,

17-hydroxyprogesterone It

and

increased

addition

bined

IN

been

steroids

is or in

biological

between

certain

not

the

the

the

at

the

may

other

metabolities

of

androgenic

follicle

significance

intermediary

hormone out

not

weather

produced

some

study,

steroid

pointed

It

time

estrogens

present

compartments

ovray.

present

the

in

anatomical

bovine

and

By

aspect has

two

progestins

for

have

than

the

estrogen

syn-

thesis. The

Discussion to The

steroidal

acetate-14C that

products

in

the

the

bovine

present

androgenic

than

steroids.

estrogenic in

the

the

the

the

upon

major

the

steroids

mature

while

and

formed

follicle

and

major

identical

and

Savard,

changes for

the

bovine

to

obtained

in

reflect

that

bovine

ovary,

that

the

follicles

of

the

the

steroidogenic

the

study

could

to

sampling

of

an

follicles,

profile

in to

be

the

and

bovine by

an

corpus in

1965).

luteum

vitro It

products,

study

elaborates

has

predominantly,

(Savard

and

out

to

of

and

differing

Telegdy,

among to

the

reflect

proportions

physiology formation

the

increase

but

of

of

without

into

proge-

of

of

of these

remains

tissue

steroids

aspect

period

The

the

individual

the

multi-

FSH

to

significant

incorporation

varying

over

androgen

ovine radioactive

17-hydroxypregnenolone

response

and

elaborates

of of

17-hydroxyprogesterone.

the

produce it

sterone

using, bovine

responded

with into

increase

steroidal

bovine

yield

been

(1968)

isolated

presence

tissues

hCG

20ƒÀ-hydroxy-4-

contrast,

although

the

established

simplest and

In turned

been

the

progesterone

pregnen-3-one. follicle

has

of

of

has

dehydroepiandrosterone,

any

action

testosterone-14C.

incorporation

histological

significance

of

leading

the

Stitch

the

the

substrate,

pathway

of

the

follicular

addition

excluded

and

reduced from

The

of as

by

system

in may

similar finding

Oakey

where

vitro

estrogen

the

specimens. Histophysiological

by

incubation

atretic

the

synthesis A

increase

dilution added of

be

reactions

testosterone

subsequent

vitro.

could

An

testosterone

reported

rather

hCG

of

stimulation

in

estradiol-17ƒÀ

sequential

stimulation.

testosterone

hCG

presence

not

with

following

of

pool

radioactive

(Mori

of

hCG

radioactive

and of

result

endogenous

under

seems

follicle

concomitant

follicles

the

account

human

atretic

although

the

may

by

the

atretic

which

present

of

mature

observed

pattern,

Therefore,

to

follicle

The

caused

a

result.

observed

of

estrone

hCG

the

The

addition

as

of

with

incorporation

the

interpreted

occur

conditions

steroidogenic

resembles

1961),

are

atretic

1975).

follicle.

due

in

the

in

Smith,

dehydroepiandro-

experimental

of

the

acetate-14C

and

17-hydroxyprogesterone

products

the

are

This

effect

compatible

into

following

structure

from

(Ryan

in

the

radioactive

examined.

experiment.

reduction

with

of

stimulatory

be

testosterone

exist,

estradiol-17ƒÀ

44-androstenedione,

sterone

of

histological

Estrone

of

to

to

the first

responded

vitro

steroids

the

seems

of

diffe-

appears

the

follicle.

and

rather

in

incoporation

the

indicates

tissues

hCG

of

into

fact

capabilities

follicle

depending of

steroids Qualitative

of

increase

acetate

elaborate

follicular

addition

overall

indicates

tissues

steroidogenic

human

from

study

follicular

predominantly

rence

formed

minced

the

of

the

two

cells be

would

relative

follicular

to

to

in

types

hCG seem.

activities of

cells

atresia.

Cellular

steroid

hormone

examined.

366

Endocrinol. Japon. August 1975

MORI

Acknowledgements This work was done at the Endocrine Laboratory, University of Miami, Miami, Florida, in collaboration with Dr. Kenneth Savard. Dr. J. M. Marsh of the laboratory is gratefully acknowledged for his suggestive discussion. The cooperation of Mr. J. Cidre in collecting bovine ovaries is also acknowledged. The author is grateful to Mrs. Blanca Fukunaga for her correction of the manuscript.

References

Baggett, B., L. L. Engel, K. Savard and R. I. Dorfman (1956). J. Biol. Chem. 221, 931. Barton, G. M., R. S. Evans and J. A. F. Gardner (1952). Nature 170, 249. Hammerstein, J., B. F. Rice and K. Savard (1964). J. Clin. Endocrinol. Metab. 24 594. Kritchensky, D. and M. R. Krik (1952). Arch. Biochem. Biophys. 35, 346. Mason, N. R., J. M. Marsh and K. Savard (1962). J. Biol. Chem. 237, 1801.

Mori, T. and K. Savard.(1975) unpablished data. Oakey, R. E. and S. R. Stitch (1968). Acta Endocrinol.(kbh). 58, 407. Oertel, G. W. and K. B. Eik-Nes (1959). Anal. Chem. 31, 98. Rice, B. F., J. Hammerstein and K. Savard (1964). J. Clin. Endocrinol. Metab. 24, 606. Rice, B. F. and K. Savard (1966). Ibid. 26, 593. Ryan, K. J. and O. W. Smith (1961). J. Biol. Chem. 236, 2207. Savard, K. and P. J. Casey (1964). Endocrinology

74,599.

Savard, K., R. I. Dorfman, B. Bagget, L. L. Fielding, K. L. Engel, H. T. McPherson, L. M. Lister, D. S. Johnson, E. C. Hamblen and F. L. Engel (1960). J. Clin. Invest. 39, 534. Savard, K. and G. Telegdy (1965). Steroids 5: Suppl. II, 205. Smith, O. W. and K. J. Ryan (1961). Endocrinology

69, 869.