Steroid secretion by ACTH-stimulated human fetal adrenal tissue during the first week in organ culture

2678

563

STEROID

SECRETION TISSUE

BY ACTH-STIMULATED

DURING

Bruce

THE

R. Carrl,

John C. Porter, The Cecil

FIRST

C. Richard

WEEK Parker,

Paul C. MacDonald,

H. and Ida Green

and the Departments

Jr.,

and Gynecology Southwestern Texas

CULTURE

and Evan R. Simpson

of Obstetrics

Dallas,

ADRENAL

Leon Milewich,

for Reproductive

of Texas

FETAL

IN ORGAN

Center

The University

Received

HUMAN

Biology

Sciences

and Biochemistry

Medical

School

75235

6-20-80 ABSTRACT

Fetal adrenal tissue has been reported to lose its in vivo secretory pattern by virtue of a loss of fetal zone cells after the-fiTweek in culture. Consequently, we studied the steroidogenic capacity and the responsiveness to ACTH of human fetal adrenal tissue during the first week in organ culture. The culture medium was removed daily and assayed for cortisol and dehydroisoandrosterone sulfate (DS). First, as the concentration of ACTH in the medium was increased from 0 to 1 &ml steroid secretion increased. When tissue fragments were maintained in the absence of ACTH for 3 to 4 days, there was a striking increase in steroid secretion upon addition of ACTH to the medium, with Second, the larger rates of secretion of cortisol than DS being observed. steroidogenic capacity of the separate zones of the fetal adrenal gland was assessed. Tissue from the fetal zone secreted large amounts of DS and small amounts of cortisol, whereas neocortex tissue secreted similar quantities of DS and cortisol. Third, fetal zone tissue was maintained the absence of ACTH for 4 days and thereafter ACTH was added to the media for an additional 6 days. In this experiment, there was a marked increase in DS secretion rate after the addition of ACTH and a smaller increase in cortisol secretion.

INTRODUCTION

The size advances

with

intrauterine number for

of the the

life

computed

Volume

This

of adrenocortical

adrenal that

36,

80%

cortex

rate

of

gland

of growth in size

comprising

the

produces

5

adrenal

increase cells

the adrenals

Number

fetal

greatest

[2].

approximately

fetal

hunan

total large

of some

S

occurring

the fetal of

quantities

TIIROXDD

the

the to

fetal

at term

as pregnancy last

6 weeks

an increase

zone which

of steroid

fetuses

strikingly

during

is due primarily

volume

human

increases

at term

adrenal

gland

hormones secrete

of

in the accounts

[A.

and it

The can be

100 to 200 mg

November,

1980

S of steroids

daily,

most

of which

androsten-3

8-yl

sulfate)

[4,

responsible

for

glands

of the

secretion

the

fetal

culture.

for

Adrenal in culture

fetal

(DS, 17-0x0-5-

is considered since

is associated

it has been

decreases

with

with

obtained

to

its absence

a much

human

elected

to study

from

on explants

fetal the

the first

be

primarily

in the

lower

rate

adrenal

of steroid

shown

that

in culture,

of

the

the

ratio

and that of the

adrenal

tissue

[El.

fragments

Most

tissue

has been

found

in vivo --

state

after

of fetal

ACTH

in culture

steroidogenic

capacity

week

culture.

zone

treatment

of the

and

the

to

lose

response

[9-

steroid

in culture.

to neocortex

its “fetal

evaluated

the

6 days

or adult

to remain

of culture

zone

of the tissue

neocortex

soon loses

have

6 to 8 days

been

in organ

found

investigators

after

in the

maintained

has

cells

in organ

of steroidogenesis

abortuses

in culture

MATERIALS Adrenal

human

of adrenal

tissue

control

tissue

up to ‘28 days

proliferation

Because

of the

employed

characteristic

time

to further

during

we

adrenal

Moreover,

Fetal

zone

secretion,

investigation

for periods

of ACTH

Human

the

tissue

pattern

ACTH

fetus

gland,

secretory

leads

fetal

in steroid

anencephalic

adrenal

effects

11].

The

sulfate

[6, 71.

human

the

is dehydroisoandrosterone

51.

increase

As a model

viable

TEIROXDS

fragments

zone

[9, lo].

appearance” of this

cells

tissue

we to

AND METHODS

Tissue.

Fetal adrenal glands were obtained from mid-trimester human abortuses Tissues were following spontaneous abortion or abortion by hysterectomy. obtained in accordance with the Donors Anatomical Gift Act of the State of Texas after obtaining consent in writing from the next-of-kin employing a consent form and protocol approved by the Human Research Review Committee of the University of Texas Health Science Center at Dallas, Texas. Within 1 h of fetal death, the adrenal glands wer; removed under sterile conditions and minced into pieces of approximately 1 mm . In experiments in which steroid secretion by the various zones was examined, the adrenal capsule was removed and a portion of both the capsule and remaining tissue were examined microscopically. The capsule contained mostly neocortex or adult zone tissue together with some remaining fetal zone tissue. The fetal zone uniformly was free of contamination by neocortex.

S

TDROXDB

Tissue Culture. Four fragments of fetal adrenal tissue (approximately 1 mg tissue protein) were placed on lens paper which was supported by a stainless steel grid. One ml of culture medium consisting of 50 vol of Waymouth’s MB 752/l medlum, 40 vol of Gey’s balanced salt solution, 1 vol of a 1% solution of antibiotics and antimycotics (Grand Island Biological Company, Grand Island, NY), and 10 vol of pooled human serum was added to each organ culture dish [60 x 15 mm (Falcon Plastics, Cockeysville, MD)]. Synthetic ACTH I-24 (Cortrosyn), obtained from Organon (W. Orange, NJ), was added to the culture medium when indicated to achieve were maintained ~ncentratio~ varying from 0 to 1 l.g/ml. The tissue fragments at 37“C in a humidified atmosphere of 95% air and 5% COP. The medium was changed daily and stored at -2O’C until assayed for steroid content. Steroid Measurements. Cortisol and DS in the culture medium were quantified, without extraction, cortisol, as described by Gomezby RIA employing the following methods: Sanchez et et al. [13j. At the end of -- al. [K!], and DS by the method of Milewich -the culture period, the tissue fragments were rinsed and homogenized in 0.5 ml of NaCl (0.15 M). Aliquots of the homogenates were assayed for protein by the method of Lowry -et al. [14]. For each experimental condition five replicate dishes of tissue were employed and the media and tissues of each dish were analyzed separately except in the case of tissue from the neocortex, in which three dishes of tissue were The results presented are the amount of steroid secreted into the employed. culture medium x mg-‘tissue protein x 24 h-‘(mean 2 SE).

RESULTS Steroid

secretion The rates

tissue

ranging

of steroid

obtained

experiments,

in response

from

secretion

a 24-week

ACTH was present

from 0 to 1 ug/ml.

days in the absence

the commencement at approximately high concentration tissue

abortus

are

presented

in the culture

medium

of ACTH, after secretion

of organ culture, initial

rates

adrenal

human

in various

maintained

1-5.

In all

concentrations for O-4

In the absence

but when ACTH was present

the rate of steroid

was added

adrenal

tissue was maintained

which time ACTH was added. declined,

fetal

in Figures

secretion

for the 7 days of study (Figure

(0.5 to 1 m/ml)

previously

by ACTH-stimulated

In some studies,

of ACTH the rate of steroid

adrenal

to ACTH.

1).

was maintained When ACTH in

to the culture.medi~

for 24 h in the absence

from

of fetal

of ACTH (Figure

21,

steroid

secretion

added

48 h after

when

ACTH

when

ACTH

marked rate

rise

the

was

added

culture

of ACTH

presented than

after

addition

secretion

DS,

the

to

secretion

was

to

medium.

to ACTH

by human

fetal

adrenal

ACTH

the

results

the

was

of which

rate

of cortisol

the

In response

to

ACTH,

and

a

96 h in the

within

period.

declined

was

in the

when

in the

culture

tissue

there

occurred

the

and then

However,

preceeding

study

increase

in secretion

3).

In this study

was found

and in the

throughout

4).

was

obtained

concentration

for the

a greater

the

increase

secretion

study

increase

in response

first

24 h

cortisol

DS seer etion

on days 6 and 7.

by separated

fetal

andneo-

tissues.

The rates

are

4, there

of ACTH

initially

tort ex zone

In this

(Figure

ACTH

those

(Figure

of ACTH

maintained

to

of culture,

to ACTH

in steroid

of tissues

5).

and

continued

increased Steroid

(Figure

in Figure

secretion the

increase medium

of culture

as a function

when

similar

commencement

in response

increased

The greatest

absence

adult

secretion

the

secretion

were

commencement

72 h after

secretion

of steroid

of culture

24 h after

added

in steroid

to the

The rates

commencement

was added

of steroid

medium.

are

was stimulated.

of steroid

zone)

and by minced

presented

in Figure

are presented

in Figure

declined.

Cortisol

presence

of ACTH

secretion whole

6.

by tissues

adrenal

The rates

6, panel

secretion

A.

the

tissue

In the

zone,

from

absence

by fetal

of ACTH, the

of DS secretion

neocortex

a 20-week

of steroids

low throughout

rate

fetal

obtained

of secretion

remained

(1 &ml)

of the

abortus,

zone

DS secretion culture

was

tissue rapidly

period.

greater

(or

than

In the that

of

cortisol. The rates presented

of.secretion

in Figure

cortisol

secretion,

ACTH,

DS secretion

of steroids B.

In the

initially

were

6, panel which

was maintained

by adrenal absence low,

at initial

tissue

from

of ACTH,

remained rates,

the

low.

which

the neocortex

were

rates

In the

are

of DS and presence

of

less than the rate

S

'x-R&01:1,11

567

8---•No

0

ACTH

Figure 1. The daily secretion rates of cortisol and DS by minced whole human fetal adrenal tissue maintained in the presence of human serum (lo%, V/V) and ACTH in various concentrations ranging from 0 to 1 &ml during the first 7 days in organ culture. ACTH was in the medium at the commencement of culture. The medium was changed daily and assayed for steroid content. The results presented are the mean 2 SE of ~intuplicate replicates.

0,

, 1234567

,

,

,

,

,

Days Figure 2. The rate of steroid secretion by fetal adrenal tissue which . . . malntalned in organ culture for the first 24 h in the absence of ACTH. After 24 h in the absence of ACTH the medium was changed daily with medium that contained ACTH.

568

S

TDROXDCII

u

No ACTH

1234567 DW

The rate of steroid secretion by fetal adrenal tissue which was Fiqure 3. maintained in organ culture for 48 h in the absence of ACTH. After 48 h in the absence of ACTH, the medium was changed daily with medium that contained ACTH.

rate of steroid secretion by fetal adrenal tissue which was Figure 4. The maintained in organ culture for 72 h in the absence of ACTH. After 72 h in the absence of ACTH, the medium was changed daily with medium that contained ACTH.

569

The rate of steroid secretion by fetal adrenal tissue which was Figure 5. maintained in organ culture for 96 h in the absence of ACTH. After 96 h in the absence of ACTH, the medium was changed daily with medium that contained ACTH. of DS secretion cortisol

by tissue

secretion

less than those attained

by fetal

Steroid

zone adrenal of steroid

6, panel

utilizing

by neocortex

C.

secretion

week abortus the culture

tissue

of steroid are depicted

medium

There was insufficient

However,

increased

in response

to ACTH

to values which were only slightly secretion

rate

was 2.5 times

that

tissue.

The steroid

by fetal

zone.

The cortisol

secretion

tissue from the fetal

The rates

the fetal

of DS secretion.

The rate Figure

from

by minced secretion

zone (Figure

zone tissue secretion

to achieve neocortex

rates

final

were similar

in response

7.

tissue

tissue

is shown in

to those

observed

to ACTH. from the fetal

In this experiment,

concentrations

tissue

adrenal

6, panel A).

by adrenal

in Figure

whole

to conduct

ranging a parallel

zone of a 16-

ACTH was added to from 0 to 100 ng/mI. experiment.

ACTH

S

570

Figure

6.

The daily

secretion

TDROXDB

rates

of cortisol

and DS by adrenal

tissue

from

a ZO-

week human abortus. The medium in one-half of the dishes contained ACTH (1 &ml) and the medium in one-half of the dishes contained no ACTH. The medium The results was changed daily and assayed for content of cortisol and DS. presented are data obtained utilizing tissue from: A) fetal zone, 6) neocortex, or C) minced whole fetal adrenal and are the mean + SE of quintuplicate replicates except for B) (neocortex) which was performed in triplicate.

was not included rates

of steroid

medium found (data times within

in the secretion

when

the

In fetal

of cortisol

24 h when

the

was found

The rate

of steroid

declined.

of

until

obtained

of

ACTH

zone tissues

the 4th day of culture.

when

in the the rate

secretion.

An increase

concentration

of ACTH

within secretion

24 h when reached

ACTH

the

was present

No increase

100 ng/ml.

concentration

not shown). that

medium

were

in a concentration

secretion

then

culture

culture

in steroid medium

of DS secretion in cortisol

concentration

a maximal

value

in the

<0.5

was more

was ng/ml than

4

was observed

and an increase

of ACTH

culture

secretion was

secretion

was 50 ng/ml

The highest

in DS

was 0.5 ng/ml.

at 7 to 8 days in culture,

S

571

'PXlROXD6

figure 7. The daily secretion rates of cortisol and DS by fetal zone tissue of a 16week human abortus maintained in organ culture for 10 days. The tissues were maintained for the first 96 h in the absence of ACTH. Thereafter ACTH was added to achieve various concentrations (0 to 100 ng/ml). The arrow is placed to indicate the time of the first addition of ACTH to the culture medium. The medium was changed daily and assayed for content of cortisol and DS. The results presented are the mean + SE of quintuplicate replicates.

DISCUSSION

Kahri secretion

et al. (10) -by

human

transformation conjugates cortisol culture

from of

lo].

secretion medium.

Thus,

fetal

[9,

adrenal

a fetal

adrenal

A5-3S-hydroxysteroids) becoming

with the

tissue

after

in

pattern

the

secretion

of fetal

the pattern culture

(high

adrenal

after

of steroid undergoes

amounts

secretory

addition

is said to occur

in the ratio

of steroid

that

maintained

secretory

predominant

a decrease

pattern

11, 151 suggested

to an adult

This transformation

and is associated [9,

and others

of

of

sulfo-

pattern

with

ACTH

to

the

6 to 8 days in culture

zone to neocortex

of fetal

a

adrenal

tissue,

zone cells during

the

s

572 first

week in organ

culture

TXJROX131

may be more representative

vivo than will be the steroid

secretory

pattern

of that which occurs h

of the same tissue

after

1 week in

secretion

by fetal

culture. It has been reported adrenal

tissue

However,

in response

in those

the absence present

previously

fetal

the

in adrenat

beginning

response the

of culture

that

ACPH

utilization

adrenal

(Figures

of ACTH

steroid

that

l-5).

in lower

previously

concentrations.

in culture.

In the present

low doses of ACTH were used to stimulate of steroid

secretion

culture,

there

whereas

cortisol

(Figures

Z-5).

differences

was an initial secretion This finding

increase continued

pools in various in steroid

secretion

that

The delayed

steroidogenic

could

of the fetal

observed

zone (Figure

steroids

5).

commencement

which declined

the intracellular

7),

with higher doses

for the remainder

cell types for these rates

during

might have been due to

48 h after

in DS secretion

is suggestive

medium.

from the fetal

medium

to increase

to ACTH

in the tissue

of ACTH for 96 h (Figure

When ACTH was added to the culture

in the

study we also found that when

tissues

deprived

response

such as aging

was much less than that

of ACTH even in tissue previously

to ACTH

previously

The delayed

causes

during

exposed to ACTH from

precursors

by others

in

In the

secretion

in response

Such an augmented

in the incubation

in culture

with ACTH.

had been incubated

also be due to other

tissue fragments

the precursor

was maintained

secretion

of steroidogenic

was not present

to ACTH might

the rate

tissue

days than in tissue fragments

to ACTH that was observed

response

for 2 to 3 days [8, 9, 111.

in the rate of steroid

In fact,

tissue fragments

of ACTH for several

time

adrenal

we found an increase

could be due to an acc~ulation the

in steroid

of ACTH for 6 to 9 days prior to treatment

the first 24 h of ACTH treatment.

absence

an increase

to ACTH may be delayed

experiments

investigation

was greater

that

thereafter, of the 7 days

precursor

pools or

are different.

be due to a) different

of

cell

These types

(neocortex esters

versus

fetal

S

z'I1ROID#!b

zone),

or b) different

of long chain fatty

573 precursor

acids in the case of cortisol,

molecules

(cholesteryl

and cholesteryl

sulfate

in

the case of DS). The rates

of steroid

neocortex

of fetal

Branchaud

et al. --

DS and

[16].

smaller

approximately

different

than

that

of

cortisol

secretion

pattern

of steroid

week,

we suggest

investigation

fetal

qualitatively

Tissue

zone.

pattern

l-7)

response

during the first secretion

of cholesterol

this

metabolism

may

observed serve

and steroid

of

secreted

was known to

tissue from three initially

probably

was

is rep-

in vivo. -~ fetal

was defined.

week in organ culture

preparation

which

by

quantities

neocortex

of DS secretion

of human

in vivo than that --

larger

of adrenal

adrenal

and by the

to those reported

the

a finding

of the fetal

zone

but the neocortex

In studies

week in organ culture

that

from

ages, the rate

(Figures

fetal

zone tissue secreted

cortisol.

the steroi~~nic

the first

of the

of DS and cortisol,

of various

of the secretory

ACTH during

fetd

by cells of the fetai

In summary,

steroid

of

by tissues

were similar

In generaI,

amounts

abortuses

resentative

tissue

equal quantities

be contaminated

higher

adrenal

secretion

adrenal

tissue

to

Since the pattern

of

may better in cultures as

secretion

a model

represent

the

older than for

L

further

by the human fetal

adrenal.

ACKNOWLEDGMENTS The authors gratefully acknowledge the skilled technical assistance provided by Dru Ann Miller, Debra Bradfield, Sara Innis, George Crowley and Loretta Dice, and the authors thank Becky McKinney-Reese, Vicki Rankin, and Millie Arnold for expert editorial assistance. This investigation was supported, in part, by USPHS Grants 5-P50-HDlll49 and l-ROl-HDI3234. REFERENCES 1. 2.

Dr. Carr is a postdoctoral trainee supported by NIH Training Grant HD07190. Spector, W.S., ted>. In: HANDBOOK OF BIOLOGICAL DATA. Saunders, ~ii~~lphia~956).

l-T32p* 353,

574

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16.

S

TDROXDE

Benirschke, K. OBSTET. GYNECOL. E&412 (1956). Siiteri, P.K., and MacDonald, P.C. J. CLIN. ENDOCRINOL. METAB. Z& 751 (1966). Simmer, H.H., Easterling, W.E., Pion, R.J., and Dignam, W.J. STEROIDS 4, 125 (1964). Easterling, W.E., Simmer, H.H., Dignam, W.J., Frankland, M.V., and Naftolin, F. STEROIDS 8, 157 (1966). Cooke, B.A., Shirley, LM., Dobbie, J., and Taylor, P.D. J. ENDOCRINOL. 2, 533 (1971). CAN. J. PHYSIOL. Stark, E., Gyevai, A., Szalay, K., and Acs, Zs. PHARMACOL. 43, l(1965). Milner, A.J., and Villee, D.B. ENDOCRINOLOGY 87, 596 (1970). Kahri, A.I., Huhtaniemi, I., and SaImenperz, M. ENDOCRINOLOGY E, 33 (1976). Voutilainen, R., and Kahri, A.I. J. ULTRASTRUCT. RES. e, 98 (1979). Gomez-Sanchez, C., Milewich, L., and Holland, O.B. J. LAB. CLIN. MED. e, 902 (1977). Milewich, L., Gomez-Sanchez, C., Madden, J.D., Bradfield, D.J., Parker, P.C. J. P.M., Smith, S.L., Carr, B.R., Edman, C.E., and MacDonald, STEROID. BIOCHEM. 2, 1159 (1978). Lowry, O.H., Rosebrough, N.J., Farr, A-L., and Randall, R.J. J. BIOL. CHEM. 193, 265 (1951). Huhtaniz, I., J. STEROID BIOCHEM. 3 491 (1977). Branchaud, C.T., Goodyer, G., Hail, C. St. G., Arato, J.S., Silman, R.E., and Giroud, C.J.P. STEROIDS g, 557 (1978).