Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

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ANDROGEN REGULATION OF PROGESTIN BIOSYNTHETIC ENZYMES IN FSH-TREATED RAT GRANULOSA CELLS IN VITRO

T. H. WELSH, JR., P.B.C. JONES, C.M. RUIZ DE GA~RRETA, L.F. FANJUL AND A.J.W. HSUEH

Department of Reproductive Medicine, M-025 University of California, San Diego La Jolla, California 92093 Received 12-20-82 ABSTRACT The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied --in vitro. Granulosa cells obtained from immature, hypophysectomixed, estrogen-treated rats were cultured for 3 days in a serum-free medium co _(rtai$ng FSH (20 ngfml) with or without increasing concentrations (10 -10, M) of 17$-hydroxy-5a-androStan-s-one (dihydrotestosterone; DHT), 5a-androstane-3a,l7R-diol (3aor the synthetic androgen 17R-hydroxy-17_methyl-4,g.l dial) , l-estratrien-s-one (methyltrienolone;Rl881). FSH treatment increased progesterone and 2Oe-hydroxy-4-pregnen-3-one(20a-GH-P) production by 10.2and 1l-fold, respectively. Concurrent androgen treatment augmented FSHstimulate progesterone and 2Oa-GH-P production in a dose-related manner (RI881 > 3a-diol > DHT). In the presence of an inhibitor of 36hydroxysteroid dehydrogenase (3S-HSD), the FSH-stimulated pregnenolone (3B-hydroxy-5-pregnen-20-one)production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3a-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3R-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20a-hydroxysteroiddehydrogenase (LOU-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of prggegterone to 20a-GH-P, but involves an enhancing action upon 3$HSD/A ,A -isomerase complexes and additional enzymes prior to pregnenolone biosynthesis. INTRODUCTION The pituitary gonadotropins, follicle-stimulatinghormone (FSH) and luteinizing hormone (LH), have long been recognized as the predominant physiological regulators of ovarian steioidogenesis-in vivo. Establishment of primary cultures of rat and porcine granulosa cells facilitated in vitro studies which suggested an intra-ovarian role for endogenously -December, 1982

produced tion

androgens

(I-4).

tic

in the regulation

Recent --in vitro

actions

of

may be related

the to

ther

androgens

studies

androgens

further

in the

(5,6).

augment

In the

present

regulation

tic

enzyme,

3R-hydroxysteroid

dehydrogenase/A5

3g-HSD)

progesterone

hydroxysteroid lation with the

of

dehydrogenase

pregnenolone

cyanoketone, various

biosynthesis

and

FSH-stimulated

progesterone

by stimulation

of

without

affecting

of

of natural

pregnenolone

of

production

in

production

production

20a-HSD)

and the

(EC 1.1.1.

cells

20amodu-

treated

androgens

that augment

granulosa

activities

vitro

biosynthe-

demonstrates

rat

whe-

--in

Possible in

study

cultured

cleav-

enzyme,

.

studied

non-aromatizable

production

synergis-

we investigated

,A4-isomerase

This

produc-

the side-chain

metabolizing

was also 3g-HSD.

the

the progesterone

(EC 1.1.1.149;

an inhibitor

synthetic

activities

and the

that

progesterone

a direct

progestin

progesterone

study,

via

511 EC 5.3.3.1;

the

cell

suggested

activities

FSH-stimulated of

granulosa

on FSH-stimulated

an increase

age enzyme complexes

of

of

cells 3R-HSD

20a-HSD activity.

MATERIALS AND METHODS Reagents and Hormones Ovine FSH (NIH-FSH-S14; FSH activity 9 x NIH-FSH-SI units/mg; LH PRL activity
Animals Immature female Sprague-Dawley rats (21-23 days old) wart? hypophysectomized by Johnson Laboratories (Bridgeview, IL) and delivered on Silastic capsules (10 mm) containing DES the third postoperative day, Animals were provided a mixture were implanted at the time of surgery. of bread, milk, dog food, water and physiological saline --ad libitum. Steroid

Production by Cultured Rat Granulosa Cells Four to six days after surgery, granulosa cells were obtained from DES-treated rats as previously dethe ovaries of hy~physect~ized, tailed (7). These cells were cultured in 35 x 1 OmmFalcon tissue culture dishes (~~2-4 x IO5 viable cells/dish)) in 1 ml of MccOyts 5a mediun sup200 U/ml penicillin and 100 pg/ml plemented Mith 2 mM L-glutamine, streptomycin. Granulosa cells were cultured for 3 days at 37’C in a humidified, 95% air-5% CO2 incubator with appropriate combinations of of the incubation period, media androgensand FSH. 1 At the conclusion were collected and stored at -2O’C until analyzed for pregnenolone, procells were scraped from Additionally, gesterone and YOU-OH-P content. culture dishes and homogenates prepared to evaluate the influence of androgens on the FSH-stimulated activities of 3R-HSD and 20a-HSD. Radioimmunoassays and Enzyme Assays progesterone and 2Ou-OH-P Bedium concentration of pregnenolone, were determined by r~ioi~unoas~y with specific antisera as previously described (8,9). The measurement of 2Ge-HS.D activity was based on a proby us (II) in cedure developed by Eckstein et al . 110) and modified which 20a-HSD activity was measured as the rate of conversion of [ 3B1The assay of 3$-BSD activity was based on 20a-OH-P to C3H1progesterone. a procedure developed by Murono and Payne (12) and modified by us (I 3) in which 3R-HSD activity was measured as the rate of conversion of pregnenolone to progesterone. -Data Analysis Radioimmunoassay data were analyzed with a program which utilizes a weighted logit-log regression analysis (14). Enzyme activities were calculated by the Wilkinson method (I I). Experimental data are presented as the mean f S.E. of measurement of triplicate cultures. Comparable Data were results were obtained in 3-5 replicates of each experiment. analyzed by analysis of variance and Dunnett’s test (151, Comparisons with P > 0.05 were not considered significant. RESULTS

-Dose-dependent gesterone

effect

of androgens

FSH-stimulated

granulosa presence

production

of

pro-

and 20a-OH-P

The dose-dependent upon

on FSH-stimulated

cells

for

of increasing

influence progestin

3 days

of

synthetic

production in medium alone

concentrations

and

natural

was investigated or with

of Rl881,

by culturing

FSH in

3a-dial

androgens

the

absence

or DHT (Fig,

or 1).

Treatment with FSH increased progesterone production 10.2-fold relative to control cultures (Fig. IA). concentrations (lo-'-10%~

Conourrent treatment with increasing

of androgens augmented FSH-stimulated pro-

gesterone production in a dose-dependentmanner (Rl881 > 3a-dial > MT). At 10s6M, DHT, 3a-dial and R1881 augmented FSH-stimulated progesterone production by 6.4-, 9.6- and 12.2-fold, respectively. Similarly, production of 20a-GH-P was increased II-fold by FSH treatment. Concomitant addition of increasing doses of androgens also augmented FSH-stimulated 20aaH-P production in a dose-related fashion (Fig. ID). FSH-stimul.ated production of 2Oa-OH-P was further increased 4.3-, 5.5

and 7.9-fold

with 10m6M of DHT, 3a-diol or Rl881, respectively.

IO

I-

Figure 1. Effect of androgens on FSH-stimulated production of progesterone and 20a-OH-P. Granulosa cells (4 x 10s viable cells/dish) were cultured for 3 days in mediun alone (C; control), or with 20 n&ml FSH in the absence or presence of increasing concentrations of R1881, 3adiol or DHT. Mediun concentrations of progesterone (panel A) and ~OW-GHP (panel B) were measured by radioimmunoassay.

Effect

of androgen

treatment

on FSH-stimulated

production

of

pregneno-

lone The’ influence of pregnenolone 3 days

inhibit

with

of synthetic production

various

3B-HSD activity 20-fold

increasing

concentrations

production

increased 10W8M and augmented

was examined

hormones

production

lone

and natural

in

(9,161 .

relative

to

by c2.4-fold

presence

cultures enhanced

granulosa

of

cells

for

10F6M cyanoketone

to

increased (Fig.

production

(P
At

pregnenolone

2).

Addition

FSH-stimulated

= DHT) in a dose-related

pregnenolone

10m6M, respectively FSH action

control

on FSH stimulation

by culturing

FSH treatment

of androgens

CR1881 > 3a-diol

FSH-stimulated

the

androgens

3.410w6M,

pregneno-

manner. and

of

RI881

6.6-fold

3a-diol

and

at DHT

(P
ANDROGEN(M) Fs+

Figure 2. Effect of androgens on FSH-stimulated production of pregnenolone. Granulosa cells (2 x IO5 viable cells/dish) were oultured for 3 days in mediun alone (C; control), or with 20 ng/ml FSH in the absence or presence of increasing concentrations of Rl881, ja-diol or DHT. All cultures were treated with lOWeM,cyanoketone to prevent conversion of pregnenolone to progesterone, Medium concentration of pregnenolone was measured by radioimmunoassay.

Effect

losa verts

of androgens on ASH-stimulated We demonstrated

earlier

cells

the activity

inoreases

pregnenolone

that

3$-HSD and 20u-HSD activities

FSN treatment

of

of 3$-HSD (13).

cultured

the enzyme which con-

Granulosa cells

to progesterone.

were incubated

days in medium alone or with FSH in the absence or presence ing concentrations the enzyme activity of

3e-diol

activity

of

the androgens

(Fig.

in a dose-dependent

of

FSH further Specifically,

manner.

was augmented 2.3-,

2.%-

with 70W714 of DHT, Rt881 and 3a-dial,

of

FSH treatment

e4, 4-fold. Treatment with increasing

and DHT in the presence

zyme activity

31.

rat granu-

and 3.0-fold

3

inoreasincreased

concentrations

increased

the 3$-HSD

FSH-stimulated for

for

cells

en-

treated

respectively,

98

T

ANDROGENtM) &I

Figure 3. Effect of androgens on FSH-stimulated 38..hydroxysteroid dehydrogenase activity, Granulosa cells 6~4 x 10s viable cells/dish) were cultured for 3 days in medium alone CC; control)., or with FSH t2b ng/mU in the absence or presence of lnoreasing concentrations of R1881, 3a-dial or DHT. Enzyme activities were determined as described under Waterials and Methods 0I1

S

TSJX%.OfXS~

697

r

Effect of androgens on FSH-st~mul%t~ boa-hydroxyst@roid deFigure 4. hydrogenase activity. Granulosa cells ($4 x 10’ viable cells/dish) were cultured for 3 days in medium alone (C; control), or with FSH in the presence or absence of 10-*N of various androgens. !Xnzyme activities were det~rrn~n~ as described under Waterials and ~~~ds.~

We also activity cells

examined

whether

of ~OU-HSD, which were

treatment

treated

administration stimulation

converts

3 days

for

increased

androgen

progesterone

with

2Oe-HSD activity

of any of the

three

of ZOa-HSD activity

treatment

various

affects

to 20u-OH-P. honaones

1. j-fold. androgens

FSH-stimulated

at

(Fig.

However, 10%

did

not

Granulosa 4).

FSH

aonc~itant affeot

FSH

(P>O.OS).

DISCUSSION The present l&ted activity list,

progestin of

the

The synthetic

study

investigate

production enzymes

whether

In cultured involved

(R1881)

in

and natural

androgens

granulosa ~ogestin

a~ment

cells

FSH-star

by affecting

the

androgens

each

biosynthe

(DHT and 3a-dial)

increased

FSH-stimulate

androgen

of

accumulation

creased

of

au~en~ation

the androgens

action

involves

studies

Since

(l-3).

it

is thought

by the granulosa ized

production treatment

cell

further

demonstrated

36-HSD activity

that

and pregnenolone

biosynthesis and

effect

a~rnen~~

rat and porcine

progesterone

granu-

suppresses

the

prodUctiOn --in vivo

of androgens is mediated

(IT).

The present

study util-

a~drogen Rl381 which s~c~fica~~y

in a variety

the

of the stimu-

synergistically

with an antiandrogen

androgen receptors

to the. androgen receptor

addition

enzyme complexes

by cultured

FSH-stimulated

eon-met&bo~~~ble

in-

biosynthesis.

androgens

that the stimulatory

the synthetio,

binds

of

that

with

3134SD activity

progesterone

upon 36~SD/isomerase

reported

This

concomitant

Thus, the mechanic

to pregnenolone

progestin

associated

Furthermore.

20a-HSD activity.

androgen au~entat~on (4),

is

of androgens upon ~~-stimulated

FSH-stimulated cell3

production

production.

augmented FSH-stimulated

an enhancing action

Rarly

and 2%-OH-P

of pregnenolone.

es prior

lose

progestin

significantly

but did not affect latory

progesterone

androgen

of

cell

types

augmentation

of

(18-20).

This

FSH-stimulated

production

may be an androgen receptor-

production

is also

mediated event, FSH-st~u~at~ itant

treatment

ulosa

cells

substant~el thetic ent

progesterone

with estrogens

(unpublish~ increases

observations~

stimulatory

(3a-dioL

be aromatized

actions

.

in aromatase aotivfty

(R1881) and natural

study cannot

in cultured

to

receptors.

A~t~ug~

(21,22f

and rat gran-

FSH trea~ent

of granulosa

cells,

induces the syn-

and DHT) androgens used in the presform estrogens.

appear to be direct,

are not mediated by estrogen

porcine

augmented by concom-

specific

Thus, the observed androgen effects

and

S A stimulatory to

pregnenolone

effect via

was proposed

synthetic

and natural accunulation

increase

progesterone

i.e,, P.

pregnenolone,

in

activity

pregnenolone

in vivo __-

and --in

enhanced

the FSH-stimulated

do not activity affect of

increase (25). the

after

the conversion

of

zymes

steroids, requisite

provides

the

basis

involved

in follicular

it

data

20a-HSD.

provide

progestin for

as of

may

substrate,

progesterone

and 20a-GHfor

be regulated

androgen

the

preg-

hormones

additional

was shown to

con-

by FSH

treatment

has been demonstrated

accunulation

androgens, for

to

enzyme

further

3R-HSD activities.

androgen

data

These

of

Concomitant

progesterone

The present ovarian

(23.24).

of

cleavage

FSH stimulation

provision

conversion

catabolism,

activity

the

cholesterol

suggestion

manner.

progesterone,

The present

this

of

of 3$-HSD, the enzyme responsible

progesterone

progesterone

by

side-chain

confirm

a dose-related

verting

to

the

augmented

enzymatic

vitro

of

. Our data

production

to

upon the conversion

activity

androgens

the

With regard

androgen

(5,6)

for

Furthermore,

of

enhanced

complexes

nenolone

TPEOXD-

further

via

inhibition

demonstrate

that

Consequently,

the

treatment

is

not

that of

5a-reductase

androgens enhanced due

to

androgens

also

do not

accunulation

a diminution

in

to 20a_OH-P. insight affect

regarding gonadotropin

biosynthesis understanding

how one class regulation

and catabolism. of

the

cellular

of of

intrathe

This

en-

study

mechanisms

steroidogenesis.

ACKNOWLEDGMENTS This work was supported by NIH Research Grant HD-14084 and Program Project Grant HD-12303. A.J.W.H. is the recibient of Research Career Development Award HD-00375. is the recipient of a Giannini T.H.W. Medi.cal Research Foundation Postdoctoral Fellowship. C.M.R.G. and L.F.F. were supported by the Spanish-American Joint Conznittee for Scientific

and Technological Cooperation. We thank J.P. Raynaud of Roussel-UCLAF for provision of R1881. We also thank C. Valk, E.M. Tucker and K. Watts for excellent technical assistance. LITERATURE CITED 1. Schomberg, D.W., Stouffer, R.L., Tyrey, L. BIOCHEM. BIOPHYS. RES. COMMON. 68_,77 (1976). 2. Armstrong, D.T., Dorrington, J.H. ENDOCRINOLOGY99, 1411 (1976). 3. Lucky, A.W., Schreiber, J.R., Hillier, S.G., Schulman, J.D., Ross, G.T. ENDOCRINOLOGY 100, 128 (1977). R.F., Tyrey, L. Ulberg, L.C. ENDOCRINOL4. Schomberg, D.W., Wimams, OGY B, 984 (1978). Nimrod, A. MOL. CELL. ENDOCRINOL.8, 201 (1977). :: Nimrod, A. MOL. CELL. ENDOCRINOL. 2l_,51 (1981). 7. Hsueh, A.J.W., Wang, C. and Erickson, G.F. ENDOCRINOLOGY108, 1697 (1980). 8. Wang, C., Hsueh, A.J.W. and Erickson, G.F. J. BIOL. CHEM. 254, 11330 (1979). 9. Jones, P.B.C. and Hsueh, A.J.W. ENDOCRINOLOGY111, 713 (1982). 10. Eckstein,B., and Nimrod. A. J. STEROID BIOCHEM. 8, 213 (1977). 11. Jones, P.B.C. and Hsueh, A.J.W. J. BIOL. CHEM. 256, 1248 (1981). 12. Murono. E.P. and Payne, A.H. BIOL. REPROD. 3, 911 (1979). 13. Jones, P.B.C. and Hsueh, A.J.W. ENDOCRINOLOGY'I&, 1663 (1982). 14. Rodbard D. CLIN. CHEM. 20, 1255 (1974). 15. Snedecor, G.W., and Cochran, W.G. Statistical Methods, Iowa State Univ. Press, Ames, (1967). 16. Goldman, A.S., Yakovac, W.C., and Bongiovanni, A.M., ENDOCRINOLOGY 77, 1105 (1965). 17. Schreiber, J.R., and Ross, .G.T.ENDOCRINOLOGY99-1590 (1976). 18. Bonne, C., & Raynaud, J.P. STEROIDS 26, 227 (1975). 19. Bonne, C., and Raynaud, J.P. STEROIDS a, 497 (1976). 20. Asselin, J., and Meancon, R. STEROIDS 2, 591 (1977). 21. Veldhuis, J.D., Klase, P.A., Hammond, J.M. ENDOCRINOLOGY109, 433 (1981). 22. Veldhuis, J.D., Klase, P.A., Hammond J.M. ENDOCRINOLOGY111, 441 (1982). 23. Zeleznik, A.J., Midgley, Jr., A.R. and Reichert, Jr., L.E. ENDOCRINOLOGY 95, 818 (1974). 24. Dorrington, J.H., Armstrong, D.T. REC. PROG. HORM. RES. 3, 301 (1979). 25. Nimrod, A., Rosenfield, R.L., Otto, P. J. STEROID BIOCHEM. 13, 1015 (1980).