Influence of High-Density Lipoprotein on Estradiol Stimulation of ...

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BIOLOGY

OF REPRODUCTION

32, 96-104

(1985)

Influence of High-Density Lipoprotein on Estradiol Stimulation of Luteal Steroidogenesis1 IQBAL

Y.-D.

KHANZ3

IDA

ALAIN

CHEN5

Department

of Physiology College

University

BELANGER,4

and GEULA

of Illinois

Department Le Centre

GIBORI3

and

Biophysics3

of Medicine

at Chicago Health Chicago, Illinois

Sciences

of Molecular Endocrinology4 Hospitalier de L ‘Universite Quebec,

Center,

Laval

Canada and

Department

of Medicine5

Sanford Palo Alto,

University California

ABSTRACT The

aim of this investigation was to determine whether luteal cells utilize cholesterol from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different human HDL resulted in a dose-dependent increase in progesterone production. HDL

enhanced

the overall

derived luteal doses of in vitro

steroidogenic capacity. However, the percentage of increases in 1 7o-hydroxy-

progesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.

rat.

INTRODUCTION

The produces

corpus not

androgen

and

1976;

Gibori

1983;

Sridaran

to

be

the

luteum only estrogen

et a!.,

most

of the progesterone

and active

pregnant but

(Elbaum

1982;

Taya

Gibori,

and

and

1983)

steroidogenic

Days

rat also

organ

corpus

12 and

Keyes,

terone

appears

This tion

in the

tial nance September 11, May 4, 1984.

1984.

doubles

significantly

output

(Morishige

acquires a greater from androgenic increase appears

capacity substrate

Gibori

requirement

and

gism

11119.

‘Reprint requests: Dr. Iqbal Khan, Dept. of Physiology and Biophysics, College of Medicine, University of Illinoisat Chicago, Health Sciences Center, P0 Box 6998, Chicago, IL 60680.

of luteal

with (Gibori

1979)

and

half Greenwald, Estradiol

96

and independent

(Gibori 1973; increases

1978).

estradiol

function

prolactin

nancy

ond

between and

Roth-

increases its progeset a!., 1973) and to produce estradiol (Gibori et al., 1982).

Keyes,

for

by

Richards,

the

The

essenmainte-

pregnant

a number

shown

in

produc(Gibori

in the

in the

been well documented in which estradiol was

‘This work was presented in part at the 64th Annual Meeting of the Endocrine Society (Abstr. #398). It was supported in part by NIH grant HI)-

in size (Pepe

in size and in progesterone to be due to lutea! estradiol

et aL, 1977;

Accepted Received

luteum

15 of pregnancy

child, 1972),

Greenwald, and

The

to first

1978;

rat

has

of studies act half

Gibori

in of

synerpreg-

et al.,

of prolactin in the secet a!., 1977; Takayama and Ochiai and Rothchild, 1981). progesterone synthesis with-

ESTRADIOL,

out

affecting

the

metabolism

child

1980;

Belanger

low

and

LH

estrogen

and

receptor by

to

nancy,

which

terol

synthesized

thesis

both

appear de of in

vivo and

1982;

Schuler

et

in conjunction of high-density receptors in

and

from and

steroidogenic

important

may

increase

to the

HDL.

This

larly

attractive

et

al., al.,

possibility

estradiol

rats

cells,

Incubat

lutea

it

from

be a particuhas

the

Serum

Lipoprotein

cholesterol

of

derived

hormone action

examined from

biosynthesis, on

luteal

exogenous

HDL

and cell

supply

of

possibilities

pregnant

2)

function

1)

rats

utilize

for

steroid

that

Human nonpyrogenic,

to

the

Beckman

sterol.

MATERIALS

AND

METHODS

Day

tomized

stereotaxic tomy was the time remnants

Hormonal

of in

operation the fossa

approach

and the Steroids

using

a

of hypophysecof the pituitary at

the

recovery and by at autopsy.

of

Extraction

rats were hypophysec-

Completeness the

from each individual rat were at 37#{176}Cin 1 ml of freshly CO,) Medium 199 containing Grand Island, NY). Following was terminated by placing the

Preparation

60 Ti rotor at 45,000

rpm

for 20 h. The

absence of Rats showing

any in-

Millipore gentamycin

filter,

placed

in a

(10 Mg/mI) and migrated as a single band on 5%

gel electrophoresis HDL used has et al. (1984).

and Purification

One ml of medium

a transauricular

instrument. judged by

agarose sition Reaven

Treatment

12 of pregnancy,

through

adhering

(d=1.090-1.215) pooled human

stored at 4#{176}C. HDL

Pregnant Sprague-Dawley rats were purchased from Holtzman Co. (Madison, WI) and were housed under controlled environmental conditions with 14 h light per day at 24#{176}C.Purina Rat Chow and tap water were available ad libitum. The day sperm was present in the vagina was designated as the firstday of pregnancy.

On

HDL

passed through a 0.45-Mm sterile tube containing

and

from

final lipoprotein fractions were dialyzed exhaustively against 0.9% NaCI, 5 mM Tris, pH 7.4, and concentrated by loosely packing the outside of the dialysis bag with Aquacide. The concentrated samples were

Animals

Operations

free

were isolated from plasma by sequential centrifugation (Havel et al.,1955) using solid KBr for density adjustment (Radding and Steinberg, 1960) in a

estrogen’s

is related

dissected

a stereomicroscope.

sample vials in an acetone-dry ice bath. Samples were stored at -20#{176}C until processed for radioimmunoassay

been

1984).

lutea

15 of

ions

analysis.

we

were

tissue under

(RIA)

Thus

Day

Preparation

Three corpora lutea cut in half and incubated gassed (95% 0, and 5% 25 mM Hepes (Gibco, incubation, the reaction

estradiol

estradiol

and corpora

of HDL receptors rats (Gibori et al.,

corpora

killed on

15, rats were killed with an overdose of the ovaries were quickly removed. Indi-

found to increase the number in luteal cells of pregnant that

The third group received Control rats were intact

the

cholesterol to

Tissue

Day

nonluteal

orig-

luteal

whether

since

ether vidual

luteal

affects

of

appeared

one,

for

and

On

steroidogenesis

of

daily. oil).

progroup (100

of Blood

Autopsy

the possibility used

ascertain

to

observation,

(HDL)-specific of pregnant

utilization

designed

A 0.75-mi sample of blood was collected daily between 0900-1100 h by tapping the jugular vein through the skin under ether anesthesia. The serum was collected and stored at -20#{176}C until assayed.

characterization

capacity

were

animals. Rats were

Collection

biosynet

supports

Because

and

al.,

exoge-

Bruot

substrate

mm)

pregnancy.

(Azhar

This

3.18

ml sesame oil) (0.25 ml sesame

pregnant

vitro

recent

and

an

steroid

lipoprotein corpora lutea

HDL.

was

Mg/O.25 vehicle

1981;

1981). the

et

utilize

in

al.,

with

biogenesis

inates

size

choles-

(Kovanen

o.d.

surface area of 100 mm’. Another received subcutaneous injections of 1713-estradiol

of preg-

lutea

for

Menon

cholesterol

membrane size

novo ovaries

et al., 1984),

the

602-305, vide a

an

in

principally

cholesterol

Azhar

that

to

both

corpora

to use rat

1981;

(Gibori

increasing

rabbit

luteinized

source

AMP

cyclase

respond

97

Silastic capsules filled with testosterone were implanted s.c. in the dorsal part of the neck. These capsules were prepared from Dow-Corning (Midland, MI) Silastic medical grade tubing (cat. no.

of

secretion.

contrast

nous

independent

content

challenge

1978),

unpub-

hormone (LH)-cyclic desensitized adenylyl

progesterone In

Gibori,

STEROIDOGENESIS

complete hypophysectomy were excluded from the experiment. Hysterectomy was done through a midline abdominal incision. Immediately after the operations, rats were divided into three groups, each group consisting of 5-8 rats. In one group of rats, 1-cm long

Roth-

(Sridaran et al., 1983) also augments the in

of progesterone

the luteinizing system. CL with

LUTEAL

to

and

and

Our recent work that estradiol

production

vivo

(Rodway

Khan,

AND

of progesterone

20a-hydroxyprogesterone

lished). indicates

HDL

(Nobel, 1968). been previously

The comporeported by

of Steroids

was extracted solvent was evaporated were separated on LH-20

twice with ether under nitrogen. columns before

measurement as previously reported (Belanger et al., 1979). Briefly, LH-20 columns (lx 10 cm) were packed and washed with 25 ml of benzene-methanol. They were then washed with 20 ml of the firstsolvent system used for elution (isooctane-benzene-methanol;

98

KHAN

90:5:5,

by volume).

Calibration of LH-20

columns

was performed following known tritiated steroids

addition of 10,000 cpm of to aliquots of the methanol extract and collection of 1-mI samples. Progesterone

elutes firstin Fractions 6 to 12. Pregnenolone is found in Fractions 13 to 24, while testosterone and 17ahydroxyprogesterone elute in Fractions 28 to 42. The polarity of the solvent was then changed to 62:20:18 for the elution of estradiol.

ET

AL.

progesterone nant rats, 15

ml

pregnant medium

trations ml

for pregnenolone,

17-hydroxyprogester-

one, testosterone and estradiol were performed after chromatography using highly specific antisera. The specificity, validity and reliability of these RIAs have been previously described (Belanger et al., 1979). Progesterone was assayed with or without chromatography by two different RIAs (Gibori et al., 1977; Belanger et al., 1979).

little

Analysis

increased

ng.corpus

luteum’#{149}3

crease

in HDL

used to determine treatment groups. A

P

Student’s test were

significant differences between value below 0.05 was considered

significant.

Effect

from of

Production To

HDL

on

Progesterone

by Luteal evaluate

Cells

increase of HDL

of Pregnant

the role of HDL

Rats

in supporting

Day

The

15

medium

with

protein.

Media

progesterone,

Luteal

500

or were

335

greatest

in-

pg

when

protein/mI.

increased

there

no

was

con-

secretion.

Steroidogenesis

the effect of corpora pregnant

as

HDL of

occurred

in progesterone on

of value

were

protein/mi

,yet of

Progesterone

concentrations

To examine steroidogenesis

RESULTS Effect

was

1

by HDL

presence

function

h’.

concentration

pg

of

a maximal

secretion

500

comitant

a

Fig.

h

the

progesterone

HDL

absence

protein/mi. as

reaching

1

protein! in

luteum’.3 in

I-IDL

concentration,

beyond

Values presented are the mean ± SEM. test, analysis of variance and Kruskal-Wallis

pg

pregDay

secretion

the

significantly

secretion

When Statistical

in

± 11 ng.corpus

as 65

pg HDL depicted

progesterone

maintained

increased

the

0-1000

Results

that

135

luteal cells of obtained from

maintained for 3 h in HDL in various concen-

from

medium.

indicated was

Assays

by lutea

rats were containing

ranging of

tissue RIA

secretion 3 corpora

of HDL lutea, rats

without assayed

on the overall tissues obtained

were

incubated

500

pg

for

in

of

HDL

pregnenolone, testos-

1 7ct-hydroxyprogesterone,

400-

300-J

C) C

w

200-

C

0 I-

a, Co

a’ 0)

0

100-

I-

0I 0

I

65

I

125

I

250

h-HDL

500

(jiG

1000

Protein/ML)

FIG. 1. Effect of various doses of human high-density lipoprotein (h-HDL) on progesterone production by corpora lutea (CL) of pregnant rats.Three corpora lutea were isolated from Day 15 pregnant rats, cut in half, and incubated in the presence of HDL at 37#{176}C for 3 h. Progesterone was analyzed in the medium. Each point represents the mean ± SEM of values from 5 rats.

ESTRADIOL,

HDL

AND

LUTEAL

99

STEROIDOGENESIS

terone

and

indicate

estradiol.

Results

depicted

that in the presence

in

of HDL

Fig.

2

the overall

steroidogenic capacity of the luteal cell was enhanced. However, the percentage increase in

300

I-

1 7a-hydroxyprogesterone,

testosterone

and

es-

Ui

tradiol

was

less than the increase in This suggests that the

significantly

‘Ii

progesterone

production.

limiting

200

luteal

I

factor cells

enzymes terone

for is

the

involved

testosterone

production

by

activation

of the

in the conversion

of proges-

stage

to aromatizable

of

androgen.

ioo Effect of Estradiol and Testosterone Administration on Progesterone and Pregnenolone

Production

As depicted

In

Vivo

in Figs. 3 and

and

pregnenolone

and

FIG. 2. Effect of HDL on steroidogenesis by isolated corpora lutea (CL). Three corpora lutes were isolated from each day 15 pregnant rat and were incubated in the presence or absence of h-HDL (500 g protein/ ml) for 3 h. Steroids were analyzed in the media as described in Materials and Methods. Levels of steroids

cantly

reduced

in hypophysectomized-hysterec-

tomized ment

rats with

in the absence of HDL

circulation.

noo

(PREG),

7

±

1 ng/CL;

TEST

170-01W

ESTR

were as follows: pregnonolone progesterone

(PROG),

134

±

30 ng/CL; 17a-hydroxyprogesterone (l7cs-OHP), 160 testosterone (TEST), 70 ± 37 pg/CL; estradiol (ESTR), 86 ± 34 pg/CL. Values are mean ± SEM of observations obtained from 5-8 rats. pg/CL;

testosterone progesterone vivo,

corpora

that

progesterone

received

sustained

dramatically and pregnenolone In contrast lutea

of

only.

Treat-

levels peripheral

results obtained vehicle-treated

of

signifi-

estradiol

increased in the to

of

Vitro levels

were

vehicle levels

In

4, serum

or

of in rats

secreted as much progesterone and pregnenolone in vitro as the luteal tissue of rats treated with steroids.

60

10

E

I.

60

w z

50

UI5

40

z4

z 0 -J 0

0

w I-. 0)

E C

C

UI

UI

I

30

a-

20

UI 0)

10

C

ii V

4 E

T

FIG. 3. Effect of in vivo treatment of estradiol or testosterone on serum levelsof pregnenolone and progesterone. Rats were hypophysectomized and hysterectomized on Day 12 of pregnancy and treated from Day 12 through Day 15 with either vehicle (V), a 1-cm capsule filled with testosterone (7) or with 100 g of estradiol daily (F). Intact pregnant rats were used as controls (C). On Day 15 blood was collected and progesterone and pregnenolone were analyzed as described in Materials and Methods. Each bar represents the mean ± SEM. The number at the base of each bar refers to the number of determinations.

KHAN

100

ET

AL

150

140

-

120

2

12

-J

o

a

100

10

Ui

z

a

80 S

4

20

2

C

V

E

I

FIG. 4. Effect of in vivo treatment of estradiol or testosterone on subsequent in vitro secretion of progesterone or pregnenolone. Rats were treated as described in the legend of Fig. 3. On Day 15 corpora lutea (CL) were isolated and incubated for 3 h. Progesterone and pregnenolone were analyzed in the incubation media as described in Materials and Methods. Each bar represents mean ± SEM and the number at the base of each bar designates the number of animals.

Effect

of HDL

Lutea and

and

manner.

Corpora

previous

on Progesterone

Pregnenolone

Production

of Rats

by

Treated

with

HDL

Estradiol

determine

whether

luteal

estradiol

affects

HDL incorporation into luteal cells and to find out if the stimulatory effect of estradiol could be demonstrated in the presence of exogenous HDL, with

corpora lutea either steroids

obtained or vehicle

from were

rats treated incubated in

the presence of 500 pg of HDL protein/mi for 3 h. Addition of HDL to the medium had little effect on progesterone production by corpora lutea of rats that received

vehicle only (Fig. 5).

In contrast,

of HDL

the presence

increased progesterone lutea of steroid-treated whether

the reduced

by corpora to an capacity

dramatically

production animals.

production

To

by corpora determine

of progesterone

lutea of vehicle-treated

rats was due

accumulation of pregnenolone, the of luteal tissue to increase pregnenolone

output in the presence of HDL was determined. The data (Fig. 5) revealed that pregnenolone production was similarly increased in all groups in the presence

of HDL.

The production

on

the

present

findings

cholesterol in

increases a

dose-dependent

to

combined

with

of specific

luteal

plasma

bind

and

indicate luteal

that

HDL-

progesterone and

saturable

metabolize

our

receptors

membrane

for

(Gibori

et

of pregnant machinery

lipoprotein.

3-hydroxy-3-methylglutaryl

Although

coenzyme

A reduc-

tase activity is substantial in this tissue (Khan et al., 1983), corpora lutea failed to secrete progesterone This

maximally

indicates

that

synthesize

in luteal

cholesterol

in

the

absence

of

cells

of

pregnant

vitro

at

a rate

HDL. rats that

is

inadequate to support maximal steroidogenesis. In fact, not only is progesterone production enhanced

by

overall Corpora output

the

in

these

comparison to one production. previous

findings

activation conversion

Thus,

HDL

but

is

increased.

is

indicate

testosterone substrate

rats

and formation is dependent

substrate earlier

estradiol of the limited

observations

that

the

synthesis but also

of the enzymes of progesterone

androgenic our

steroids

the

in

3-fold increase in progesterThese results corroborate our which

(Sridaran estradiol

of pregnant

two

the

limiting step for only progesterone

of

of

pathway

lutea increase testosterone and by 50%. However, the magnitude

increase

of the

presence

steroidogenic

androgen contrast,

DISCUSSION carried

findings,

al., 1984), indicate that luteal cells rats are equipped with the necessary

Testosterone To

These

demonstration

rate-

is not the stage

responsible to aromatizable Gibori, by

1983). corpora

on the (Gibori

et that

for In lutea

availability al., activation

1982). of

ESTRADIOL,

HDL

LUTEAL

AND

500

STEROIDOGENESIS

101

PREGNENOLONE

-I

I

400 UI C)

z

UI 0) UI

300 UI

I

I-

z UI

20O UI

‘.2

z

.1i:i

100

V

E

I

V

E

T

FIG. 5. Effect of h-HDL on progesterone and pregnenolone production by corpora lutes of rats treated with estradiol or testosterone. Rats were treated as described in the legend to Fig. 3. On Day 15, three corpora lutea were isolated and incubated for 3 h in the presence or absence of h-HDL (500 g protein/mi). Progesterone and pregnenolone were measured in the incubation media. Values are mean ± SEM with the number of animals at the base of each bar.

the enzymes progesterone chorionic increases

involved in to testosterone

gonadotropin the capacity

testosterone respectively,

conversion of with human

the

(hCG) of luteal

and estradiol in media devoid

administration cells to produce

10and of HDL

200-fold, (Sridaran

treatment

of

either

estradiol

or

and Greenwald, 1973; Rodway 1982). The results indicate that

and HDL tion

Gibori, 1983), whereas incubation with has only a limited effect on the producof both steroids (Fig. 2). These results sug-

testosterone not only secretion

gest nous

that although source of

ences vehicle-

found in vivo, or steroid-treated

corpora rats

specific

levels

of

and

remains

incubated of luteal

the availability cholesterol is

steroidogenesis, enzymes

the

an important Since cholesterol

activation

the

in

synthesis,

as

pathway

in steroid

biosynthesis.

cells derived

of pregnant rats also utilize from HDL for steroid

reported

for

serum gonadotropin-hCG-treated al., 1981; Azhar and Menon, 1982; Schular et al., 1981), determine whether estradiol tion of cholesterol from were obtained hypophysectomized Day

12.

tase et

cal

These

enzyme(s) al., doses

from

of

lutes and

highly

mare’s

rats (Azhar et 1981; Bruot et al.,

Day 15 pregnant and hysterectomized

corpora

estradiol,

pregnant

it was of interest to affects the utilizaHDL. Corpora lutea

(Elbaum

are

1981),

of some

steroidogenic

step luteal

of an exogeimportant for

are Keyes,

sensitive and

respond

rats on

progesterone in the cells

luteal

and Garris, estrogen or

lutea produce

of

pregnenolone

of HDL. vehicle-treated

The

either similar when

capacity rats to

as luteal cells be related to sterol (Gibori

of its et

al., 1984), which may be capable of supporting steroidogenesis in lipoprotein-deficient media. Thus, progesterone production in the absence of HDL capacity

may not of luteal

of different commencement

the

between of

to the steroidogenic due to the presence

of steroid of

production by others Terranova

be related cells but

amounts

discrepancies

vivo

absence from

secrete as much progesterone steroid-treated animals may higher storage of preformed

1976; Azhar to physiologiin

in

(Gibori et Takayama

administered to these rats increases progesterone but also pregnenolone in vivo. However, despite the differ-

rich in aroma-

to

aromatizable

androgen with a dramatic increase weight and progesterone production al., 1977; Gibori and Keyes, 1978;

have and

at the Similar

the in vivo and

progesterone

(Takayama and Greenwald,

precursor

experiment.

been

Greenwald, 1981). Corpora

in vitro reported 1973; lutea

KHAN

102 which

cease

the

end

to

treatment

were

terone in very active

found

progesterone

in vivo

or after LH

antiserum

to produce

lipoprotein-deficient corpora lutea

yams and Greenwald, tained lutes

secrete

of pregnancy

Greenwald, 1981). In

progesterone

administered suggesting

of

one

production

by

or utilization cells, delivery The HDL

of

and for

of

affecting

the

thus allowing steroidogenesis.

vehicle-

HDL

does not seem apolipoprotein

substrate

and Hesse, proposed HDL

to require degradation

1980). Winkel that cholesterol particle

depending movement

into

et the

on the force of cholesterol

cell

from How-

complete (Gwynne

al. may

(1980) move

have from

vice

and

versa

action. Net cell may be

of mass into the

possible only in cells which actively catabolize cholesterol. Based on the in vivo results, it can be envisioned that corpora lutea of rats treated with steroids are increasing in size and actively producing progesterone, and thus ously metabolizing cholesterol for ogenesis and membrane biosynthesis.

are both

continusteroidThus, an

increase in HDL-cholesterol incorporation occur. However, the finding that corpora of vehicle-treated rats secrete as much terone

in vitro

as corpora

rats strongly cholesterol-HDL to

an

increased

measuring [Iiiodo-HDL corpora (Gibori

lutea

lutea et al.,

increase is not solely

number in the

catabolism.

By

of binding sites plasma membrane

of pregnant rats, we 1984) provided direct

that estradiol increases receptors. Thus, estradiol

the number appears to

steroidogenesis

a

through

in due

the

cholesterol

the

may lutes proges-

of steroid-treated

suggests that incorporation

of of

previously evidence of HDL stimulate

mechanism

gives rise to an increase in the number receptors in the plasma membrane.

steroidogenic the pregnant receptors

precursors. The direct correlation activity

rat previously

and

present between

(Gibori

et

triples progesterin the presence the may

effect

but

and et al.,

on

may

also

since estradiol in luteal cells

affect

Both

hormones

on

in the

luteal

Hwang both

et al., 1981; Strauss appear to promote

cell

receptors

(Gibori

et al.,

(Azhar et production.

progesterone

nisms

of

totally

action

of

different.

by cyclic dent of

AMP,

the

maintains

al.,

two

LH

and increase the mechahormones

action

adenylyl

cyclase

responsiveness

its

from

et al., 1983). It remains whether both hormones act

adenylyl

to induce/activate for steroidogenesis. the corpora

cholesterol

steroidogenesis estradiol may sis,

on

at least

cyclase to be detervia different

the

proteins

results lutea

of this investigation of pregnant rats can

from

HDL

and stimulate in part,

to

1983); howis indepen-

(Sridaran mined

utilize

effect

al.,

dent

In summary, indicate that

are

is mediated

the effect of estrogen is indepencyclase system. Luteal estradiol

LH in luteal cells (Sridaran et ever, its action on steroidogenesis

messengers responsible

and of

and Boyd, the incorlipoprotein-

1981), However,

these

While

1984;

et al., 1982) the conversion

cholesterol to pregnenolone (Leaver 1981; Toaff et al., 1983), accelerate poration and the metabolism of cholesterol

function.

lipoprotein

membrane

rats

the effect of LH

luteal

increase cell

choles-

pregnant

that

of to

also depletes of hypophysec-

to compare

estradiol

lipoprotein The effect solely due

hysterectomized 1984).

of

action of be largely

further luteal by

affecting

tion of cholesterol from thus possibly allowing delivery for steroidogenesis.

for

maximal

indicate that cell steroidogene-

HDL into increased

the

incorporathe

cell and substrate

ACKNOWLEDGMENTS

study the

of the corpus luteum the levels of lipoprotein reported

its

with

which of HDL In this

fashion, the supply of cholesterol to the rat luteal cells is optimized to meet the demand for steroidogenic suggests a

by

uptake

tomized (Gibori

the

and

cholesterol delivery. not, however, be

It is interesting

into

increased

and may

terol utilization cholesterol ester

incorporation

from

for

receptors estradiol lipoprotein

of HDL, progester-

estradiol in vivo

in vitro suggests that on luteal cell function

accounted

and ob-

mechanism of cholesterol uptake is not yet completely understood.

ever, it lysosomal

the

those

cholesterol

(Taka-

corpora markedly

the presence stimulates

1984). The fact that one production both of HDL estrogen

than

Terranova to results

media, produced

than

animals in that estradiol

proges-

medium pregnancy

1973; contrast

in lipoprotein-deficient of steroid-treated rats

more

more

at

ET AL.

We are grateful to Dr. G. D. Niswender, Colorado State University, for the progesterone antiserum, to Rosemary Ciepper and Linda Alaniz for animal care, and to Suzanne Glass for the preparation of the manuscript. The expert editorial assistance of Linda Glaser is also gratefully appreciated. REFERENCES

of Azhar, al.,

S. and Menon, K.M.J. (1981). Receptor-mediated gonadotropin action in the ovary. Rat luteal

ESTRADIOL,

HDL

AND

LUTEAL

cells preferentially utilize and are acutely dependent upon the plasma-lipoprotein supplied sterols in gonadotropin stimulated steroid production. J. Biol. Chem. 256:6548-6555. Azhar, S., Menon, M. and Menon, K.M.J. (1981). Receptor-mediated gonadotropin action in the ovary. Demonstration of acute dependence of rat luteal cells on exogenously supplied steroid

precursor (sterols) for steroidogenesis. Biochim.

gonadotropin-induced Biophys. Acta

Gibori,

Endocrinology 99:573-579. G. and Keyes, P. L. (1978). Role of intraluteal estrogen in the regulation of the rat corpus luteum

during

pregnancy.

Endocrinology

102:

1176-1182. Gibori, G. and Richards, J. S. (1978). Dissociation of two distinct luteotropic effect of prolactin: regulation of LH receptor content and progesterone secretion during pregnancy. Endocrinology 102:767-774. Gibori, G., Antczak, E. and Rothchild, I. (1977). The

role of estrogen terone secretion

in the regulation in the rat after

of luteal

proges-

pregnancy. Endocrinology 100:1483-1495. Gibori, G., Richards, J. S. and Keyes, P. L. (1979). Synergistic effects of prolactin and estradiol in the luteotropic process in the pregnant rat:

regulation of estradiol Biol. Reprod. 21:419-423. Gibori,

G.,

Control

Sridaran,

of

R.

receptor and

aromatase

day

12

by

Basuray,

activity

of

prolactin. R.

in

(1982).

luteal

and

ovarian non-luteal tissues of pregnant rats. Endocrinology 111:781-788. Gibori, G., Chen, Y.-D.I., Khan, I. and Reaven, G. M. (1984). Control of luteal cell lipoprotein receptors and steroidogenesis by estradiol in the pregnant rat. Endocrinology

114:609-617.

Gwynne, J. T. and Hesse, B. (1980). The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis. J. Biol. Chem. 255:10875-10883. Havel, R. J., Eder, H. A. and Bragdon, J. H. (1955). The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34:1345-1353. Hwang,

J.,

Rajendran,

K.M.J. (1981). tein receptors pins.

Endocrinology

K.,

Johnston,

S.

and

Annual

Menon,

Regulation of luteal cell lipoproand steroidogenesis by gonadotro108:A518.

Khan, I.,Chen, Y.-D.I. and Gibori, G. (1983). Regulation of cholesterol metabolizing enzymes and cholesterol synthesis by estradiol in the corpus luteum of pregnant rat. Program of the 65th

Meeting

of

The

Endocrine

Society,

San Antonio, TX, (Abstr.). Kovanen, P. T., Goldstein, J. L. and Brown, M. S. (1978). High levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol synthesis in the ovary of pregnant rabbit. J. Biol. Chem. 235:5126-5132. Leaver, H. A. and Boyd, G. S. (1981). Control of

steroidogenesis in rat corpus luteum: the rate of access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme. J. Endo-

665:

362-3 75. Belanger, A., Caron, S. and Picard, V. (1979). Simultaneous radioimmunoassay of progestins, androgens and estrogens in rat testis. J. Steroid Biochem. 13:185-190. Bruot, B. C., Wiest, W. G. and Collins, D. C. (1982). Effect of low density and high density lipoproteins on progesterone secretion by dispersed corpora luteal cells from rats treated with aminopyrazolo-(3,4-d) pyrimidine. Endocrinology 110: 1572-1578. Elbaum, D. J. and Keyes, P. L. (1976). Synthesis of 1 7j3-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum.

103

STEROIDOGENESIS

crinol. Morishige,

91:123-133. W. K., Pepe,

G. J. and Rothchild, 1. (1973). Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinology 92:1527-1530.

Noble,

R.

plasma Ochiai,

P.

(1968).

lipoproteins

9:693-700. A. and

Electrophoretic

in agarose

Rothchild,

separation

gel. J. Lipid

1. (1981).

The

of Res.

relation

number and the lueotropic effect of estrogen in rats after hypophysectomy and hysterectomy on day 12 of pregnancy. Endocrinology 109:1111-1116. G. J. and Rothchild, 1. (1972). The effect between

conceptus

of hypophysectomy on day 12 of pregnancy on the serum progesterone level and time of parturition in the rat. Endocrinology 91:1380-1385. Radding, C. M. and Steinberg, D. (1960). Studies on the synthesis and secretion of serum lipoproteins by rat liver slices. J. Clin. Invest. 39:1560-1569. Reaven, L. P., Chen, Y-D.l., Spicher, M. and Azhar, S. Pepe,

(1984). Morphological evidence that high density lipoproteins are not internalized by steroids producing cells during in situ organ perfusion. J. Clin. Invest. 74:1384-1397. Rodway, R. G. and Garris, D. R. (1982). Potentiation by prolactin of the luteotrophic effect of estradiol in the pregnant rat. Acta Endocrinol. 101:287292. Rodway, R. G. and Rothchild, I. (1980). Effect of estradioi on steroid dehydrogenase activities in

rat corpora Schuler, L. A.,

lutea. J. Endocrinol. Langenberg, K. K.,

84:391-395.

Gwynne, J. T. and High density lipoproluteal cells. Biochim.

Strauss, J. F., III. (1981). tein utilization by dispersed Biophys. Acta 664: 583-601. Sridaran, R. and Gibori, G. (1983). Intraovarian localization of luteinizing hormone/human chorionic gonadotropin stimulation of testosterone and estradiol synthesis in the pregnant rat. Endocrinology 112:1770-1776. Sridaran, R., Hunzicker-Dunn, M. and Gibori, G. (1983). Testosterone stimulation of progesterone synthesis by gonadotropin desensitized corpora lutea. Endocrinology 112:610-617. Strauss, J. F., III, MacGregor, L. C. and Gwynne, J. T. (1982). Uptake of high density lipoproteins by rat ovaries in vivo and dispersed ovarian cells in vitro. Direct correlation of high density lipoprotein uptake with steroidogenic activity. J. Steroid Biochem. 16:525-531. Takayarna, M. and Greenwald, G. S. (1973). Direct luteotropic action of estrogen in the hypophysectomized-hysterectomized rat. Endocrinology 92: 1405-1413. Taya, K. and Greenwald, G. S. (1981). In vivo and in

KHAN

104 vitro

ovarian

steroidogenesis

Biol.

Reprod.

25:683-691.

Terranova,

P. F. and

Greenwald,

in the

pregnant

rat.

Endocrinol.

G. 5. (1981).

Acute

90:19-30.

M. E., Strauss,

(1983). immature

effect of an antiserum to luteinizing hormone on ovarian steroidogenesis in the pregnant rat. J. Toaff,

ET AL

J. F., III and

Hammond,

J. M.

Regulation

of cytochrome

450scc

in

porcine

granulosa cells by FSH and estradiol. Endocrinology 112:1156-1158. Winkel, C. A., Gilmore, J., MacDonald, P. C. and Simpson, E. R. (1980). Uptake and degradation

of lipoproteins culture.

primary

by human trophoblastic cells Endocrinology 107:1892-1898.

in