Effect of Luteinizing Hormone Deprivation In Situ on Steroidogenesis ...

18 downloads 36 Views 3MB Size Report
inhibit. LH production were incubated with a saturating concentration. (2 jim) of pregnenolone. Leydig cells from control and. T-E-implanted rats produced. 537.
BIOLOGY

OF REPRODUCTION

36,

Effect

GARY

769-783

(1987)

of Luteinizing Hormone Deprivation In Situ on Steroidogenesis of Rat Leydig Cells Purified by a Multistep Procedure1’2

R. KLINEFELTER,3’5

PETER

Division Department

of Reproductive of Population

School

of Hygiene

The Johns

and

and

LARRY

L. EWING5

Biology5 Dynamics Public

Hopkins

Baltimore,

The

F. HALL,4’6

Health

University

Maryland

21205

and Worcester Foundation for Experimental Shrewsbury, Massachusetts 01545

Biology6

ABSTRACT Depriving

rats

diminishes physectomized adopted in the Percoll

their

of

a multistep procedure Leydig cell. Our method gradient centrifugation.

trifugation

contains

structural

characteristics

cells

luteinizing

when

hormone

(LH)

P450 C17-hydroxylase/C1720-lyase rats prevents these changes

95%

incubated

Leydig

in Leydig

well-preserved 3 h with

These

and

without

to lose

smooth

dehydrogenase

Leydig

cells

produced

a maximally

and

stimulating

29

tively, from

suggesting T-E-implanted

enzyme, protein of

LH

immunoblots from control withdrawal

diminished lyase content.

capacity

of one-dimensional and 4- and 12-day continues. to

convert

These

reactions antibodies

to

sodium dodecyl T-E implanted results

pregnenolone

show to

and

to hypo1983). We

converting the P450

Leydig

testosterone

cells and

from

with 06

of ovine

LH.

and cenultra-

Leydig Purified

(T-E) implants for 4 days to of pregnenolone. Leydig cells Leydig cells3 h, respec-

pregnenolone to testosterone C17 -hydroxylase/C17 20 -lyase

sulfate polyacrylamide rats revealed a continued that

cells

ng of testosterone/i

concentration

cells obtained from control rats and rats treated with testosterone-estradiol LH production were incubated with a saturating concentration (2 jim) control and T-E-implanted rats produced 537 and 200 ng of testosterone/106 in the steroidogenic By using rabbit

reticulum

LH administration (Ewing and Zirkin,

(3-HSD)-staining

248

Leydig inhibit from

a defect rats.

endoplasmic

et al., 1984). and function

to study the trophic effects of LH on steroidogenesis enzymatic dissociation, centrifugal elutriation, fraction obtained after Percoll density-gradient

3!3-hydroxysteroid cells.

cells

activity (Wing cell structure

of rat Leydig cell isolation employs vascular perfusion, The purified Leydig cell

of Leydig for

causes

gels of Leydig loss of enzyme animals

reduced

P450

in Leydig cells pig microsomal cell microsomal as the period

deprived

of

LH

had

C17-hydroxylase/C1720-

INTRODUCTION

Changes in levels, reflecting

Accepted October 7, 1986. Received May 19, 1986. ‘This research was supported in part by NIH Grant HD-07204, The Population Center (Grant HD-06268), an EPA cooperative agreement (CR81-2765), an NSF equipment grant, and a Mellon Foundation Postdoctoral Fellowship for Gary Klinefelter. 2Although the research described in this article has been funded

puberty, in Leydig

circulating varying

breeding season, cell morphology

luteinizing physiological and and

aging, induce in the cell’s

synthesize and secrete testosterone 1970; Neaves, 1973; Pahnke et al.,

in part by the U.S. Environmental Protection Agency through cooperative agreement (CR81-2765) to the Division of Reproductive Biology at Johns Hopkins University, it has not been subjected to the agency’s peer and policy review, and therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred. Reprint requests. Present address: Department of Medicine, Prince of Wales Hospital, Randwick, N.S.W. 2031 Australia.

1978). Similar experimental concentration example, physectomy 769

trophic alteration

hormone states,

(LH) such as changes ability to

(Knorr et a!., 1975; Pirke et al.,

effects are demonstrated of endogenous serum

by LH

(O’Shaughnessy and Payne, 1982). For withdrawal of endogenous LH by hypo(Morat, 1977) or by implantation of

770

KLINEFELTER

testosterone-estradiol (Ewing the

(T-E)

et a!.,

volume

1977)

causes

of smooth

Silastic

a coincident

Leydig testis

cell and to secrete

it

shown

that

capsules reduction

endoplasmic

in the perfused was

filled

reticulum

the ability testosterone.

restoration

of

of

in (SER)

causes

centrifugation. With our approach, obtain a preparation that is 95% Leydig 3a-hydroxysteroid and light microscopy.

the in vitroSubsequently,

LH

ET AL.

a co-

Leydig

cells

produce

testosterone cells in the

of LH is to regulate the ylase/C17,20-lyase enzyme

activity contained

Ewing, 10-fold

1979) increase

et al.,

earlier

to

Leydig

1984),

confirming

observations

obtained

for only desirable

LH-directed processes over synthesis and/or degradation lated molecule, for example, to eliminate precursor(s) highly

enriched

Various

Leydig

cell

single-step

cells from dissociation

To ascertain rates of of a specific LH-reguit would be advantageous

uptake cells

of by

radiolabeled providing

other testicular have been

cell

fraction

contaminating

and sperm. More isolating rat Leydig utilized centrifugal zimide centrifugation We were described findings, reported ness of (Bordy

isolating

Leydig

that tion,

al.,

approach

cells from In this multistep

cells,

significant

numbers

condensed

spermatids,

of

recently, a two-step method of cells has been reported, which elutriation followed by metri(Aquilano and Dufau, 1984).

1984),

prompted to

the

adult rat testes. paper we report isolation scheme

us

to

purification our success to isolate rat

employs vascular perfusion, centrifugal elutriation,

and

antiserum neonatal

to this pig testes

Male

cells following enzymatic described (Janszen et al.,

unable to attain the Leydig cell enrichment for the centrifugal elutriation step. These coupled with the great disparity in the testosterone production and LH responsiveLeydig cells isolated by various methods et

another

blood

isolated

40%

numbers rat testis

of

the

of Leydig (Chubb and

approximately production in response

by our

method

from

a

rats

microsomal by Nakajan

enzyme and Hall

AND

METHODS

rats

weighing

purified (1981).

in in an from

Animals

of

containing

red

similar

and demonstrate in testosterone

MATERIALS

1976; van Beurden et al., 1976; Conn et al., 1977; Payne et al., 1980; Browning et a!., 1981; Dehejia et al., 1982; Gale et al., 1982; Cooke et a!., 1983). However, in our hands, these methods resulted in a Leydig

approximately

produced by in vitro-perfused

cells

Leydig cells The isolated

a

preparation.

methods

that these integrity.

1984). Finally, we have demonstrated a decrease the amount of P450 C17-hydroxylase/C17,20-lyase purified Leydig cells after LH withdrawal using

a few hours, and to follow specific,

time.

nonspecific by contaminating

LH.

(313-HSD) staining dye exclusion and

implanted with (T-E) capsules for 4 days show a marked decrease in their ability to convert pregnenolone to testosterone, similar to results obtained with the in vitro-perfused rat testis (Wing et al.,

with Leydig cells from hypophysectomized rats (Purvis et al., 1973). A highly purified and viable preparation of Leydig cells is required to study these trophic effects of LH on Leydig cell steroidogenesis. The in vitro-perfused testis remains viable ultimately it would be

blue

electron microscopy indicates have maintained morphological

incident temporal recovery of SER and the ability of the testis to secrete testosterone (Ewing et al., 1983). Finally, it was shown that one of the trophic effects of P450 C17-hydroxin the SER (Wing

dehydrogenase Trypan

we routinely cells based on

enzymatic Percoll

adopt of

Sprague-Dawley

275-300

g

were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). The rats were housed four to six per cage under controlled temperature (22#{176} C) and light (14L: 1OD) conditions and were given food and water

ad libitum.

Luteinizing For

Hormone some

Withdrawal

experiments,

testosterone

(T)

and

estradiol (E) were administered via s.c. Silastic implants. Details of the fabrication of these steroid implants have been described previously (Ewing et a!., 1977). Briefly, a 2.5-cm T and a 0.3-cm E implant were placed s.c. into the interscapular region of anesthetized, Implants

remained

adult,

to Leydig

cell

male in the rats

Sprague-Dawley for 4 or 12 days

rats. prior

isolation.

yet Leydig

in using a Leydig cells dissociagradient

Chemicals N-2-Hydroxyethyl piperazine-N’-2 ethansulfonic acid (HEPES, H-3375), soybean trypsin inhibitor (STI, T-9003), ethylenediamine-tetraacetic acid (EDTA, ED255), heparin (H-3125), DNase (D-4527), Nitro Blue Tetrazolium (NBT, N-6876), etio-

STEROIDOGENESIS (E-5 251),

cholan-33-ol-1

7-one

dinucleotide

(j3-NAD+,

sulfonyl from

fluoride Sigma

f3-Nicotinamide

N-7004),

(PMSF,

Chemical

(St.

199 (M-199, 400-1100), Solution (HBSS, 310-4185), phate-buffered purchased NY). chased

serum

from

albumin

NP-40

Tetroxide Research

(5323) were Corp. (Rockville,

melting

phoresis

were

(Richmond,

purchased MD).

point.

CA).

I’25-Protein Dr. Joel

while After

M-199

without

and All

shaking at dissociation,

M-199

was

ferous

tubule

90

added mass

doubled,

100-jim

MULTISTEP

PROCEDURE

cycles/mm 30 ml to

Ovine

LH

Institute Kidney

ylase/C17,20-lyase

was

kindly

at 34#{176}C.After

at 34#{176}C for 10 of coilagenase-free flask,

removed

nylon

with 0.50 mg/mi were dissociated

and

Cooper deter-

mesh.

The

(oLH-4)

prepared anti-pig

FOR

RAT

LEYDIG

provided

CELL

Collagenase

Testicular Artery

Extravascular Collagenase

for

Dissociation

to electro-

Laboratories was

a gift

Diabetes, (Bethesda,

D

from

Dr.

Dissociated

Cell

Suspension

(-)

RBCs

and MD).

provided C17-hydroxby

PURIFICATION

via the

by

Peter

Centrifugal

E-1 Sperm Condensed Spermatids Round Spermatids Endothelial Cells

Small Germ Cells

Elutriation

E-2 Leydig

Cell

was

Perfuse Testes with

Hall. Leydig

filtrate

from

used

and P450

through

Calbio-

recrystalized

Bio-Rad

semini-

by filtration

pur-

purchased were

the

mm.

NY).

from

of Arthritis, Diseases

A was kindly Shaper. Rabbit

each

was

Percoll (17-0891Inc. (Piscataway,

reagents

from

collagenase

an equal volume of M-199 was added, and the testes

from Tousimis Pregnenolone and

were NY)

purchased

the National Digestive and

of

771

CELLS

resin (51002) and Cacodylic Acid purchased from Ernest F. Fullam Glutaraldehyde (10575) and Osmium

(Pawling,

constant

was

(Orangeburg,

purchased

20-hydroxycholesterol Steraloids

were Island,

was obtained from PA). The nonionic was

White were NY).

Salts phos-

802249)

(La Jolla, CA). from Pharmacia,

NJ). LR (50040) (Latham,

Medium-

450-1300) (Grand

Mann

(492015)

MO).

ml

10 mm, collagenase

purchased

Balanced Dulbecco’s

(BSA,

(CLSII, 4176) Inc. (Malvern,

chem-Behring Co. 01) was purchased

Louis, Hanks’ and

LEYDIG

15

adenine

phenylmethylwere

(DPBS, Laboratories

Schwarz

Collagenase Biomedical, gent,

saline Gibco

from

Bovine

and

P7626)

Co.,

BY PURIFIED

Cells

Macrophages

Isolation

Spermatocytes Multinucleated Pachytene

Interstitial

cell

preparation.

cervical dislocation and the removed and placed on ice 0.71 g/L sodium bicarbonate

Rats

were

killed

by

containing 0.1% BSA and 25 rng/L STI, pH 7.4. The multiple steps used to isolate Leydig cells are shown diagramatically in Figure 1. To remove red blood cells and increase the number of purified Leydig cells obtained, fat, the testicular micropipette, and

each testis was artery cannulated the vasculature

trimmed free of with a glass of each testis

flushed with 0.3 ml of M-199, buffered as above, but containing 1 mg/rn! collagenase. The testes were placed in fresh, ice-cold M-199 without collagenase until 20 testes were collected. They were then decapsulated incubated

and 6 to in a 250-ml

Germ

testes were immediately in M-199 buffered with and 2.21 g/L HEPES

7 decapsulated tissue culture

flask

testes were containing

Cells

Centrifugation

p-i Pachytene Spermatocytes Multinucleated

Through

Percoll

Germ Cells Macrophages P-2

Viable Leydig

Cells

FIG. 1. A schematic flow diagram of the sequence of steps used to purify rat Leydig cells. D = cell suspension following collagenase dissociation; E-i = a 300-mI fraction of cells collected with an elutriator rotor speed of 2000 rpm and a 16 or 20 mI/mm countercurrent buffer flow; E-2 = a 100-mI fraction of cells collected from the elutriation chamber after decreasing the rotor speed to 0; P-i a lighter-than1.068 g/ml fraction of E-2 cells recovered from a continuous, linear Percoll gradient formed from 60% Percoll during centrifugation for 1 h at 20,000 ga,; and P.2 = a 1.068 g/ml-and-heavier-fraction of E-2 cells recovered after Percoll gradient centrifugation.

772

KLINEFELTER

centrifuged ture. Centrifugal Fig.

1)

at 250

elutriation.

60-cc

The

resuspended

consisting

containing heparin,

10 mm

at room

of

0.5% 25 mg/L syringe

dissociated in

M-199,

60

cells

ml

of

buffered

used

to

load

cells into (Masterfiex;

the

(see

as above,

of

buffer

in

the

chamber

approximately elutriation 900-197,

bubble

trap

of

elutriator

the

but

50,000 U/L I; pH 7.4. A 3.0

X

system. A Cole Parmer,

Chicago, IL), set to pull buffer at a flow rate mi/mm, aspirated the cells from the syringe. from the syringe were diluted into approximately separation

D,

elutriation

BSA, 1 mM EDTA, STI, and 12 mg/L DNase

was

108 dissociated peristaltic pump

ml

fixed

tempera-

before

entering rotor

of 6 Cells 25

Inc., Palo Alto, rpm was maintained. the buffer flow

while a When the rate was in-

creased

each

a flow

2-3

ml/min

was

speed,

all

mi/mm, established. sperm,

minute, At

any

this

until flow

remaining

rate red

of 20

and blood

rotor cells,

cells are Once a fraction-i, decreased

to 0, those cells that had been retained in the chamber previously were washed out by the buffer still flowing at a rate of 20 mi/mm. When 100 ml were collected (eiutriation fraction-2, E-2), the cells in both fractions were pelieted by centrifugation (250 X g, 10 mm) at temperature.

To elutriate

cells

from

rats

treated

with

T-E

Silastic

implants for 4 and 12 days, the elutriation flow rate was decreased from 20 mi/mm to 16 mI/mm to prevent losing Leydig cells due to decrease in their size resulting from LH withdrawal (Ewing et al., 1983). All other conditions were the same as for control rats. Percoll centrifugation. osmotic by dilution

after

1984).

elutriation Mg+-free

Percoll 11: 1; v/v of

Ca++, Mg++free The Leydig (E-2) HBSS

was Percoll

made with

isolOX

HBSS (Vincent and cell fraction obtained

was resuspended buffered with

in 0.35

14 g/L

ml of sodium

bicarbonate and containing 0.25% BSA and 25 mg/L STI, pH 7.4. The suspension was thoroughly mixed with 21 ml of iso-osmotic Percoll. A similar 60% Percoll solution was prepared that contained only 1.062 g/ml (17-0459-01, cells.

The

(JA-20,

and 1.075 Pharmacia 2 Percoll

g/rnl Inc.,

solutions

density Piscataway, were

marker NJ) centrifuged

Beckman)

types

at 20,000

become

g,

partitioned

for

due

to

the various buoyant densities while a continuous, linear density gradient is generated. After centrifugation, the gradient was divided into a lighter-than1.068 g/ml fraction (Percoll fraction-i, P-i) consisting of germ cells, macrophages and damaged Leydig cells, and a 1.068 g/ml-and-heavier-fraction 2, P-2). Each fraction was diluted free HBSS to remove the Percoll 0.10).

production relative without LH (E-1; results suggest that The rotor

fraction after

pachytene and celis

to cells incubated Table 2). Taken Leydig cells in E-l

(E-2) of cells retained elutriation contained

spermatocytes,

313-HSD control

testes,

cells,

19 and 21% from control

of the and for

(E-2; Table 1). The Leydig cells in E-2 and 4-day T-E-implanted testes produced

from 124

respectively,

ng of T, respectively, with LH (E-2; Table

T production 2) for Leydig Leydig (E-2) were

when maximally 2). The stimulation

by LH was 2.6- and cells in this fraction

T-E-implanted

testes,

3.3-fold from

(E-2; control

of

Table and

respectively.

cells in purified

centrifugation.

The

lighter-than-1.068

production

by

g/ml

Leydig

cells

purified

fraction

from

Fig.

1)

obtained

of

the

cells

in

this

fraction

gradient

produced

very

little

(-)

D E-1 E-2 P-i P-2

43 13 47

LH

±

1i

±

3.6 13 10 8C

±

10 ±

29± aValues b100

represent ng/ml

ovine

or

are significant

ng/106

Leydig

cells-

68 * 22 15 ± 2.3 124 ± 10 10 ± 10 248±5oy

1.6 1.1 2.6 1 9

±

SEM,

the

Percoll

T in the

Percoll when

gradients, obtained

contained from control

rats

and

rats

of

implanted

(P-2; type from

again

or in

suggesting

(P-2; Fig. i), region of the

95 and 91% and from

Leydig cells T-E-treated

Table 1). No predominant was present in Leydig cell control rats (Fig. 4A). The

3a-HSD-positive

with

absence

cells

testosterone-estradiol

in

(T-E)

a field

Silastic

of

capsules

LH/(-)

LH

(-)

LH

17 ± 4 ± 17 ± 6 ± 806d

(+)

1.4 0.6 4.3 2.8

LH

(+)

27 ± 1.6 5 ± 1.0 56 ± 10.7 10 ± 10 9l*18Z

LH/(-)

LH

1.6 1 3.3 2.6 11.0

N=3.

LH. in unstiniulated

(p