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