BIOLOGY
OF
REPRODUCTION
30,
Identification
564-572
(1984)
of a Porcine
Follicular
Follicular
0.
RAY
PATRICK
KLING,4
C.
ROBERT
ROCHE,3
M.
Fluid
Response
JOSEPH
D.
NAKAMURA
Livingston
and
Reproductive
Department
of
of
of
Los
Pathology3
School
California
Oklahoma
and
Health
City,
of Medicine
90033
Gynecology
Oklahoma
NISHIMURA,5
Gynecology
California
of
of
KAZUNORI
Laboratory
and
Experimental
Angeles,
Department
University
Suppresses
S. diZEREGA2
Biology
Southern
Which
CAMPEAU,
GERE
Obstetrics
Department
University
Fraction
to Gonadotropins1
Obstetrics4
Sciences
Oklahoma
Center
73190
and
The
Second
Yamagata
Department
of
University
SurgeryS
Medical
Yamagata,
School
Japan
ABSTRACT
To
evaluate
the
saturated
ammonium
0.025
M Tris/HC!,
role
of
nonsteroidal,
sulfate
follicular
fluid
proteins
in folliculogenesis,
the
10-55%
of pooled
porcine
follicular
fluid (PFF)
was dialyzed
against
pH 7.5,
using
10,000
molecular
weight
exclusion
membranes,
then passed
through
agarose-immobilized
textile
dye. Activity
was determined
by test fraction
inhibition
of
human menopausal
gonadotropin
(hMG) [2 U human luteinizing
hormone
(LH)/follicle-stimulating
hormone
(FSH)
per dayl induced
ovarian
weight
and serum
estradiol
increase
in hypophysectomized,
diethylstilbestrol
(DES)-treated,
25-day-old
female rats. Specific
inhibition
(84 ± 7.4%) of
ovarian
weight
increase
was found
in the material
(5 ml) eluted from the orange A column
with
KCI (1.5
M, pH 6.8).
Inhibitory
activity
of the orange
A-bound
material
which
eluted
through
a
standardized
Sephadex
G-100
column
corresponded
to a molecular
weight
of 12,000-30,000.
Isoelectric
focusing
(IEF)
on a Sephadex
G-15
support
bed of orange
A-bound
material
demonstrated
inhibitory
activity
at pH 3.7-4.0.
Serial dilutions
of active material
from IEF preparations
demonstrated
a dose-response
relationship
in the bioassay.
No demonstrable
activity
was found in
similar fractions
eluted
through
a Concanavalin
A-Sepharose
4B column
with or without
addition
of alpha-methyl
mannoside
(2 M, pH 7). When active fractions
were heated
(56#{176}C,
1 h) or exposed
to trypsin
(10 mg%),
activity
was lost. When aliquots
of the saturated
ammonium
sulfated
precipitated,
dialyzed,
orange
A-bound,
Sephadex
G-100
(Ve/Vo
1.3-1.7)
eluent
were
separated
by
high-performance
liquid chromatography
(HPLC)
using gel exclusion
columns,
activity
in the bioassay
was recovered
in the
Although
confirmatory
may be an important
inter-
fraction
18,000-35,000
molecular
weight
range.
data await further
studies,
it is tempting
and/or
intraovarian
regulator
of follicular
INTRODUCTION
The
ovulate
follicles
fact
that
are
derived
drawn,
ovarian
from
in turn,
liferating
follicles
a cohort
from
a pool
destined
of
fetal
to
primordial
development,
mental
growing
mammals
of nonpro-
successive
Accepted
November
14, 1983.
Received
June
14, 1983.
Supported
in part
by NIH
Clinical
Investigator
Award
HD-00401
and a grant
from
the University
Southern
California
Faculty
Research
and Innovation
Reprint
requests:
Hospital,
Rm.
L1013,
les,
CA
Gere S. diZerega,
1240
N. Mission
that this protein(s)
to gonadotropins.
follicles
is perhaps
similarity
of
(diZerega
reduction
follicles
during
characteristic
formed
the
of
Hodgen,
90033.
564
fundaamong
1981).
potentially
This
ovulable
each
ovarian
cycle
to a number
of
the
species
implies
some
presence
secreted
the
human
ovarian
venous
drainage
ovary
containing
the
preovulatory
which
suppressed
follicular
response
MD, Women’s
Rd., Los Ange-
during
most
folliculogenesis
and
process
of selection.
Recently,
we reported
the
heatand trypsin-labile
substance
of
fund.
2
to speculate
response
of a
into
from
the
follicle
to gonado-
OVARIAN
tropins
(diZerega
not
secreted
ovaries
inhibitory
et al.,
in
large
as evidenced
activity
1982).
This
amounts
by
INHIBITORY
protein
was
detect
from
and
hyperstimulated
(diZerega
et a!., 1983a,b)
media
of human
granulosa
et al.,
1983b).
follicular
human
similar
gel
filtration
points,
as
Sephadex
molecular
using forward
flow.
molecular
fractions
weights,
and
by
MATERIALS
ampholyte
Since
response
to
in a monotocous
similar
species
containing
this
gonadotropins
species,
activity
like the
AND
from
serum
as determined
chromatography,
determined
tioned
whether
in a polytocous
cycles
might
pig.
by
isoelectric
displaceinhibitor
of
was
we
ques-
also
occur
METHODS
Preparation
ceptive
Development
Branch,
National
Institute
of
Child
Health
and
Human
Development,
was
slowly
thawed
and fractionated
by drop-wise
addition
of an
equal
volume
of saturated
ammonium
sulfate
(SAS)
during
persistent
agitation
at 4#{176}C.Following
a 12-h
incubation
at 4#{176}C,the
precipitate
was pelleted,
the
supernatant
discarded,
and
the
pellet
resuspended
(2:1,
vol:vol)
with
10% SAS.
An additional
12 h of
agitation
was followed
by centrifugation
at 3000
X g
for
30 mm.
The
resulting
supernatant
was dialyzed
(Visking
dialysis
tubing,
10,000
molecular
weight
exclusion)
against
3 changes
of 0.025
M Tris/HCI,
pH
7.5 (buffer
A, 3000:
1), for 16 h. Insoluble
material
was
removed
from
the
retentate
by centrifugation
(3000
X g; 30 mm).
further
Ligand
Elution
(1.6
X
A. All
of the manufacturer,
and
buffer.
Elution
profiles
ISCO
absorbance
meter
estimation
of molecular
same
Sephadex
G-100
(2.6
X 70 cm, Vt’#{176}280,
and
developed
with
ribonuclease-A,
chymo-
of the PFF
(50 mg in 10 ml buffer
A) were
passed
through
a Concanavalin-A
linked
Sepharose
4B (Con
A) column
(5 ml, Pharamacia
Fine
Chemicals,
Piscataway,
NJ) which
was washed
with
5 volumes
at 0.5 M
NaCI,
0.05
M Tris/HCI
pH 7.4,
then
further
eluted
with
2 M alpha-methyl-D-mannoside
in buffer
A at a
flow
rate of 20 mI/h,
Both
Con A-bound
and unbound
fractions
were
assessed
for
activity
in the bioassay.
Chromatography
fractions
containing
inhibitory
activity
were
heated
(56#{176}C for
30 mm)
or trypsin
digested
(10
mg%)
for
3 h and
then
retested
for
activity
in the bioassay.
iso electric
A series
of agarose
(triazine
ring)
immobilized
textile
dyes
(Dyematrex
Screening
Kit,
Amicon,
Lexington,
MA)
were
prepared
according
to
the
manufacturer’s
instructions
(Fulton,
1980).
Columns
(9 X 32 mm,
2 ml bed volume)
containing
matrex
gel
blue A (triozinyl
dye cibacron
blue
3GA),
red A
(procion
red HE3B),
blue
B (cibracron
brilliant
blue
FBR-P),
orange
A, or green
A were equilibrated
with
20 mM Tris/HCI,
pH 7.5, and then charged
with 10-mi
aliquots
of the dialyzed
retentate
(75 mg). Unbound
material
was eluted
with
10 ml of buffer
A. The
bound
protein
was eluted
with
1.5 M KCI added
to
buffer
A.
Eluent
fractions
were dialyzed
overnight
against buffer A, and protein
concentration
determined
(Lowryetal.,
1951).
column
buffer
trypsin,
and
ovalbumin
in buffer
A at 10 mI/h
4#{176}C.
Fractions
were
assessed
for bioactivity
in the bioassay.
Kay for each standard
and active
fraction
was calculated
using
Vt280,
Vo90
(KavVe-Vo/VtVO).
Additional
aliquots
from
the 10-55%
SAS fraction
Pooled
porcine
follicular
fluid
(PFF;
600
ml),
kindly
provided
by Dr. Gabriel
Bailey of the Contra-
Dye
G-100
4#{176}C)with
A-bound
in 10 ml
weight
seiving
was performed
Sephadex
G-100
was
prepared
according
to the instructions
equilibrated
in the
elution
were
determined
using
an
(Lincoln,
NE) at 280 nm. For
weight
by gel filtration,
the
column
used
for purification
Vo=90
ml)
was
equilibrated
molecular
weight
standards
the spent
(diZerega
activity
extracted
and ovarian
vein
chromatography.
fo!licular
identified
Sample
protein
inhibitory
follicular
fluid
had
ment
The
ovarian
and from
cell cultures
containing
active material
(orange
were further
separated
(45 mg
buffer
A, 4#{176}C)on a Sephadex
50 cm,
Vo=60
ml, 5 mI/h,
the
contralatera!
ovary.
Similar
biological
activity
was
recovered
from
pooled
human
follicular
fluid
aspirated
during
spontaneously
occurring
565
Chromatography
Eluents
fractions)
anovulatory
by our inability
to
in the venous
drainage
PROTEINS
Orange
Focusing
A-bound
purified
fractions
by isoelectric
containing
focusing
activity
using
were
a Sepha-
dex
G-15
support
matrix
as previously
described
(O’Brien
et al., 1976).
The apparatus
consisted
of a 4
X 30 cm water-jacketed
glass column
containing
a 2.5
X 20 cm C-iS
Sephadex
bed supported
by a 2.5 X 8
cm Teflon
elution
plug under
a 25-mm
Millipore
filter.
The column
was previously
equilibrated
with two-bed
volumes
of a solution
containing
8% carrier
ampholytes
(4% of pH 3-10
and 4% of the pH 2-4;
Pharmacia)
in
12.5%
glycerol.
Cytochrome
c was used
as an internal
marker
protein
(p1=10.5).
The fraction
(50 mg)
was
then
washed
into
the
column
with
20 ml of the
ampholyte-glycerol
solution.
A second
Millipore
filter
was placed
on the top of the Sephadex
bed.
A 10-mI
polyacrylamide
solution
of 14% acrylamide,
0.3% Bis,
and 0.07%
ammonium
persulfate
(polymerized
by the
addition
of 50 I TEMED)
was then
poured
over the
filter.
Upon
completion
of polymerization
(approx.
20
mm),
the
column
was
inverted,
the
Teflon
plug
removed,
and
a second
acrylamide
plug
layered
over
the bottom
filter.
After
polymerization
of the bottom
plug,
the column
was returned
to its upright
position
and lowered
into
anode
buffer
containing
1% sulfuric
acid.
The
remaining
upper
portion
of the
column
was
filled
with
1% ethanolamine.
The
column
was
cooled
by recirculating
water
at 1-4#{176}C throughout
the procedure.
Isoelectric
focusing
was initiated
at 800
constant
volts (16 mA)
and
allowed
to proceed
to
equilibrium
as monitored
by an eventual
decline in the
milliamperageto
2.5 mA (approx.
8-12
h). Thereafter,
column
fractions
(3 ml) were dialyzed
against buffer A
to remove
ainpholines
prior
to bioassay.
KLING
566
AL.
of the specific
Bioassay
Twenty-three-day-old
(45-55
g) were received
Chicago,
IL
Sprague-Dawley
from Hormone
female
rats
Assay Labs.,
after
hypophysectomy,
and were
intervals
of 14L: 1OD (Goldenberg
et al., 1972;
Louvet
et al., 1975;
Zeliznik
et al., 1979).
Animals
were
caged
in groups
of three
and given
rat
chow
and
water
ad lib. Silastic
implants
containing
kept
2 days
at 25#{176}Cwith
diethylstilbesterol
(DES) were prepared
as follows:
10
g Silastic polymer
(elastomer
brand 382, Dow Corning
Co., Midland,
MI) were mixed
with 3 g DES (Sigma
Chemical
Co., St. Louis,
MO) for 30 mm at room
temperature;
thereafter,
4 drops of stannous
octoate
(Catalyst
M; Dow Corning)
catalyst
were added with
an additional
10 mm of mixing.
The material
was
passed
steaming
through
a Luer-lock
syringe
(56#{176}
C) 0.9%
NaCI
water
for 2 h. DES-containing
Silastic
were
inserted
subcutaneouly
incision
48 h before
assay.
each
dose
sectomy,
of unknown.
animals
gonal;
Serono
dissolved
in
in
Assay
(i,d,
bath
1 mm)
into a
and
anealed
implants
(1 X 5 mm)
the hypophysectomy
design
was 3 rats
Forty-eight
h after
at
hypophy-
were
given
2 lU LH
Labs.,
Inc.,Boston,
MA;
buffer
A with
1%
two
and FSH (Per1:1,
LH:FSH)
bovine
serum
albu-
divided
daily doses.
The
hypophysectomized,
immature,
DES-treated,
female
rat challenged
with LH/FSH
is referred
to here
asHIFR-hMC.Twenty-fourh
after the initial injection,
animals
were
sacrificed
by decapitation,
ovaries
removed,
trimmed,
and weighed
on a Roller-Smith
balance.
Rat trunk
serum
estradiol-17j3
concentrations were determined
by methods
previously
described
(Kling and Westfahl,
1978).
Control
determinations
(no injected
test fractions)
for unstimulated
ovarian
weight
were
34.7
± 3.2
mg
min
ET
and/or
test
fractions
in
per
rat and
192.0
±
30.5
mg per
rat for LH/FSH
stimulated.
Control
levels
of trunk
serum
estradiol
were
12.5
pg/mI
±
0.7 pg/mI
for unstimulated,
and
118.5
± 21
pg/mI
for LH/FSH
stimulated
rats.
Where
indicated,
100%
inhibition
equals
ovarian
weight
and/or
serum
estradiol
concentration
of mean
unstimulated
control
values.
Zero
percent
inhibition
equals
ovarian
weight
or serum
estradiol
concentration
of
LH/FSH-stimulated
control
rats.
These
results
are
similar
to those
which
have
been
obtained
with
previous
applications
of this
bioassay
procedure
(Coldenberg
et al., 1972; Louvet et al., 1975; Zeliznik
et al.,
1979;
diZerega
analysis
was performed
Kletzky
et al., 1975),
statistical
significance
test
and
Duncan’s
1968).
High.Performance
Protein
et al.,
1982,
1983a,b).
Data
using
rankit
plots
(Bliss,
1967;
and,
where
indicated,
tests
of
were
performed
by Student’s
multiple
range
analysis
(Kirk,
Liquid
separation
fluid
and bovine
serum
Aromatase
Activity
albumin
proteins
were performed
weight
chromatography
chymotrypsin,
ovalbumin
(Pharmacia).
Replicate
0 1-mi portions
of each rat granulosa
cell
(5 X 10 cells) were pipetted into 17 X 100
mm
polystyrene
tubes
(Falcon
5052;
Falcon
Plastics,
Los Angeles,
CA).
Greater
than
80% of the cells were
viable
prior
to assay as determined
by exclusion
of 1%
trypan
blue. Androstenedione,
the referent aromatase
substrate,
was
added
in 0.1
ml medium
A [final
concentration,
1.0 X 10
M (Armstrong
and Papkoff,
1973; Dorrington
et al., 1975; Erickson and Hsueh,
preparation
1978a)].
All incubations
were performed
in triplicate
for 3 h at 37#{176}C
in a shaking
water bath (120 cycles/
mm),
The reaction
was stopped
by transferring
the
tubes to an iced water bath before centrifugation
for 5
mm at 1000
X g. The supernatants
were decanted
and
stored
at -20#{176}C until the measurements
of estradiol
and estrone
were performed
by radioimmunoassay
(RIA),
as described
previously
(Kling
and Westfahl,
1978).
Control
incubations
(no
androstenedione
added)
were processed
in the same way. Blank estrogen values obtained
for the controls
were subtracted
from the corresponding
values for incubations
in the
presence
expressed
of androstenedione.
as estrogen
production
activity was
granulosa
Aromatase
(ng/viable
cell).
RESULTS
Table
1 summarizes
the
matrix
chromatography
PFF
fraction.
Although
13%
of
the
charged
results
of the
orange
protein
of
dye-ligand
10-55%
A bound
(1.2
Chromato
was
graphic
performed
using
Procedures
a Waters
chromatograph
equipped
3000
SW (Varian
Assoc.,
column.
A 100-gil
(2 mg)
orange
A
dye-matrix
eluent
HPLC
run.
The proteins
were
eluted
from
the TSK
column
using
an isocratic
gradient of buffer A at a flow of 0.5 mI/mm,
350 psi. The
protein
peaks
were
detected
by absorbance
at 280 nm
with
a Waters
variable
wavelength
detector
(Model
M-450,
Waters
Assoc.)
and molecular
weight
estimates
SAS
only
mg/ml
equiva-
lent of whole
PFF),
1.5 M KC1 elution
recovered
bioactive
material
which
contained
the greatest
inhibition
of hMG-induced
rat ovarian
weight
response
(1
7.4%).
A-bound
mg
injected/rat;
Dose-response
fraction
of 400
weight
P<0.05;
84
±
studies
of the
orange
indicated
that
an ID50
dose
pg inhibited
by 50%. When
the hMG-induced
these
same fractions
ovarian
were
treated
with
heat
(56#{176}C)or trypsin
(10 mg%),
no suppression
in ovarian
weight
was found.
Figure
1
depicts
the
chromatographic
elution
profile
of
PFF
at
280
nm
developed
through
Sephadex
G-100
following
55%
SAS
cut,
dialysis,
and elution
ALC/GPC
Model
244 liquid
with a 0.75
X 50 cm TSK
Palo Alto,
CA) gel exclusion
aliquot
of the dialyzed,
was separated
on each
follicular
using
highly
purified
molecular
standards
of ribonucleas-A,
A-bound
peak
can
thereafter
with
be
at
seen
M KC1.
followed
second
biological
HIFR-hMG
1.5
a Ve/Vo
trailing,
ascending
When
material
of
plateau
at Ve/Vo
was
(2.5
assessed
mg injected/rat),
inhibition
of both
serum
estradio!-17j3
ovarian
weight
levels
were found
of
mg/rn!
1.42-1,55
Dose-response
(0.36
studies
equivalents
using
An
1.1
by
activity
bioassay
the
10of orange
this
initial
to
1.17,
a
gradual
1.7
to
1.9.
in
the
95%
and
trunk
at Ve/Vo
of PFF).
fraction
OVARIAN
TABLE
1. Protein
mg of the 10-55%
recovery
saturated
and inhibition
ammonium
INHIBITORY
of ovarian
sulfate-dialyzed
567
PROTEINS
weight
response
from
porcine
follicular
dye-matrix
fluid
fraction.
chromatography
Unbound
from
75
Bound
Protein
Dye-matrix
(mg)
(%)
Inhibition
(%)
(mg)
(%)
Inhibition
(%)
Control
Blue A
Blue B
RedA
Orange
A
Green
A
70.5
26.0
60.7
24.7
66.0
27.7
94
34
81
33
88
37
78 ± 6.1
22 ± 8.3
0
12±4.2
0
0
4.5
48.7
15.0
48.7
9.7
47.2
8.8
65.0
20.0
65.0
13.0
63.0
0
0
39
0
84
22
aSignificandy
to
other
greater
dye-ligand
indicated
an
inhibition
eluents
ID50
(P<0.05
as determined
of
58 pg
Student’s
by
for
50%
1-mg
of hMG-induced
t)
doses
of each
(IEF)
and
with
developed
saline
(PBS),
the
Kay
taining
weight
molecular
with
pH
0.02
7.4,
weight
for
(log
Vo-Ve/Ve-Vo)
inhibitory
activity.
of
be between
inhibitory
12,000
of
activity
and
estimation
estimated
from
were
pH
7.4
±
7,4a
±
3.8
compared
(0.14
isoelectric
rat
within
mg/rn!
for
response
the
orange
bioassay
the
activity
in
0.5
was
(0.5
range
of
PFF)
3.7
con-
of both
rat ovarian
serum
estradiol-1
7$3
respectively).
PFF
fractions
A dye-matrix
from
relationship
extraction
equivalent
inhibition
and trunk
recovered
focusing
PFF
by
fractions
the
the
IEF
with
column
bioassay.
apparent
1.5
were
A
(Fig.
dose3) with
B E2
E
0
the
A-bound
evaluated
only
from
KC1
tested
to
daltons.
response
(83.6
± 9.4%
and 47.2%,
dilutions
of the extracted
eluted
M
weight
orange
obvious
response
levels
Serial
of
fractions
conThe
molecular
was
30,000
4.0
tained
weight
5 M phosphate-buffered
allowing
the
2)
mg/rat),
standards
ovarian
eluents
of
(Fig.
to
rat
±
fraction.
When
inhibition
of hMG
induced
increase
in rat ovarian
weight.
To
guard
against
nonspecific
effects
of the
eluents,
we only
considered
active
those
fractions
with
both
serum
estradiol
and
ovarian
weight
inhibition.
The
same
column
was then
equilibrated
Protein
0 OVARIAN
WEIGHT
0.4
cJ
w
()
z
0.3
100
0.2
E
80
0
60
C’)
40
0.1
20
I
1.0
I
I
I
1.25
1.5
1.75
2.0
I
2.25
I
I
2.5
2.75
mol.
wt.)
3.0
I
3.25
ILl
o
I
3.5
Ve/Vo
FIG.
1. Chromatographic
elution
profile
of
dialyzed
fate fraction
of pooled
porcine
follicular
fluid
(PFF)
raphy.
Maximum
inhibition
of LH/FSH-stimulated
bars)
in immature,
hypophysectomized,
DES-treated
ing to a molecular
weight
of 12,000-30,000
daltons
which
ovarian
rats
(mean
(>10,000
10-55%
saturated
ammonium
sul-
bound
to orange
A after
Sephadex
G-010
chromatogweight
(open
bars)
and trunk
estradiol
levels
(closed
by eluent
fractions
occurred
at 1.5 Ve/Vo
correspond± SEM
of triplicate
determinations,
5 rats/assay).
KLING
568
T
ET AL.
0.5
1000
0.4
80
0
C’1
0.3
60k,.
Ui
I
I
2
a.
0.2
40
0.1
20
z
10
20
30
ISOELECTRIC
40
FOCUSING
50
ELUENT
60
70
mI/fraction)
(1
FIG.
2. Isoelectric
focusing
chromatogram
fate fraction
of pooled
PFF
(5 ml) after
ovarian
weight
inhibition
(hatched
bars)
was found
in the 3.7-4.0
pH range
(mean
of dialyzed
(>10,000
mol. wt.)
10-55%
saturated
ammonium
sulelution
(1.5 M KCI) from
an orange
A dye-matrix
column,
Maximum
in immature,
hypophysectomized,
DES-treated
rats by eluent
fractions
± SEM
of rats/fraction).
fractions
(1D50=22
7.2
in
pg/m!),
the
3.5-4.0
while
other
pH
pH
range
ranges
from
the
column
were without
inhibitory
activity.
When
aliquots
from
the ammonium
sulfate
10-55%
cut eluted
from
the
orange
A dyematrix
were passed
through
concanavalin-linked
Sepharose
4B (eluted
3 x Vo with
by 2 M alpha-methyl
activity
in the
rat
noted
with
in the Con
mannoside)
of
activity
weight
orange
after
100
in
(i.e.,
inhibition
of
material
eluted
the
eluted
recovery
A-bound
separation
column
extracted
through
in the
PFF
material
the Sephadex
(Ve/Vo=1.3-1.7).
This
chroma-
is representative
of a highly
reproducible
procedure
facilitated
by the automated
HPLC
(N=15).
The
HPLC
eluent
was
divided
into
5 fractions
based
on peak
absorbances
retention
time
25.0-27.8
mm; 2: 27.8-3
mm;
3:
3 1.0-36.0
mm; 4: 36.0-39.0
mm;
39.0-43.0
mm). These fractions
corresponded
to
the
following
molecular
>100,000-74,000;
absorbances
lated
to
highly
4:
The
after
molecular
reproducible
ranges:
5:
1:
3:
18,000-12,000;
retention
times
and
5:
of peak
HPLC
elution,
when
weight
standards,
(5
(1:
1.0
74,000-36,000;
2:
36,000-18,000;
12,000-5,800.
weight
of
(2
G-
tograrn
elution
runs).
HPLC
bioassay
correwere
fractions
(0.2
were
mg/rat;
Fig.
tested
in
the
inhibition
5),
of
ovarian
weight
gain was evident
in rats injected
with fraction
3 (0.11
mg/rn!
equivalent
of PFF;
74
± 8%,
P<0.001)
and
fraction
4 (0.008
mg/mi
equivalent
of PFF;
58 ± 9% P<0.005)
as
compared
to the
other
3 fractions
(1, 2, 5).
Dose-response
studies
using
these
fractions
indicated
ID50 of 18 pg and 13 pg, respectively,
for 50% inhibition
of the hMG-induced
increase
in rat ovarian
weight.
Inhibition
of androstenedione
ovarian
PBS fraction
(data
not shown).
4 depicts
the HPLC
elution
profile
Figure
the
mg)
(20-30%
followed
no inhibitory
mg/rat)
was
A-bound
fraction
with
only
marginal
response)
unbound
mannoside),
bioassay
(3.5
PBS
When
±
IEF
conversion
estrogen
to
(Fig.
by
6)
total
from
the
HPLC
fraction-treated
(n=6
HPLC
ovaries/l-IPLC
fraction
3
fraction)
(11.1
±
no
test
cells/rn!).
When
into
the
aromatase
HIFR-hMG
assay,
44
of total
cells/ml
Table
procedure
in rat
variety
was
3
pg
fractions
on dose-response
inhibition
of
ovarian
of
folliculogenesis
fluid
including
granulosa
harvested
rat’s
ovaries
evident
per
were
in
10,000
injected
bioassay
rat prior
to
± 8 pg (range
of 30-60
the
pg)
cell
the
ID50s
of each
hMG-induced
weight.
DISCUSSI
A
cells
immunoreactive
estrogen
per
10,000
was present
in the control
incubates.
2 summarizes
the partial
purification
and
resultant
yields
and
recovered
activities
based
active
fraction’s
increase
immunoreactive
granulosa
ON
nonsteroidal
have been
inhibitors
maturation
regulators
of
identified
in follicular
and
stimulators
of
and
luteinization
OVARIAN
INHIBITORY
PROTEINS
569
WEGT
0V**N
100
80
(::
6o
z
z
40
w
<0
>
Ow
o_
#{149}
0
I
60
20
ISOELECTRIC
FOCUSING
(g
FIG.
3. Dose-response
relationship
weight
augmentation
bioassay
recovered
tion
(1.5 M KCI) and dialysis
(>10,000
sis of ovarian
weights
from
dilutions
(ID50 =26 ± 7.2 Mg/mI PFF equivalent).
(Ledwitz-Rigby
tion
FSH
et
inhibitor
receptor
al.,
(Tsafrir
binding
Abou-Issa,
1977;
1977),
oocyte
and
Reichert,
and
and
1978;
statin,
by
independently
bovine
1976;
al,,
from
follicular
Welschen
1977;
regulating
LH,
has
FSH
been
fluid
(Dejong
et a!., 1977),
PFF
Marder
et
al.,
1977;
discussed
1981;
reported
in
and
Sharpe,
(Welschen
et
and
(Franchimont
et al., 1982).
The biophysical
characteristics
of
extracts
described
here are different
mentioned
above,
Data
derived
protocols
employed
suggest
that
the
follicular
from
those
from
the
the
active
material
tography
elutes
with
pH
3.7-4.0
40,000,
does
through
gel permeation
chromaa molecular
weight
of 15,000
to
not
thermo-labile.
secretion
Schwartz
elsewhere
Channing
FRACTION
bind
to
an apparent
isoelectric
and 4.0, binds
to orange
Channing,
1977)
and
human
follicular
fluid
(Chari
et al., 1981),
as well as in the culture
medium
of rat granulosa
cells
(Erickson
and
Hsueh,
1978b).
These
substances
have
been
reviewed
and
and
Channing,
2
injected/rat)
matura-
In addition,
a widely
studied
to as inhibin
or folliculo-
acts
6
PFF
fractions
which
contained
inhibitory
activity
in the rat ovarian
from
isoelectric
focusing
(pH 3.7-4.0)
after
orange
A dye-matrix
elumol. wt.) of 10-55%
saturated
ammonium
sulfate,
insert
is rankit
analytested,
the 0 intercept
of which
was plotted
on the dose-response
curve
Sato
et al., 1982).
substance
referred
which
I
of
and Channing,
1975),
inhibitor
(Reichert
Darga
I
I
The
concanavalin-A,
studies
reported
rule
out the possibility
that
of the fractionation
procedures,
itself,
may
have
inhibitory
activity
fractions
activity
were
has
fo!licular
media
(diZerega
effluent
et a!.,
addition
variety
secretory
contribute
fluid
human
et
from
al.,
the
of
A similar
et
a!.,
observations
nonsteroidal
products
of
to the regulation
of human
spent
cell
cultures
ovarian
ovary
venous
(diZerega
suggest
steroida!
compounds,
the
biological
1983a,b),
granulosa
intrafollicular
cannot
for
the
all active
in extracts
1983b)
and
preovulatory
These
to the
here
3.7
and
a toxic
byproduct
or the bioassay
responsible
however,
reproted
(diZerega
from
1982).
been
observed;
thermolabile.
been
has
point
between
pH
A, and is trypsin-
that,
in
milieu,
a
perhaps
granulosa
of folliculogenesis.
cells,
570
KLING
ET AL.
What
might
substance
the pig?
activity
po!ytocous
occurrence
and
the
number
U,
is
of
this
proteinaceous
of folliculogenesis
in
with similar
inhibitory
both
monotocous
and
ovulatory
well
granulosa
berg
et al.,
and
Hellbaum,
cell
FSH
a!.,
1972)
and
1955),
LH
1979;
enhance
1976;
et
a!.,
1975;
et
Armstrong,
1980),
Hillier,
a!., 1975;
La
=
FIG.
4. High-performance
liquid-gel
permeation
chromatogram
of the orange
A-bound
fraction
of 1055% saturated
ammonium
sulfate
of PFF
recovered
from
Sephadex
G-100
elution
(Ve/Vo
1.3-1.7,
2 mg).
Retention
times
corresponded
to the following
molecular weight
ranges:
25-27.8
mm, >100,000-74,000;
27.8-31
mm,
74,000-36,000;
31-36
mm,
36,00018,000;
36-39
mm,
18,000-12,000;
and 39-43
mm,
12,000-5800.
a!.,
that
1981),
Erickson
Erickson
1978;
McNatty
aromatization
of follicular
aromatization
maturation
we
found
and
of
(Hillier
others
and Hsueh,
1976)
24
by
h
Hsueh,
new aromaand resultant
(Leung
and
et
al.,
1980;
(Dorrington
1978a;
Moon
et a!., 1979)
have
may
be a central
et
et
suggested
regulator
maturation,
and that
inhibition
of
would
suppress
continued
follicle
to ovulatory
status,
In this study,
a reduction
in the rats
treatment
extracted
obtained
RAT
Hillier
and
et
a!.,
secretion
phase
of
1978b)
sufficient
for induction
tase
activity
(Wang
et al., 1982)
estrogen
production.
Armstrong
U-
Payne
granulosa
Goldenberg
(Richards
induction
of estrogen
cells requires
a lag
(Dorrington
estrogens
(Golden-
Hillier,
and
et a!.,
and
is not
that
proliferation
Ross
(Richards
between
the
protein(s)
follicles
known
cell
1972;
binding.
The
rat granulosa
co
role
of
It
stimulate
‘0
the
species,
a correlation
of this fo!!icular
inhibitory
apparent.
‘0
be
in the regulation
Since
proteins
are present
in
used
with
PFF.
from
in serum
estradiol
levels
in the bioassay
after
concurrent
exogenous
gonadotropins
and
Furthermore,
granulosa
cells
the
bioassay
rats
demonstrated
a
BIOASSAY
60
z
o
AROMATASE
ASSAY
E
0
I
50
40
z
La
C.)
30
0>
z
0.
20
0
0
1
2
HPLC
3
FRACTION
PORCINE
CCo
La
4
OF
FOLLICULAR
weight
augmentation
hypophysectomized,
(mean
DES-treated
FLUID
±
SEM)
rats
nn
1
EXTRACTED
2
HPLC
FIG. 5. Bioassay
results of high-performance
liquid
chromatography
(HPLC)
fraction
of orange
A-bound
PFF
after separation
by Sephadex
G-100 chromatography (Ve/Vo
1.3-1.7),
Inhibition
of LH/FSH-induced
ovarian
ture,
10
in imma(n#{176}6/
fraction
tested)
was measured
after
treatment
with
2
ml of HPLC
eluents
which
corresponded
to the following
molecular
weights:
1, >100,000-74,000;
2,
74,000-36,000;
3,
36,000-18,000;
4,
18,00012,000;
and 5, 12,000-5800.
PORCINE
FIG.
derived
treated
6.
3
FRACTION
Aromatase
from
immature,
rats (n=6/fraction
4
5
OF
EXTRACTED
FOLLICULAR
FLUID
activity
of
rat
granulosa
hypophysectomized,
tested)
which received
cells
DESLH/
FSH
stimulation
and
injection
of HPLC
fractions
(2
ml) of extracted
PFF
(mean
± SEM).
HPLC
fractions
corresponded
to the following
molecular
weights:
1,
>100,000-74,000;
2, 74,000-36,000;
3, 36,00018,000;
4, 18,000-12,000;
and 5, 12,000-5800.
Control
(no
HPLC
fraction)
44.7
±
8 pg estrogen
per
10,000
cells/mI.
OVARIAN
INHIBITORY
PROTEINS
marked
Co
C)
.‘
.
.
C
Co
reduction
vitro.
other
Co
-
.
,*In
InN
,-CN
C
N
-
C
en
0
‘0
‘0
‘0’0
.0
activity
remains
of
C)
different
E
Co
C)
confirmatory
U
C
tempting
C)
0
‘-C
C)
-C
bE
C.
0
cC-
C)
C
aromatase
activity
in
in follicle
selection
or
can be ascribed
to this
to be determined
be
an
biological
phases
fluids
through
serial
of
data
to
speculate
Although
further
that
this
inter-
of
during
folliculogenesis.
await
important
regulator
collected
studies,
and/or
follicular
it is
protein(s)
may
intraovarian
response
to
gonado-
tropins.
Co
C
0
ACKNOWLEDGMENTS
Co
C)
CO
0
‘-C
th
their
any role
function
studies
N
Co
C)
C-
of
Whether
biological
CrC
-
.c
‘0
571
N
N
00
In
C
ON
C’
-
-
*
CO.
“‘0
Co
C)
Co
0
E
C
We would
like
to thank
Dr. Griff
Ross
for his
timely
suggestions
during
the course
of this study,
Ms.
JoAnn
Brown
for her excellent
technical
assistance,
and Ms. Kay Moon
for her diligent
preparation
of the
manuscript.
C)
‘0
REFERENCES
C)
N
Armstrong,
0
crC
of
U
C)
androgens
CO
-
bE
be
C
bE
0.
Co
bE
0.
N
N
CO
In
bEbS
t
COon
-
-
.0
C.
‘-C-..
C)
Co
Co
C)
.
.
-
CrC
C.
C.
N
-
CrC
Cfl’0
*
Ifl5..
0
‘O
C)
C
‘C-.
C
C)
C)
Co
0
-C
U
C’
0
4-
C
U,;
C)
C’
0
C.
C
0
0
Co
,0
000
‘0
*
.0
-C
C
,-CCO
on
0
Cn”l.t-.
C)
-O
V
Co
‘0
C)
0
C)
C)
C
0
Co
4..
V
Co
Co
4-
E
0
C
‘O
C)
N
0
CO
U
CO
CO
00
,
.0
C.
0
Co
C
0
‘0’
#{149}O
C’
Co
U
Co,
Co
4-
C’
0
0
‘C)
.9 Z
II
IrCb
InC
“0
CI
‘CCo
0
.0
(*
4-
en
‘C-
0
-
.
C)
bE
Co
E-’o
U,;
C)11
‘
bECo
e->
0
C
00
OO
00
C
0
-
I
vivo
crinology
C-
‘O
‘0
0
in
0
.0
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H. (1976),
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Stimulation
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in the control
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15 7-166.
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N. C. and Reichert,
L. E. (1978).
Some
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of
the
interaction
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stimulating
hormone
with
bovine
granulosa
cells and its
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by
follicular
fluid.
Biol.
Reprod.
19: 23 5-241.
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R. M. (1976).
Evidence
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diZerega,
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Identification
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secreted
by
the preovulatory
ovary which suppress
follicular
response
to gonadotropins.
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R. P., Campeau,
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human
follicular
fluid
which
suppress
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response
to gonadotropins.
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G. S., Marrs,
R. P., Campeau,
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of protein(s)
which
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response
to gonadotropins.
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572
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and
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