antz. SG, Tanabee. K, 1982. The role of nonsteroidal regulators in control of oocyte and follicular maturation. Recent. Prog. Horm. Res 38:331-408. Channing.
BIOLOGY
OF
REPRODUCTION
Purification,
(1989)
40,407-415
Measurement,
from
and Tissue
Low-Molecular
Weight
M. SLUSS,
of a Dansyl-Derivatized
Follicle-Stimulating
Fractions PATRICK
Distribution
of Porcine
ANDREW
and
LEO
Hormone-Inhibitor-Containing
Follicular
J.
A. BRANCA,
JOE
Fluid’
FORD,
E. REICHERT,
Department
Glycopeptide
KRISHNASWAMY
A. KRISHNAN,
JR.
of Biochemistry
Albany
Medical
Albany,
College
York
New
12208
ABSTRACT We tives
have
used
weight
(MW)
was
tissue even served
found
of
in follicular
of follicle-stimulating
detection of the nature of the dansyl porcine
from
chromatography
was from
(5-dimethylamino-1-naphthalenesulfonyl
compounds
purified
(thin-layer that D15
choloride
inhibitor
lowed sensitive hydrophobic tions
dansyl
of amine-containing
purified follicular adult
follicular
fluid.
and
high
rats,
lesser
amounts
were
Based
cells
(241
ng/mg)
was
highest
estrous cycle. in fluid from MW FSH-BI gonadal that
this
(15.6
tig/mI)
particularly
factor
is a local
on
in fluid
luteinizing modulator
in kidney
and
tissues
nonsteroidal, the
actions
presumably
1982;
1982;
Sluss
been
from
tubules
small
follicles
cell
of FSH
al.,
hormones
to
action
et 1987).
a!., These
that in vivo
1982; factors
using the
and
62
In porcine
inhibit and
Grady
in
1981; and et
appear
types.
cows
On
different
systems
factor
(D15)
ng/mg,
respectively)
and
of D15 as much
ovarian
were D15
tissue,
during
the
basis
further
the
luteal
phase
No D15 was data indicate concentrations
of these
data,
the
obwas
granulosa
contained at least 4-fold of D15 material were also MW FSH-BI. The concentration
from
althe
for quantification of was found in ovarian
High concentrations approximately twice
ng/mg).
deserves
four derivatized
was developed ng/mg) of D15 (93
approach
higher deterof of
the
detected that low exist in
hypothesis
study.
be relatively hydrophilic molecules and have been partially purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Efforts to purify gonadal factors have been hampered by their low
contain
1981; Jagiello and Vogel, Channing, 1981; Channing
Channing et
factors
(136
obtained
hormone-responsive
shown
criteria
tissues
This
derivamolecular
By taking advantage of in low MW FSH-Blprepara-
the highest concentration of low MW FSH-BI. pregnant cows or from cystic follicles. These after dansylation and suggest that high local
(antagonist)
peptide,
of gonadotropic
vitro (Hammond, Franchimont and Segal,
have
liver
a low
(FSH-BI).
chromatography), assay (153
dansyl
containing
receptor
91 ng/mg, respectively) Relative concentrations shown to contain low
INTRODUCTION
Gonadal
to
chromatographic liquid
as in seminiferous
These follicles also contained large or small follicles from can be quantified by TLC
tissues,
binding
to form
fractions
fluorescent properties. (RF = 0.15) identified
(5 and 0.5 ng/mg, respectively). from immature rats in which
cells from large follicles and corpora lutea (69 and concentrations than follicle wall tissue (14 ng/ml). mined in pools of bovine follicular fluid previously D15
purified
A direct, chemical highest concentration
observed
and heart tissues extracts of testes
(FSH)
on their derivative
performance
appeared to be homogeneous. fluid or tissue extracts. The
less in diaphram in derivatized in Leydig
based a dansyl
choloride)
or highly
hormone
derivatives group,
[TLC]
fluid
concentrations in gonadal extracts or follicular fluid and low yields by the methods employed. In addition, detection of such factors by available in vitro or in vivo biological assays consumes large quantities of
al., to
material. We have
used
dansyl
nathpthalaenesulfonyl derivatives of
Accepted September 15, 1988. Received June 20, 1988. This study was supported by NIH Grant HD-1 9302 (P.M. S.), USDA Cooperative Agreement-58-3244-3-612 (L. E. R.), and a Grant from the Mellon Foundation (L. E. R.). 2 Reprint requests: University of Rochester School of Medicine, Urology Department, Box 656, 601 Elmwood Avenue, Rochester, NY 14642.
purification.
The
by adsorption the hydrophobic additional 407
chloride
chloride) amine-containing resultant
of this
to form fluorescent compounds before
derivatives
chromatography, nature of
advantage
(5-dimethylamino-1-
can
be purified
taking advantage the dansyl group. approach
is that
extreme-
of An
SLUSS
408 ly
sensitive
because
detection of
their
methodology, highly purified stimulating
of
the
fluorescence we identified low molecular
hormone
derivatives
is possible
Purification
properties.
Using
from
a dansyl weight
this
derivative of (MW) follicle-
receptor-binding
inhibitor
(F SH-
B!) by its migration position (RF = 0.15) on Silica G thin-layer chromatography (TLC) plates. Thus, native, biologically active low MW FSH-BI appears to be dansylated RF
of
to give
0.15
in the
concentration fluid. The is reported.
in
a derivative TLC
system
characterized used
tissue homogenates distribution of the
tissue
ET AL.
by an
to quantify and dansyl
its
follicular derivative
at
Identification of Dansyl
AND
and Quantification Derivatives
Tissues
were
homogenized
tris(hydroxymethyl)aminomethane 7.5, using a Polytron Instr., Westbury, NY) with
a motor-driven
enates
were
mm at Tissue
4#{176}C to extracts
lated
as
(40%,
tissue followed Potter
centrifuged obtain or
follows,
wt/v)
homogenizer. at
aqueous follicular
according
to
mM
(Tris) buffer, pH grinder (Brinkman by homogenization 30,000
HomogX
for
g
extracts of fluid were previously
procedures (Seiler and Weichmann, Herbst, 1970). Samples (300 i.zl) were by incubation with an equal volume loric 2000
in 50
tissues. dansy-
established
1967; Dion and acid-precipitated of 0.4 N perch-
acid for 24 h at 4#{176}C.After centrifugation x g for 10 mm, 200 il of supernatant fluid
at was
to
was
received
use.
We
frozen
are
Development Research, National
and
indebted
to
Branch, Center Institutes of follicular fluid was
fluid. filtered
through cheese cloth and clarified by centrifugation (1500 X g for 30 mm at 4#{176} C). Clarified follicular fluid was mixed with an equal volume of 0.4 acid and precipitate X
g for
stirred overnight was removed by
10 mm
at
4#{176}C).The
ants were then dansyl chloride
dansylated (added as
0.15 The
(added stirred
g Na2CO3 mixture was
ture for L-proline mixing maining (2000 flash
30
fluid
for a gift of the porcine thawing at 4#{176}C,follicular
perchloric resulting
METHODS
Material Fluid
-20#{176} C prior
Contraceptive Population
Health, After
0.15
follicular
stored the for
=
Follicular
Porcine
(2000 MATERIALS
of RF
Porcine
X
acid
supernat-
by addition of 20 mg 10 mg/ml acetone) and
ml
16 h after supernate
continued for an additional solid Na2 CO3 was removed g for 10 mm) and acetone at
at 4#{176}C.The centrifugation
as solid) per ml supernate. in the dark at room tempera-
approximately (as solid) per
evaporation
40#{176}Cunder
which 0.15 g was added, and hour. The by centrifugation was removed
vacuum.
swollen
adsorption LH2O,
in isopropanol
packed
into
a 1.5
by for was at was
column chromatography then on Silica G. LH2O
and
re-
Residual
aqueous material was then extracted with benzene 30 mm in a separatory funnel. The benzene layer removed, concentrated by flash evaporation 40#{176}C under vacuum, and stored at -20#{176} C. Purification of the RF = 0.15 derivative accomplished by first on Sephadex
N
x
was
85 cm
removed and combined with 400 p1 dansyl chloride in acetone (10 mg/ml) and 30 mg solid Na2 CO3. The mixture was incubated in the dark for 16 h at room temperature; 100 p1 proline (100 mg/mi) in water was added and the incubation continued for
Cheminert glass column (LDC/Milton Roy, Riviera Beach, FL) using a flow rate of 0.2 ml/min. The benzene extract was thawed at room temperature, dried under nitrogen at 40#{176}C,and redissolved in isopropanol at 80#{176}Cto achieve a saturated solution. This solution was injected onto the LH2O column and
1 h. Acetone
eluted
samples (1-20
was
then
were extracted p1) of benzene
preabsorbant
layer
of prechanneled
(Analtech, Newark, plates were then acetate dried,
removed
under
DE) and developed
Silica
G Uniplates
(3:2). After chromatography, the plates sprayed with triethanolamine:isopropanol
and
cobalt
filter.
and
Aliquots to the
air-dried. The Silica in cyclohexane:ethyl
(1:4), and dried again. Fluorescence was using a scanning densitometer (Kontes Vineland, NJ) equipped with a 351-nm filter
vacuum
with 500 /.Ll benzene. extract were applied
G were
quantified Glass Co., excitation
with
chromatography Six-millimeter intervals and
isopropanol (HPLC) fractions subjected
by
high-performance
at a flow rate were collected to quantification
cence using a Turner fluorometer (relative cence; Turner Assoc., Palo Alto, CA). The were also subjected to TLC on Silica G described below to identify those containing 0.15
material.
Fractions
containing
liquid
of 0.2 ml/min. over 30-mm of fluores-
this
fluoresfractions plates as RF = substance
were then pooled, dried under nitrogen, and stored at -20#{176}C. Pools from several LH2O runs were resuspended in cyclohexane:ethyl acetate (3:2) and
DANSYL
pooled
for
further
purification
This material was 25 cm, Cheminert
injected glass
cyclohexane:ethyi ment
at
were
a flow
rate
quantification
of
G
RF gen,
plate
(3:2)
of
over
using
2 ml/min.
2-mm
G columns. (1 x with
below).
HPLC
and
(4
ml)
subjected
to
fluorescence
and
on
Fractions
containing
TLC
0.15 material were pooled, dried under nitroand stored at -20#{176} C. Pooled follicular fluid obtained from bovine ovaries was generously provided by Dr. Guy Kiracofe (Kansas State University, Manhatten, KS) for the quan-
ular
MW
been
0.15
FSH-BI
reported
material. Details and concentration
in these previously
of follicular
(Sluss
et al.,
fluid
were
then
ture
100 and for
Purified
RF
=
mide
have
2 h.
Samples were both normal normal phase Silica column Milton Roy).
sheets
under
Silica
systems. G plates
RF using
Samples were subas described below.
ml/min. Eluant was fluorescence detector
monitored (Fluorometer
Roy) nm
370 nm excitation The reverse-phase
with filters.
volved elution (Excalibar, 4.6 gradient sodium isopropanol
of X
sample 25 cm,
continuously with III LDC/Milton
were
(Pierce
(Weiner and Tishbee, 1981) from 0.05 M acetate (pH 6.3), 12.5% acetonitrile, 5% (Buffer A) to 0.05 M sodium acetate (pH
Chemical
Company,
and
and RF
1967; Weiner et al., fraction in benzene
Wang, 0.15
reaction
Rockford,
hydrolysis =
in
with
dansyl 1972). were
IL) chloride Samples placed
B). =
after
0.15 sheets acid
were
90
the
vacuum,
the
identified Dansyl
amino
two acids
and dried developed vacuum,
developed
acid
(9:1) sheets
in ethyl drying
were
photowere
(C 18-Silica)
HPLC
also
procedures. reverse-phase
previously. time relative
(Pierce Chemical (PTC) amino acids
and were
After
hydrolyzates by
(3:2,
under air. in 1.5% the polya-
and
sheets
chromatographic were resolved
under
acid
dimension,
in acid
HPLC as described based on elution
reverse-phase
PICO turer
dried
(20:2:2).
polyamide
under UV light. acids present by
then
degrees
second acid
graphed Amino
by
from a C 18-Silica column 5 pm) by a complex step
times.
(10 mg/ml) in at room tempera-
in benzene:acetic Finally, polyamide
amino acid standards Phenylthiocarbamyl
a
various
p1 acetone:acetic
rotated
Identificato dansyl Company). were resolved
using
a Waters
TAG system as recommended by the manufac(Waters, Millipore Corp., Milford, MA). Identifi-
cation was based on elution time of amino acid standards (Sigma Chemical Co., St. Louis, MO) derivatized with phenylisothiocyanate (PITC) using procedures identical to those applied to acid hydrolyzates of dansyl RF = 0.15 samples.
and 418/700 system in-
6.5), 50% acetonitrile, 1% isopropanol (Buffer The presence of amino acids in the RF fraction was examined by TLC on polyamide
chloride samples
samples in 100
again
(C 18-Silica) tion was
also subjected to analytical HPLC using phase or reverse-phase systems. Two systems were employed using a CN(Excalibar, 4.6 X 25 cm, 5 pm; LDC/ The first system used an isocratic
elution with cyclohexane:ethyl acetate (3:1) at a flow rate of 1 ml/min. The second normal phase system involved a curvilinear gradient from cyclohexane: isopropanol (49:1) to cyclohexane:methylene chloride: isopropanol (21:3:1) over 90 mm at a flow rate of 1
equipped emission
The
acetate:methanol:acetic
containing homogeneity
105#{176}Cfor
p1 of dansyl incubating the
(second dimension) dried under vacuum.
1983).
0.15 Material
chromatographic to TLC on
at
v:v), applied to polyamide sheets, The polyamide sheets were first formic acid. After drying under
of
Pooled Silica G column fractions 0.15 material were examined for four jected
heated
dissolved
developed, Characterization
409
After hydrolysis, HC1 was removed under vacuum and the residue was dissolved in lOOp! of 0.5 M Na2 CO3. Reaction with dansyl chloride was accomplished by
vacuum,
of the collecof low molec-
pools
FLUID
tubes and dried under nitrogen at 60#{176} C. One hundred microliters of 6 N HC1 was added and the tubes were sealed under vacuum after flushing with nitrogen.
adding acetone
the
=
tification of RF tion, characterization,
FOLLICULAR
Tubes
equip-
Fractions
intervals
relative
(see
Silica
FROM
onto Silica G columns columns) and eluted
acetate
collected
silica
on
PEPTIDE
RESULTS
The follicular
separation fluid
of dansyl by adsorption
derivatives from chromatography
porcine on an
LH2O column is shown in Figure 1. RF = 0.15 material (D15) elutes as a single peak and is partially resolved from other dansylated compounds, particularly dansyl polyamines. Fractions containing D15, as determined by TLC on Silica G plates, were pooled from several runs, dried at 60- 80#{176}C,and stored at -20#{176}C. LH20 pools were then redissolved in cyclo-
(Woods
hexane:ethyl
acetate
and
further
purified
of the in glass
G column using Cheminert glass columns D15 eluted in a single peak, as shown
on
a Silica
and HPLC. in Figure 2.
410
SLUSS
ET
AL.
Mithinol
I.opropanol
Cyclohexane:
Ethyl
acetate
Acetone
(3:2)
I
1
4,
100
0
I#{174} E Rf
0.15 ON TLC
Ri = 0.15 on TLC
#{149}
75
E
50 U Is
0
25
I’ 0
_________
Is
10
20
30
40
FRACTION
FIG Dansyl
50
NUMBER
60
70
80
90
material
appeared
on
Silica
of Figure Purity
3. of
D15
0
to
G TLC obtained
by chromatography results are shown
be
homogeneous
plates
as shown
by
Silica
G
from D15
each other (data not
10
purity power
of D15 (selectivity)
system. sorbed
It UV
was
HPLC
was
is also at 254
in weak buffer well Thus, the estimated
not limited of a single
20 FRACTION
30 NUMBER
40
50
60
70
(4 mllfraction)
FIG. 2. Purification of F = 0.15 in LH2O pooled G chromatography. LH2O fractions (22-28) containing pooled and chromatographed on Silica G columns Materials and Methods. Relative fluorescence of each
fractions by silica RF = 0.15 were as described in fraction is shown.
The elution solvent was changed from cyclohexane:ethyl acetate (3:2, v/v) to acetone at Fraction 42 (168 ml). Elution position of RF = 0.15 material was determined by TLC and is illustrated in Figure 3, Lanes C and D. Fractions containing RF = 0.15 material were pooled as indicated by the shaded area and tested for homogeneity (Fig. 3).
A
using three other systems. in Panels B through D of
and eluted shown).
/\.iur #{149} #{149} #{149} p #{149}
“
when in Panel
Figure 3. In each of these analytical systems, D15 was greater than 95% homogeneous based on area under the fluorescent profiles. Selectivity of these chromatographic systems was quite different due to the solid (CN-Silica or C18-Silica) or mobile phases used. For example, in the normal phase systems, dansy! polyamine standards eluted near or after D15, while in the reverse-phase system, dansyl polyamines were unresolved before
i
1. Separation derivatives
examined
assessed These
0
(6 mlltrictlon)
of dansyl derivatives by LH2O chromatography. from acid-extracted porcine follicular fluid were chromatographed on Ll-120 columns as described in Materials and Methods. Relative fluorescence of each fraction is shown. The elution solvent was changed from isopropanol to methanol at fraction number 50 (300 ml). The elution position of RF = 0.15 material (Fractions 22-28) was determined by thin-layer chromatography as described in Materials and Methods and illustrated in Figure 3, Lane B.
This
Is
by the resolving chromatographic
of interest to note that D15 abnm, as shown in Panel C of Figure
sis chromatographed in this system. These ly recognized (Woods ology was qualitative. the demonstration partially peptide quantify amino alone more tion
the acids
that in
purified composition.
derivatives
that
acid
standards.
DNS-amino
D15 We
various dansylated with confidence using
due to the ability of some than one dansyl derivative, of all dansyl amino acids,
dansyl
fied
similarly to dansyl amino acids results indicated that, as generaland Wang, 1967), the methodOf significance, however, was
could
is at could
least not
and hydrolyzed this methodology
amino acids to form the limited resoluand the presence of not
be
matched
Amino acids in acid hydrolysates were also by reverse-phase HPLC of dansyl or PTC
to identideriva-
Samples of Silica G HPLC pools were subjected to acid hydrolysis followed by reaction with dansyl chloride and chromatography on polyamide sheets (Data not shown). D15 was partially hydrolyzed by
tives (Fig. 4). Figure 4A shows the identification of amino acids derivatized with dansyl chloride. In addition to the amino acids Ser, Thr, Gly, Pro, Ala, Val, Arg, and Lys, several unidentified dansyl derivatives were observed. Figure 4B shows the identification of PTC amino acids after PITC derivatization of acid (6 N HC1) hydrolysates (105#{176}C, 24 h) of D15.
the
Ser,
3.
methods
employed
and
the
products
of hydroly-
Thr,
Gly,
Pro,
Ala,
Val,
Arg
and
Lys were
present,
DANSYL
PEPTIDE
FROM
FOLLICULAR
FLUID
411
B
INITIAL DANSYLATED EXTRACT OF PORCINE FOLLICULAR FLUID STEP
l
LH2O
I-PLC POOL
STEP
2
SILICA
6 HPLC
G HPLC
(10
(5 ul) SILICA
ul)
CN-SILICA HPLC OF SILICA 6 HPLC POOL
AB
CD
E
D
C
I
I
FIG. 3. Purity of RF = 0.15 material obtained by silica G chromatography. Silica G fractions containing RF = 0.15 material were pooled and dried under nitrogen. Purity was assessed with four separate chromatographic systems. Panel A: thin-layer chromatography (Silica LTLCI), Lane A, dansyl derivatives of acid-extracted porcine follicular fluid (starting material); Lane B, LH2O pooled fractions; Lanes C and D, 5 sl and 10 .sl Silica G pooled fractions respectively; Lane F, CN-Silica (see also Panel C) pooled fractions. Panel B: lsocratic normal phase high-pressure liquid chromatography (HPLC) on CN-Silica, Panel C: Gradient normal phase HPLC on CN-Silica, Panel D: Gradient reverse-phase HPLC on C18-Silica. Details for each system are given in Materials and Methods.
412
SLUSS
although it should be noted that glucosamine galactosamine PTC derivatives coelute with Ser possibly Gly. Additionally, a PTC derivative (Fig. that
eluted
which upon
appears retention
standards. Purified quantify
consistently to
be
times
Dl 5 was this
at about for
used
dansyl
PTC
7
mm,
as a reference
fluid. A typical standard curve The amount of D15 in samples of a reference material stored
detected,
ethanolamine,
derivatized
derivative
was
in tissues
and and 4B)
AL.
all assays
reported
D15 SD,
p1 of the reference 5). Thus, relative
per n
=
quantified
based
herein.
based
on
However,
the
the
dry
dry
weight
weight
of
1.3 ng ( of D15 were
was 16.7 amounts
±
of this
±
reference
material.
ethanolamine preparation
ET
to
or follicular
is shown in Figure 5. is expressed as volume at -20#{176}C and used in
Studies were conducted to quantify the tissue distribution of D15. The measurement of D15 in the rat tissues is summarized in Table 1. Large quantities of D15 were observed in ovarian tissue (Table 1), and significant kidney and amounts
of
heart
or
gonadal
A
D15
were
diaphram cells
adult
I
amounts of liver extracts.
is also
concentrations adults and
were Leydig
These
were
levels
detected
muscle.
of immature
females)
observed
D15 were In contrast,
in homogenates
The male
shown
amount rats
of
of
D15
(offspring
in Table
observed cells of
in
of the
1. The
highest
in ovarian tissue of the the immature animals.
approximately
in diaphram
also present in extremely small
100-fold
or heart
tissues.
higher
than
Distribution
of
20 a
16 E U
0 U C
0 U 0 0
B
12
0 3
U0
>8Is
0
4 a
FIG. 4. Presence of amino acids in acid hydrolyzates of RF = 0.15 material. Samples of RF = 0.15 material (“D15”) obtained by Silica C high performance liquid chromatography (HPLC) were subjected to acid hydrolysis (6 N HCL, 16-24 h, 105#{176}C)followed by reaction with dansyl chloride or phenylisothiocynate as described Methods. Panel A) Separation and identification of by reverse-phase HPLC. Amino acids and unhydrolyzed tified by retention time relative to known standards
lyzed), respectively. thiocarbamyl-amino
Panel
in Materials and dansyl amino acids D15 were idenor Dl 5 (unhydro-
B) Separation and identification of phenyacids from acid hydrolysates of Dl 5 of reversephase HPLC (PICO-TAG). Amino acids, ethanolamine, and unhydrolyzed D15 were identified relative to retention time of known standards run separately.
I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
Dose
Cult
FIG.
5. Fluorescence response curve for the quantification of dansylfactor (D15) by thin-layer chromatography (TLC). Increasing volumes (X-axis) of purified Dl 5 (Fig. 3) were applied to the preabsorbant layer of multichannel Silica G TLC plates and chromatographed as described in Materials and Methods. Fluorescence (351 nm excitation, 400-700 nm emmission) of the reference preparation (RF = 0.15) was derivatized
quantified
V-axis. to data
with
Similar
quantify reported
a Knotes
standard DIS
Tables
(RF
fiber
optic
plate
curves
were
generated
= 0.15) after 1, 2, and 3).
TLC
scanner on of
and each
dansylated
is shown TLC
plate tissues
on
the used (e.g.,
DANSYL TABLE
1. Distribution
Tissue
of RF
=
Source
0.15
material
Peak
heighta
PEPTIDE
in various
l
(cm)
rat
RF
FROM
tissues.
(Table observed
015b
=
FOLLICULAR
ng/mgC
reference
tory
FLUID
2).
In contrast, very low in extracts of follicular
follicles.
concentrations Immature
Leydig cells Seminiferous tubule ovaryd Kidney Liver Diaphram Heart
Immature Adult Adult Adult Adult Adult
aRelative mg
male male femaleC femaleC femalee femalee femalee
fluorescence
tissue extract. bvoiume of
fluorescence CCalculated
from
reference
peak
20.3
5.68
eTissues from and
D15
within
of
contained
136.0
3.61 2.19 1.46 0.12 0.01
153.4 93.1 62.1 5.1 0.5
5 tl
amount
mothers
seminiferous
corpora
licles
3.20
benzene
extract;
giving
of
by
relative
to
corpora
lutea.
immature
equivalent
comparable
fluorescence
factor from 0.4 mg tissue extract X 17 ng/d reference material. dOvaries with multiple functional
cells
both
13.7 9.1 6.8 1.4 1.0
preparation
height. based on
241.4
12.3
male
.zI
rats
to 0.4
RF
=
0.15
dansyl-derivatized reference
used
material
to obtain
Leydig
ovary
was
also
studied
(Table
2). Pig
ovaries were used to obtain substantial quantities granulosa cells from preovulatory follicles (>4 mm diameter). obtained were
Granulosa cells and follicle wall tissue from ovaries on which no corpora
visible.
Separate
with respect lutea were
ovaries
of
similar
to obtain
of in
were lutea
appearance
to follicular size and absence used
of corpora
extracts of whole
ovaries.
The majority (60%) of precursor of D15 (the undansylated molecule) present in ovaries from pigs slaughtered was
during
the
present
follicular
phase
in granulosa
2. Distribution
cells
of RF
Peak (cm)
Tissue
Follicle Granulosa Corpus
of the
estrous
cycle
of preovulatory
0.15
material
cells lutem
aRelative
mg tissue
fluorescence
height5
ovarian
of
small phase
fluorescence. cCalculations
follicles of the
5 zl
benzene
preparation
on amount
(l)
but
no
functional
the
to
the
follicular
5-fold)
less
walls
of this
large
of pregnant estrous cycle
cows (Sluss
fol-
precursor
contained
material.
conHowever, not
be
fluid. large or
or cows in the luteal et al., 1983). Table 3
receptor
assay) previously
(Sluss et al., 1983).
These
centrations of the precursor of D15 vary among follicles contributing to these pools. Concentration of precursor of Dl5 was lowest in follicles obtained from
pregnant
extract;
giving
corpora
relative follicles
equivalent
similar
quantities
lutes.
regardless of follicle size, and in
of precursor
of D15.
containing significant
In these
follicles,
small follicles contained approximately 4-fold higher concentrations of D15 precursor than did large follicles. The concentration of Dl5 measured was correlated positively with the concentration of low
3. Relative fluid POOIS.a
No.
RF
to sl reference mm)
concentration
B. Description
of
RF
C. FSH-Bl
of Pool
3 to 0.4
=
Pregnant, large follicles Pregnant, small follicles Luteal, large follicles Luteal, small follicles Cystic follicles
2
0.15
by dansyl-derivatized
(>4
cows,
cystic follicles. Follicles from ovaries corpora lutea (luteal cows) contained
=
0.15
material
in bovine
fol-
D.RF=0.15 material
potencyb
(Mg/mI)
ng/mgc
113.9 13.6 69.3 91.4
of fluorescence
factor from 0.4 mg tissue extract X 17 ngfl reference material. containing preovulatory albicantia
of
of
the quantification of D15 in these and shows the concentration of low MW determined by activity (e.g. radioligand-
4 5 aBovine
based
cells
amounts
high 2). Thus,
summarizes samples FSH-BI
A. Pool
RF = 015b Reference
2.68 0.32 1.63 2.15
from
reference
granulosa
and
contained
tissue.
extract.
bVolume
(Table
substantial
also
follicles
in porcine
10.7 3.1 7.3 9.0
wall
precursor
lutea
(4-
I
ovaryd
lutea
of D15
were preovula-
the origin of the precursor of D15 could determined from these data alone. D15 was also quantified in bovine follicular Pools of follicular fluid were obtained from
TABLE licular TABLE
Corpora
whereas
siderably
concentrations walls from
values represent much higher (500- to 1000-fold) concentrations of D15 than have been observed (data not shown) in serum and demonstrate that con-
tubules.
the
D15,
of
413
and
in columns
bFolliclestimulating
material corpora
follicular
A, B, and
defined 50% tent
fluid
1.2
0.09
1.8
0.09
3.0
3.94
100
15.64 2.1
pools
C are from hormone
0.09
have
been
described
Sluss et al., 1983. binding inhibitor
as the dose (Ml) required (Bl50 value), is shown as percent fluid (Sluss et a)., 1983).
to
inhibit inhibition
previously.
(FSH-Bl)
Data
Potency,
[‘25l]-hFSH binding relative to the most
by po-
414 MW
SLUSS
FSH-BI
of bovine
activities
(receptor
assay)
follicular fluid (Table
in these
pools
ET
AL.
tography normally
3).
to purify highly polar native would not be soluble in organic
Dl5 DISCUSSION
In previous studies (Sluss and Reichert, reported that porcine follicular fluid several inhibitors tors complicated
of FSH-BI. efforts to
1984a), we contained
These multiple inhibistudy the concentration
of the FSH-BI in physiologic fluids and tissue assess its potential significance. Measurement of FSH-BI activity in small samples with a gonadal radioligand-receptor assay is not feasible. As ously high
shown
(Sluss
(>5000)
MW
contamination FSH-BI present be due However,
fractions
l984a),
may
be due
to
et
al.,
1985),
we
and low (about
follicular fluid (Sluss have opposite (agonist
in and may
and Reichert, 1982). have been identified
have 1000)
identified
both
FSH-BIs
et
al., 1987). or antagonist)
in
cultured
rat
Sertoli
cells.
Thus,
FSH-BI
activity
in
may
et of
of a dansyl derivative (D15) characterized by an RF = 0.15 in the TLC system used in these studies to separate dansyl derivatives and reaction products. On the basis of this observation, we undertook the studies
reported
and
demonstrated
that
after
perchlor-
ic acid extraction and dansylation-D15 can be purified to homogeneity by adsorption chromatography. The novel combination of classical LH2O or Silica G column chromatography with HPLC enabled rapid, preparative scale purification of RF = 0.15 material. Using different selectivities of these two chromatographic systems in sequence resulted in purification to greater than 95% homogeneity in two steps. Using dansyl derivatization before purification was attempted resulted in extremely sensitive detection of materials of interest by fluorometry and allowed
the
use
of high
resolution
adsorption
chroma-
tion
hydrolyzates Arg, Lys,
1) and
although Finally,
be
Ser, Thr, Gly, appears to be
these the
concentration
fluid (Table 3) of the FSH-BI were positively low
contained and what
that
addition, amino sugars (assuming some unknown PTC derivatives neutral sugars (phenol-sulfuric be present in both dansylated and
samples, unequivocally.
(Table
have tissue
in pools
precursor correlated.
of D15 Thus,
a dansyl-derivatized
glycopeptide,
MW FSH-BI. The exact relationship
between
not been distribu-
of follicular and Dl5
low MW appears
possibly D15
the
and
the
low acid
MW FSH-BI is unknown at present. precipitation of samples prior to might cleave the molecule, resulting in
only partial recovery of the FSH-BI as the dansyl derivative (e.g., D15). Since the underivatized low MW FSH-BI was also purified under acidic conditions (e.g., a larger
be associated with either inhibitory (antagonist) or stimulatory (agonist) post-binding effects on FSH responsive cells (Sluss et al,, 1987). The low MW FSH-BI, having antagonist biologic activity in vitro, has been partially purified (Sluss al., 1987; Sluss and Reichert, 1984a). Dansylation highly purified inhibitor resulted in the identification
underivatized identified
acidic
These biologi-
cal activity in vitro; high MW FSH-BI stimulates whereas low MW FSH-BI inhibits steroidogenesis
Val,
underivatized Perchioric derivatization
purification of bacterial
MW
Ala,
ethanolamine. In the identity of observed) and/or acid-positive) may
to
bacterial
1984b) fractions
to be of gonadal origin. Using that would inactivate FSH-BI
(Sluss
or to total tissue previ-
FSH-BI
(Sluss and Reichert, in very low MW (25,000) porcine factors
Reichert,
to salt effects (Andersen other FSH-BI activities
that appear procedures origin
and
acid
Pro,
materials solvents.
acetone, (>2500
underivatized (Sluss and of
pH MW)
low Reichert,
follicular
3.0),
fluid
it too
may
molecule.
exist
However,
naturally since
as
active,
MW FSH-BI in follicular fluid 1984a) or acid acetone fractions (Sluss
et
al.,
1987)
passes
a 5000
MW cutoff membrane, the precursor of the dansyl derivative (D15) must be less than 5000 MW. On the basis of amino acid composition (excluding carbohydrates weight
and ethanolamine), of at least 2500,
underivatized to apparent
molecule, size (based
amino acid components Precise compositional purified
D15.
D15 which
has a molecular is comparable to the
considering the on ultrafiltration)
contributions of non-
and the hydration data is not yet
Although
amino
acids
shell. available are
for
present,
quantification cannot be achieved until the presence of amino sugars is verified and PTC amino sugars can be resolved from PTC amino acids. The presence of ethanolamine
is indicated
by HPLC
of PTC
tives. Mass spectrometry (data not shown) drolyzed samples of Dl 5 also suggested the of dansyl impact and etry showed
derivaof unhypresence
ethanolamine. Low resolution electronFast Atom Bombardment mass spectromions at M/Z
of 294
and smaller. Elemen-
tal composition measured by high resolution electronimpact mass spectrometry of the 294 ion was C14H18N203S and is consistent with the suggestion (Figure 4B) of dansyl ethanolamine in the sample. Additionally, highly purified low MW FSH-BI con-
DANSYL
tains acid
PEPTIDE
carbohydrates measured by the phenol method (Dubois et al., 1956), which
detect
amino
sugars. Analysis
and dansylated is a glycopeptide
of both
samples suggests containing
sulfuric does not
underivatized
that low ethanolamine,
MW
sugars, and amino sugars. Such a compound tively unusual and could be derived in membrane components. Similar structures reported
recently
of membrane
for
‘membrane
proteins
Saltiel, 1988). Purified Dl5
was
cation
factor
of
(Low
anchoring’
used
as a reference
after
for
dansylation
is not
cant as autocrine)
observed,
the
other
of absolute tissue activity observed
tissues
with
receptor
al.,
1988)
1983; Sluss also include for
FSH
as well
ly of receptors 1983; Further by use
the
with and
as other
for
FSH-BI antagonsignifi-
(paracrine or FSH action
and Reichert, 1984a). Such FSH-like agonists interacting (Sluss
FSH
et al., factors
(Hsueh
1987;
Schneyer
acting et al.,
a
testicular G,
Schaeffer and
of
spectrometry
Massachusetts
was
performed
Institute
of
Technology.
Spectrometry
We
are
Seller
to
PM,
Insulin-like cells
growth
growth and
Andersen TT, Reichert testis. Modulation Chem 257:11551-57
D’Ercole factors
function.
AJ, as Endocr
LE Jr, 1982. by monovalent
intraovarian Rev
VanWyk
regulators
JJ, of
Sluss
Melson by
in calf to
receptor
Florm
follicle.
Inhibition
of
Aust
J
ovarian
factor.
and
Endocrinol-
of
Reproduction.
New
York:
Biol
Reprod
LE a
31:520-30
PM, in
1988. porcine
123:487-91
einiger 1-dimethylkiessel G-schichten.
J
Biol
a
Jr,
low
ir,
Reprod 1983.
MW
FSI-l
receptor
33:925-33 of
of
to
binding
with
Inhibition
fraction
Reprod
1984a.
1985.
interacts
is related
weight Jr,
“I-hFSH
bovine
follicular
biochemical
parameters
29:1105-13
Porcine
follicular
inhibitors
1984b.
of
Secretion
binding
to
isolated
fluid
FSH
of
by
porcine
MA, both
an
receptor
from
Franke
contains T,
Weber
purified
Chem
contains
binding
to
recep-
inhibitor the
LE and
follicle-
Serratia,
follicular
Reichert agonist
of
bacteria
Jr,
fluid. 1987.
antagonist
Biol Porcine
activities.
K, on
1972.
Amino-terminal
a nanomole
scale
sequence by
gel
analysis
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electrophoresis.
247:3242-51
Tishbee
reversed
A,
phase
method KR,
LE
Serratia
120:1477-81
Platt
polyamide
Reichert
Biol
AL,
fluid
protein’s
369-70
Sluss
Endocrinology
bacteria LE
Jr,
strain
Schneyer
AM,
BE, membranes.
hormone
Endocrinology
Woods
RJ,
30:1091-1104
Reprod
tive
FSH.
the
molecular
including
S.
Ryan
immunoactivity
chromatographie auf
Reichert
LE
Reichert
PM,
M,
(FSH)
development. low
PM,
of oxytocin and follicular
59:970-73
zur
by
Reichert
follicular
granulosa
to receptors in cations. J Biol
free
Progr
ovarian
growth
Franke
concentration
Chromatogr binding divalent
testis PW,
follicular
Sluss
1985.
ir,
pituitary
JF,
stimulating
6:400-20
Follitropin salts and
Recent
Yen SSC, 1984. Presence human ovary, oviduct,
Metab
produced Fletcher
PM,
Weiner
ME,
1981.
epidermal
A1W, in
LE is not
inhibitor
Biol
Svodoba
serum
28:351-62
FSH
tor.
REFERENCES CE,
fluid
fluid:
of
Resnick
in the
PBC,
hormone
Ewing
PM,
Weiner
EY,
in
fluid.
Bioregulators
Endocrinol
Reichert
binding
Dr.
follicular
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N, Wiechmann M, 1967. aminonaphthalin-5-sulfonyl-derivate
for
Facility
indebted
1981.
J, Hsueh vasopressin
Clin
AL,
several
Mass
in
by
Follicle-stimulating
Sluss
Catherine Cosetello and Simm Maleknia for their contributions in this regard. Critical reviews of this manuscript by Drs. A. L. Schneyer, T. T. Andersen, and P. Weber are acknowledged and appreciated. Skilled technical assistance was provided by Mary Franke, Bruce Melson, and Jay Boniface, and we thank Mary Heiser, Amy Fournier and Audrey Cox for secretarial assistance.
Adashi
1
fluid.
Sluss
at the
HJ,
JM, Liu arginine
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of
Mass
Vogel
inhibitor
ACKNOWLEDGMENTS
ovarian-cells
Academic Press Low MG, Ferguson MAJ, Futerman AU, Silman I, 1986. Covalcntly attached phosphatidylinosital as a hydrophobic anchor for membrane-proteins. Trends Biochem Sci 11:212-15 Low MG, Saltiel AR, 1988. Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 239:268-75 Reichert LE Jr. 1978. Some properties of small molecular-weight tissue and serum inhibitors of follitropin binding to receptor. In: Spilman CH, Wilks )W (eds.), Novel Aspects of Reproductive Physiology. New York: Academic Press, pp. 355-68 Reichert LE Jr. Andersen TT, Branca AA, Fletcher PW, Sluss PM, 1984. FSH binding inhibitors in follicular fluid. Steroids 20:1592
Sluss
Schaeffer et al., 1984; Adashi et al., 1985). study of such possibilities will be facilitated of the quantitative assay described here.
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Chromatogr
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steroidogenesis
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cultures
38:409-56 iM, 1981. Peptide Biol Sci 34:491-504 Hsueh AJW. Welsh TU, Jones
independent1981;
FSH-suppressing
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low liver
Primary
Res Hammond
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RR,
Grady
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(Sluss system
and
concentrations of Dl 5 were observed and liver) may be involved in degradation or of the substance. It should be noted that a
similar lack MW FSH-BI
GF,
medium Endocr Franchimont
various
tissues. Gonadal concentrations of D15 (i.e., trations of the precursor of D15) were higher the other tissues. Although absolute gonadal significant (kidney excretion
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LD, Hoover DJ, Kolena J, Osteen K, 1982. The role of nonsteroidal and follicular maturation. Recent
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part have
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duction.
the
specificity
FROM
1981.
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determining
213:501 Wang
KT,
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of liquid
N-termini
DNS-amino
acids
chromatography-A of
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and
J
proteins.
-06 1967.
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of
Biochem
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133: