J. JOE

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

J

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

regulators

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

Schneyer

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.

of

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Jageillo

Chromatogr

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activity

steroidogenesis

follicular

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cultures

38:409-56 iM, 1981. Peptide Biol Sci 34:491-504 Hsueh AJW. Welsh TU, Jones

independent1981;

FSH-suppressing

ogy

low liver

Primary

Res Hammond

in which

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one component of a local regulatory system modulating

et al., may

1983.

as models of hormone-dependent differentiation. Mol Cell 30:37-50 P, Channing CP, 1981. lntragonadal Regulation of ReproNew York: Academic Press Charlesworth C, Schwartz ND, 1982. Characterization of

RR,

Grady

concenthan in tissue

homogenate, has been reported for low MW activity (Reichert, 1978). Hypothetically, low MW FSH-BI, an FSH ist (Sluss et al., 1987), may be physiologically

(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

Erickson

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of

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38:331-408 CP, Segal Si, 1982. lntraovarian Control Mechanisms. New York: Plenum Publishing Corporation Dion AS, Herbst EJ, 1970. Polyamine changes during development of Dryosophila Melanogaster. Ann NY Acad Sci 171723-34 Dubois M, Gilles KA, Hamilton JK, Reebers PA, Smith F, 1956. Coloremitric method for determination of sugars and related substances. Anal Chem 28:350-56

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Low

LD, Hoover DJ, Kolena J, Osteen K, 1982. The role of nonsteroidal and follicular maturation. Recent

Channing CP, Andersen antz SG, Tanabee control of oocyte Res

regions

et al., 1986;

415

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Channing

FSH-BI neutral

part have

FOLLICULAR

duction.

the

specificity

FROM

1981.

Separation

high-performance for

determining

213:501 Wang

KT,

layer

of liquid

N-termini

DNS-amino

acids

chromatography-A of

peptides

using sensi-

and

J

proteins.

-06 1967.

chromatography.

Separation

of

Biochem

dansyl-amino

Biophys

acids

Acta

by

133: