Identification of insulin-like growth factor-II in human seminal and follicular fluids

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

October 30, 1986

Pages 536-542

IDENTIFICATION OF INSULIN-LIKE GROWTH FACTOR-II HUMAN SEMINAL AND FOLLICULAR FLUIDS K. Ramasharma,

Claudia

M. Cabrera,and

IN

Choh Hao Li*

Laboratory of Molecular Endocrinology, University of California, San Francisco,CA 94347 Received September 16, 1986

Summary: Antisera raised in rabbits against synthetic insulinlike growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 pg/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant D 1986 Academic Press, Inc. amounts of IGF-II. Insulin polypeptides share

several

(l-3).

like

growth

with

70 and 67 amino

structural

These

growth

fetal

and postnatal

these

growth

regulators IGF-I plasma gonadal

factors

of

factors

related

the

are probably

(4,5).

growth

and function

information

fluids

is

lacking

(6).

with

should

insulin

for

normal

Recently

(6-10). only

the

levels

We report

Although in human of

here

Abbreviations: IGF, insulin-like growth factor; RIA, radioimmunoassay; HPLC, reversed-phase high performance chromatography: SC, subcutaneously; hSP, human seminal BSA, bovine serum albumin; EDTA, ethylenediaminetetraacetate; hFP, ethanol precipitate of human follicular fluid. * TO whom correspondence

and

as intra-gonadal

identified

regarding

chain

respectively

required

and development

has been

single

features

implicated

cellular

are

residues

have been

immunoreactivity (111,

acid

and biological

growth

factors

I and II

be addressed.

the

seminal

IGF-II the

liquid plasma:

in

Vol.

140,

No. 2, 1986

presence

of

and follicular

BIOCHEMICAL

significant

amounts

AND

of

BIOPHYSICAL

IGF-II

RESEARCH

in

the

COMMUNICATIONS

human

seminal

fluids.

MATERIALS

AND METHODS

IGF-II was synthesized by the solid-phase method as Synthetic peptide (2 mg) was mixed with described (12, 13). 3 mg of keyhole limpet hemocyanin (Sigma) in 1.5 ml of 0.05 M sodium phosphate buffer (pH 7.4). One ml of 20 mM buffer was glutaraldehyde (grade 1, Sigma) in the phosphate After 24 hrs, glycine added, and the mixture was kept at 4'C. was was added to a final concentration of 0.1 M. The mixture stirred overnight at 4OC and dialysed against phosphate buffered saline. After removal of insoluble material by filtration, the dialysed conjugate was mixed with equal volume of Freunds complete adjuvent prior to immunization. Two New Zealand white rabbits weighing about 3 kg were injected (SC) with the conjugate (50 11) at lo-15 different sites; 4 weeks later, one booster injection (0.5 mg peptide conjugate) was administered were bled after 10 days of the booster (SC) - The animals injection and the serum samples were tested for the presence of IGF-II antibodies. The antisera were purified by the protein ASepharose CL 4B (Pharmacia) immuno-affinity column as described The RIA buffer consisted of 0.05 M PBS/EDTA, 0.1% BSA and (14). 3 Kg/ml poly-L-lysine (3OF-5017 Sigma). The synthetic IGF-II was iodinated by the lactoperoxidase procedure (15) and the RIA was carried out in 12x75 mm polypropylene tubes using the double antibody method as described (16). Each assay tube received the following solutions: the sample/unlabeled peptide in 200 11, 125I-IGF-II in 200 ~1 and purified antiserum (2.5 kg) in 200 ~1. The reaction mixture was incubated at 22OC for 24 hrs before the addition of 200 11 of 1:lO diluted sheep anti-rabbit gamma globulins (Antibodies Inc., Davis, CA). After the addition of the second antibody, the assay was further incubated for 15 hrs and the radioactivity-bound pellet was separated by centrifugation and counted in a Beckman gamma counter (40% counting efficiency). The data were calculated for ED50, slopes and parallelism using a non-linear least-squares curve fitting program (17, 18). Fresh seminal fluid samples were collected from the Fertility Clinic at the Department of Urology, UCSF. The seminal fluid was pooled and centrifuged at 15,600 x g. Clear supernatent (30 ml) was acidified to 0.1 M by addition of 6 M HCl. Acidified fluid was mixed with 7 volumes of cold ethanol and kept at 4'C for 16 hrs. After centrifugation, the precipitate human seminal plasma (hSP, approximately 1.3 g) was dissolved in 150 ml 0.1 M HOAc and lyophilized and stored at -2O'C until use. A portion of hSP (700 mg) was dissolved in 10 ml of 50% HOAc and applied on to a Sephadex G-50 column (2.3 x 75 cm) in 0.01 M NH40Ac buffer (pH 4.6). Each fraction was neutralized and a 50 ~1 aliquot was taken for RIA. Those fractions having IGF-II immunoreactivity were pooled and lyophilized for fractionation on HPLC. HPLC was performed on a Cl8 column (Vydac, 218 TP 104, Western Analytical Products, Temecula, CA) using a dual pump system fitted with a gradient programmer (Laboratory Data Control, Riviera Beach, FL) and variable-wavelength UV detector. Absorption was monitored at Fractions 280 nm. The solvent was 0.1% CF3COOH and 2-propanol. 537

Vol. 140, No. 2, 1986

were lyophilized, 50 pl-aliquot

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

resuspended in was taken for RIA.

500 ~1 of

the RIA buffer

and a

Follicular fluid samples were collected from women undergoing in vitro fertilization examination at the Department of Obstetrics and Gynecology on this campus and kept frozen at -70°C until use. From 10 ml follicular fluid, approximately 0.5 g ethanol precipitate (hFP) was obtained by the procedure as described for hSP. The alcohol precipitate (200 mg) was subjected to gelfiltration on Sephadex G-50 column and the IGF-II immunoreactive fraction was further analyzed on HPLC as described above. RESULTS

Two rabbits high in

titer the

immunized

antiserum.

purification

with

of the

2.5

pg of

no cross-reaction including

human

cross-reaction dose

growth

to displace

(ED50) detectable

dose

of

synthetic

rig/tube IGF-II

of

(14)

the

(n=5,

was showed

or peptide it

1). bound

mean?SE).

was found

IGF-II

but

(Fig.

1251-IGF-II

of gamma

The antibody

and insulin,

a

was effective

labeled

antibody.

IGF-I

developed

70% recovery

known protein

hormone

50% of the

was 4.6Ok1.7

with

binding purified

IGF-II

column

antiserum

to any other

with

synthetic

The protein-A

A significant

globulins. observed

against

hormones

gave

some

The effective to the

antibody

The minimal

to be 0.2-0.4

rig/tube.

The

60

10 PEPTIDE

100 (ng)

1025 SAMPLE

(111)

inhibition of the binding of 125I-labeled Figure 1. Competitive IGF-II to IGF-II antibody by unlabeled IGF-II, IGF-I, insulin, human growth hormone and acid-ethanol extracts of human seminal All points represent the mean of and follicular fluids. duplicate tubes. 538

Vol.

140,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

Table Comparison Pept ide

of

54.5

IGF-II

3.3

0.526

0.332

5.9

(2.1-4.8)

0.420

0.323

limits

was

with

with

It

IGF-I

Human seminal

of

batches

antiserum

of only insulin

fluids

labeled

to

IGF-II

6% when compared and human growth

showed a dose-dependent

IGF-II

the

fluid

The amounts of

a pooled

1).

10 11 of

by RIA.

in Table

extent

recognize

for

respectively.

purified

and follicular the

(2.4-14.5)

100

variation

(n=3)

1, the

(Fig.

in displacing

two different given

to

potency*

test

of

to the

failed

volume

‘F’

and 9.5Ok1.6

at 10 pg/tube

levels

by

in Table

cross-reacted

RIA

parenthesis

coefficient

9.07fO.5

As summarized

IGF-II

in

parallelism

sample

A minimum

IGF-II

(37.9-117.6)

and inter-assay

response

in

Relative

intra-

hormone

IGF-II p**

of

IGF-II.

and

Slope

* 95% Confidence Probability

COMMUNICATIONS

1

IGF-I

EDso rig/tube

IGF-I

**

synthetic

RESEARCH

as shown in Figure was

sufficient

of

immunoreactive

human seminal

and follicular

to

1.

detect

IGF-II

fluids

in

are

2.

Table 2 Amounts

of Batch

immunoreactive No:

IGF-II

in

Seminal

seminal

fluid (IGF-II

Each

value

fluid

and

follicular

Follicular rig/ml;

Mean

fluid

f SE)

1

1575

f

66

1979

*

2

2498

+ 197

1925

2 168

represents

the

mean of

the

539

triplicate

tubes.

86

fluid

Vol.

140,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A 0.4 0.3 0.2

FRACTION

NUMBER

Figure 2. Fractionation of human seminal (A) and follicular (B) fluids. About 700 mg of hSP and 200 mg of hFP were loaded on Sephadex G-50 (fine) column in 0.01 M NH40Ac (pH 4.6). The flow rate was 30 ml/hr. 3 ml fracions were collected/tube and void volume was 145 ml. All the fractions were neutralized and assayed in duplicate (50 kl for A and 200 ~1 for B) for RIA. 0.6 - A

0.4 -300 i-7 - 200 : : ‘--1..

-100

r -L--J 1

E P

:____

0.3-B -300 1

-7 I

- 200

2 62

-100 0-

__ __I

!__

C

0.2 -

, IOF

0.1 L 0

10

20

30

40

50

60

TIME, min

Figure 3. RPLC of IGF-II immunoreactive fraction (1 mg) from Sephadex G-50 column (Fig. 2) of seminal (A) and follicular (B) fluids. The flow rate was 0.5 ml/min. All the column fractions were lyophilized and resuspended in 0.5 ml RIA buffer and 50 ~1 aliquot was taken for RIA. The elution profile of synthetic IGF-II (5 pg) under identical conditions (C). 540

Vol.

140.

No. 2, 1986

BIOCHEMICAL

AND

When hSP was fractionated immunoreactove about

1.2

IGF-II

IGF-II

similar fractions

volume

(1 mg each)

immunoreactivity This

peak

IGF-II

in a single

as shown in Figure

elution

2A.

Under

peak the

appeared

(Fig.

2B).

When these

to the

3C) under

the

a V,/Vo

of the

fractions

with

immunoreactive

by HPLC,

major

elution

with

in

were chromatographed

corresponded

column,

same conditions,

hFP also

in a single

COMMUNICATIONS

G-50

in

eluted

(Fig.

RESEARCH

on a Sephadex

appeared

immunoreactivity

BIOPHYSICAL

peak

the

(Fig.

position

IGF-II

3A and 38).

of synthethic

same experimental

conditions.

DISCUSSION

Purification

of the

column

was useful

which

may interfare

IGF-II

IGF-II

the

fluids

in Table nearly

reported fluid

(6) but

IGF-I

in

seminal

of it

2.45

gave

of

the

ED50 of

of ED50

to antiserum

20% binding

immunoreactive

ml.

of

at a labeled

displaced

by 1 pg

in normal

human

fluids

This

value

is

normal

subjects

is considerably fluids

remains

the

level

men (11).

than

the

gonadal

cell

in Table growth

the

follicular pg/ml) level

of of

clg per ml

concentration

to be investigated. 541

with

(0.02

be 0.65

IGF-II.

a concentra-

The serum to

seminal

likely

comparable

as summarized of

have

ml of porcine

was found

lower

as regulators

in men and animals

both

than

in human

is most

that

is much higher of

IGF-II

peptide

per

plasma

IGF-II

the

pg IGF-II

and follicular of

mean+SE)

RIA and was completely

2 indicate

seminal

which

components

The value

The purified

in HPLC,

2 pg per

value

IGF-II

for

serum

RIA.

(n=6;

tube

affinity

purification,

the

mean+SE).

behavior

and follicular

of

After in

kg per

protein-A

peptide.

From

role

2.5

as required

The data

RIA.

rig/tube

(n=5; of

of unlabeled

the

37.Ot4.5

rig/tube

concentration

(19)

in

by the

nonspecific

significantly

from

4.6f1.7

tion

to eliminate

improved

decreased

antiserum

in human 2.

The

and function

Vol. 140, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS

We thank Drs. Mary C. Martin and Dale R. McClure for providing clinical material. This work was supported in part grants from the National Institute of Health (AM-06079, AM-18677) and the Hormone Research Foundation.

by

REFERENCES

1. 2. 3. 4. 5.

Humbel, R.E. (1984) in Hormonal Proteins and Peptides 12, 57-79. Van Wyk, J.J. (1984) in Hormonal Proteins and Peptides 12, 82-125. Nissley, S.P., and Rechler, M.M. (1984) in Hormonal Proteins and Peptides 12, 127-203. Foresch, E.R., Schmid, Chr., Schmander, J., and Zapf, J. (1985) Ann. Rev. Physiol. 47, 443-467. Rechler, M.M., and Nissley, S.P. (1985) Ann. Rev. Physiol. 41,

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Adashi, E.Y., Resnick, C.E., D'Ercole, A-J., Svoboda, M.E., and Van Wyk, J.J. (1985) Endocrine Rev. 6, 400-420. D'Ercole, A.J., Stiles, A.D., and Underwood, L.E. (1984) Proc. Natl. Acad. Sci. 81, 935-939. Adashi, E.Y., Resnick, C.E., Svoboda, M.E., and Van Wyk, J.J. (1985) Endocrinology 116, 1125-1129. Veldhuis, J.D., and Furlanetto, R.W. (1985) Endocrinology 116, 1235-1242. Davoren, J.B., Hsueh, A.J.W., and Li, C.H. (1985) Am. J. Physiol. 249, E26-33. Baxter, R.C., Martin, J.L., and Handelsman, D.J. (1984) Acta Endocrinologica 106, 420-427. Li, C.H., Yamashiro, D., Hammonds, R.G.Jr., and Westphal, M. (1985) Biochem. Biophys. Res. Comm. 127, 420-424. Yamashiro, D., and Li, C.H. (1985) Int. J. Peptide Protein Res. 26, 299-304. Ey, P.L., Prowse, S.J., and Jenkins, C.R. (1978) Immunochemistry 15, 429-436. Thorell, J.I., and Johansson, B.G. (1971) Biochem. Biophys. Acta 251, 363-369. Schalch, D.S., and Reichlin, S. (1966) Endocrinology 79,

17.

Rodbard,

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G.R.

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De Lean, A., Munson, P.J., and Rodbard, Physiol. 235, E97-102. Zapf, J., Walter, H., and Froesch, E.R. Invest. 68, 1321-1330.

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(1978)

(1981)

J.

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