Identification and Partial Characterization of Gonadotropin-releasing ...

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preparations. Ultrafiltration and gel column chromatography of pooled extracted seminal plasma identified two compounds with apparent molecular weights.
BIOLOGY

OF REPRODUCTION

33, 370-374

(1985)

Identification and Partial Characterization Hormone-like factors in Human SOKOL,2

REBECCA

Z.

DAVID

HEBER,

Division

PETERSON,

MARGARET

and

RONALD

of Endocrinology,

S. SWERDLOFF

Metabolism

Department Harbor-UCLA 1000

of Gonadotropin-releasing Seminal Plasma’

and

Nutrition

of Medicine Medical Center

West

Torrance,

Carson

Street

California

90509

and The

UCLA

Los

Angeles,

School

of Medicine

California

90024

ABSTRACT

hormone (GnRH)-like material was measured by radioimmunoassay in acid-ethanol-extracted human seminal plasma using radiolabeled D-[Leu6 I GnRH ethylamide as labeled ligand, authentic GnRH as standard, and antibody raised against D-[Lys’ I GnRH analog. The mean amount of GnRH-like material measured in the seminal plasma of semen samples with sperm counts greater than 20 X 10’/ml was 229.0 ± 66 pg/mI, with sperm counts less than 20 X 106/ml was 213 ± 42 pg/mI, and from vasectomized samples was 252 ± 36 pg/mI. There was no significant difference among the three groups. Scatchard analysis of radioreceptor binding data demonstrated significant displacement of GnRH agonist ligand from castrated male rat pituitary membrane preparations. Ultrafiltration and gel column chromatography of pooled extracted seminal plasma identified two compounds with apparent molecular weights of 2600 and 5000 that differ chemically and immunologically from native GnRH. Further characterization using affinity column chromatography suggests that at least one of these GnRH-like factors is a glycosylated protein. Gonadotropin-releasing

INTRODUCTION Gonadotropin-releasing like

material

has

hormone been

identified

in

and GnRH receptors have been on Leydig cell membranes (Bourne Clayton et al., 1980; Dutlow and Bhasin been with

et al., used to certain

Fraser,

1980;

1983).

The

describe anti-GnRH Dutlow

term

rat

testes

demonstrated et al., 1980; Millar, 1981;

“GnRH-like”

factor(s) sera and

(GnRH)-

Millar,

son et analogs

al., 1982), in GnRH

et

1981;

al.,

stimulate

has

that this between testes

1981;Turkel-

Turkelson

luteinizing

stimulating layer cultures

that interact (Sharpe and

1This

March 5, 1985. September 24, 1984. research was supported

and

The and

Research Center (NIH RR-00425) HD-15 132. This

Federation

by

a General

Clinical Associate Physician to R.Z.S. and by NIH grant

study was presented Clinical Research,

for June 1983. 2Correspondence: sion of Endocrinology,

from

Clinical

Street,

Torrance,

a single

been

at the American Western section

material is and Leydig Fraser,

1980;

hypothalamic estimated from 5000 various

family

Immunoreactive detected

mation bioactivity

Rebecca

Medical

GnRH (Sharpe

1982), and

and follicle-

to

Sharpe

et al.,

reported by in immuno-

GnRH and have have molecular to 100,000. It is

identified

of peptides

a messenger cells in the

factors or are

stem

unrelated

compounds.

Award 1 ROl

meetings,

Harbor-UCLA

aL, (LH)

GnRH-like peptides Millar (1981) differ

if these

unclear

et

hormone

GnRH-like the Sertoli

(Sharpe

1981). Dutlow

iodinated assays

hormone (FSH) in pituitary mono(Yingetal., 1981). It is postulated

reactivity from been variously weights ranging Accepted Received

displace radioreceptor

Z. Sokol, M. D., DiviMetabolism and Nutrition, Center, 1000 West Carson

and and

370

the of

Tang, 1983). partially

GnRH-like

CA 90509.

on

in

factors

GnRH-like material human semen, but molecular

this

material

In this study characterized in human

has no

characteristics is available

also infor-

and (Chan

we have identified two species seminal

plasma.

of

GnRH-LIKE

MATERIALS

AND

FACTORS

IN HUMAN

METHODS

SEMINAL

371

PLASMA

lar

weight 1200). Eluted fractions of the seminal were lyophiized and dissolved in RIA buffer and assayed for GnRH using an antibody raised against D-ELys’ I GnRH (Heber and Odell, 1978). plasma

Volunteer

Subjects

samples were obtained from three groups of male volunteers: 1)10 men with a history of infertility of at least 1 year’s duration and sperm counts between 1.0 X 10’ and 20 X 10’ sperm/ml; 2) 9 men with no history of infertility and sperm counts of greater than Semen

20 X 10’ vasectomy

sperm/mI; at least

An additional obtained from 40 yr.

and 3) 10 men 1 yr prior to the

who had undergone present experiment.

250 ml of pooled volunteers between

seminal the

ages

plasma was of 21 and

of Samples

Extraction After

liquifaction,

a standardized

semen

analysis

performed using the methodology outlined by Belsey et al. (1980) in the World Health Organization Manual on human semen analysis. Spermatozoa were then separated from seminal plasma by centrifugation at 600 X g for 10 mm at room temperature. After was

verifying by microscopy the absence of spermatozoa, the seminal plasma was stored in 0.5 ml of 5 N acetic acid at -20#{176}Cuntil extraction with 5 vol of 95% ethanol. These conditions are similar to those recom-

mended

by Niswender

in body

fluids

and

for the

tissue

samples

measurement (Nett

of GnRH

and

Niswender,

1978). Gonadotro

pin-releasing

Hormone

Gonadotropin-releasing oassays (RIAs) were

hormone

radioimmun-

performed using either antibody raised against D-(Lys’ I GnRH (Heber and Odell, 1978) or with antibody raised against native GnRH (Nett et al., 1973) (courtesy of Drs. Nett and Niswender). The former antibody primarily recognizes the ter

carboxyl end of the GnRH molecule. antibody recognizes the carboxyl and of the authentic GnRH molecule equally antibody regularly used in most laboratories authentic standard ethylamide

GnRH. (pg/mI) as the

The

native

and iodinated

The latamino ends

and

decapeptide

radiolabeled ligand.

is the

to measure served

native

as

GnRH

Ultrafiltration

The acid,

pooled was

centrifuged

was then (Amicon,

seminal

extracted

for 10 filtered

plasma, stored in 5 N acetic with 5 vol of 95% ethanol and mm at 2700 rpm. The supernatant

through

an ultrafiltration

Danvers, MA) using a membrane exclusion limit of 30,000 Da under nitrogen of 75 pounds. The retentate was lyophiized filtrate filtered through an Amicon membrane exclusion limit of 500 DL The fmal retentate phiized.

Gel Filtration

system with an pressure and the

with an was Iyo-

Chromatography

of each lyophiized retentate were disM phosphate buffer and applied to an Ultra Gel Aca202 column (LKB Instruments, Inc., Rockville, MD) with exclusion limits of 1000-15,000 DL The column was eluted with 0.01 M phosphate buffer at a flow rate of 74 mI/h. The column was calibrated with myoglobin (molecular weight 17,800), cytochrome C (molecular weight 12,400), 1-calcitonin (molecular weight 3300), and ‘251-GnRH (molecuAliquots

solved

in 0.01

Immunoaffinity

Column

Chromatography

seminal plasma extract as well as half of the seminal plasma extract that had been retained on the 500-D Amicon filter were solubiized in 0.5 M ammonia acetate. Each was individually reacted for 2 h with cyanogen bromide-activated Sepharose 4B Beads (Pharmacia, Piscataway, NJ) covalently bound to an antibody generated against D-ILys’ I GnRH (Pharmacia Fine Chemicals, 1979). Both columns were washed with 0.01 M ammonia acetate prior to the elution of the retained material with 1.5 M acetic acid. The acid eluate was lyophiized and dissolved in RIA buffer prior to measurement. Crude

Hormone

Gonadotropin-releasing

The

Radioreceptor was

GnRH radioreceptor assay described by Heber and

previously

Odell

Assay

performed

(1979)

as

with

radioiodinated D-[Leu’ ,des-Gly-NH21#{176} I GnRH ethylamide as ligand and a 10,800 X g membrane fraction prepared from castrate male rat pituitary glands. Specific binding varied from 20% to 30% in various experiments. Nonspecific binding varied from 3% to 4%. The standard curve was established by quantitating the amount of iodinated ligand displaced by 10-10,000 pg of D-[Leu’ ,des-Gly-NH2 i01 GnRH ethylamide. At least four dilutions of pooled extracted seminal plasma were tested in the radioreceptor assay. Statistics

Values are reported as mean ± SEM for each group. One-way and two-way analyses of variance (ANOVA) were performed as well as multiple regression analysis. Displacement curves were compared by iterative nonlinear sigmoidal curve fitting using the ALLFIT program (DeLean et al., 1978). Curves were first fitted to allow four parameters per curve and then progressively forced to fit under assumptions of common slope and/or ED5, until a significant departure from goodness-of-fit of individual curves or a deterioration of overall residual mean square variance occurred. RESULTS Measurement

of GnRH-like

Individual

Seminal

20 of

x

samples 106

with

sperm/ml

GnRH-like

from pg/mi.

Material

sperm

less than 213 ± 42 pg of seminal plasma. counts

counts

contained

material,

vasectomized These results

in

Samples

samples with sperm sperm/mI contained material/ml of

Semen

20 x 106 GnRH-like Semen

Plasma

greater

229.0

and

semen

men contained were obtained

than

± 66 pg/ml collected 252 ± 36 using the

antibody raised against D-[Lys6JGnRH and Odell, 1978). The differences measured were not significant. No

(Heber in levels correlation

was

amount

found

between

sperm

count

and

of

372

SOKOL

GnRH-like plasma

in

spermatozoa

material

measured/ml

samples

that

of

initially

seminal contained

Ultrafiltration

dilutions retentate

of the containing

500-30,000-D range curve approximately GnRH against

resulted parallel

material

was

measured

taining molecules the retentate

D when the generated

30,000

antibody

Immunoaffinity

fitting

curves using

the

the

the

generated was used GnRH-like

retentate

than 30,000 molcules

assays against

Column

Displacement

in

greater containing

seminal in the

in a displacement to that of

standard when antibody the D-[Lys6] GnRH analog 1, labeled preaffinity). No

(Fig.

curve

extracted molecules

conD nor in of 500-

were performed native GnRH.

using

ALLFIT

fitted

al.,

1978)

(Fig.

1).

Preaffinity

by nonlinear

Dilutions

(DeLean

of Seminal 1:40

1:20

extract

but not with the standard curve. hand, the standard curve was

parallel materials ± 0.11),

with both such postaffinity (pooled common slope factor indicating purification of

seminal

plasma

after

eluted -0.73 extracted =

immunoaffinity

column

chromatography. Chromatography using

of extracted

concanavalin

A

assayable GnRH-like material of methyl-c-D-glucopyranoside gesting

that

at least

one

is a glycoprotein.

chromatography neutralization

Plosma 1:10

seminal

yielded

of

to of the

plasma

radioimmunoafter

the

the addition (-GMP), sug-

proteins

Incubation

plasma with the antibody D- [Lys6] GnRH analog of material measured

program

crude

extract containing molecules and 30,000 Da were parallel (pooled common slope factor =

-1.84 ± 0.47) On the other

fied

Chromatography were

et

and the preaffinity between 5000 with each other

(r=-0.039).

Serial plasma

ET AL.

identi-

of the

seminal

generated against the decreased the amount after concanavalin A

one third. protein by

This indicates the antibody.

Extrocts :5

I00-

pr e - affinity

80-

-

60-

Amicon 500

-

filtrate

Crude

Extract

30,000

0

ID’

40-

post-affinity

20-

1.0

10

GnRH

00

1000

(pg/tube)

FIG. 1. Displacement of the binding of 1251-GnRH to D-[Lys’ IGnRH antibody by extracted seminal plasma. Aliquots of pooled acid-alcohol-extracted seminal plasma, both as a crude extract (solid lines) and after filtration through 2 filters with exclusion limits of 500-30,000 Da (dashed lines) were individually chromatographed on immunoaffinity columns (cyanogen-bromide-activated Sepharose 4B beads covalently bound to an antibody raised against D-[Lys6 I GnRH). ., Pre-affinity values; 0, post-affinity values.

GnRH-LIKE

FACTORS

IN HUMAN

SEMINAL

PLASMA

373 50

M,ogI,b.,

((7.800)

40-

I

V0

[“i] co(on,

CeVVIVoo,

(3,300)

I

II

Ill 8 -.-

Mle,iI

i,,AH-Ijke

30

I

j

(‘-26000)

[“ii

#{149} .

C

300

GnRH ((2000)

I

i,s

I

II 20

I ‘

A

Se,n,,oI

#{149} #{149}

I

I

I ‘

60

60

00

120

140

leO

150

220

240

200

VOLUME

FIG.

2. Elution

without

Gel

profile

preincubation

Filtration The

260

aeo

seminal plasma. were chromatographed

125

seminal

places, corresponding The elution profile modified after incubation plasma

for

plasma to of

2600 and I-GnRH of label with

in two 5000 Da. was not extracted

38 h at 4#{176}C(Fig.

Gonadotropin-releasing

2).

Assay

Scatchard binding data

(RRA)

analysis demonstrated

of

of GnRH association

the radioreceptor significant specific

agonist constant

ligand (Ka)

with an of 3.0 x

1010M-1 compared to a Ka of 2.7 x 1010M in the same assay for the index GnRH agonist compound D-[Leu6des-Gly’#{176}] GnRH ethylamide. To verify that displacement of the iodinated

label

of the run

label

in the

in the by

RRA

a tissue

presence

5 bzg/ml soybean was not shifted

was

not

protease, of

10

I

300

320

I

I-”

340

360

380

due the

bIg/mi

to cleavage assays

were

bacitracin

and pooled on an Ultra

120

requires for

trypsin inhibitor. in the presence

Displacement of protease

DISCUSSION

plasma. GnRH

suggest factors

species of in seminal

At least one of these proteins manifests receptor binding properties. These newly

identified

factors

proteins chemically

that from

interacted

against

that two present

are

with

the

middle

appear differ native an

to

be

glycosylated

immunologically GnRH. These

antiserum

region

440

extracted

both

440

480

seminal

500

-NH2

plasma

and

GnRH decapepantiserum that

with and

binding.

with

column.

hypothalamic not react

the

effective

These

-COOH

termini

findings

suggest

that native GnRH and GnRH-iike factors seminal plasma share amino acid sequences the middle and at the -COOH terminus.

in in The

GnRH

in extracted

is directed

that

and

at

and factors

the

-COOH

seminal

plasma

for

48

h at

4#{176}C did not modify the elution profile of the 1251-GnRH; and the radioreceptor assay displacement curve was not shifted in the presence of protease inhibitors. The levels of GnRH-iike material four

reported

in our

study

than

the

Tang to

(1983). differences

This

function

and

times

greater

Chan and attributed

are approximately levels

reported

discrepancy in extraction

by can

be pro-

cedures. The factors dated.

in seminal Testicular

shown

to cell

Similar human

observations testicular

ties

of

nal

plasma

source. excreted

assay

Seminal by the

glands,

and

1981).

Numerous

identified

GnRH-like to

be have

elucibeen

hormone

the

mouse

and

Swerdioff,

(LH) in

vitro 1984).

have not been reported in tissue. Because of the difficulhuman selected

testicular as an

tissue, alternate

plasma is composed testes, seminal vesicles,

prostate in the

in

(Bhasin

procuring was

of

remain factors

luteinizing

release

Leydig

origin

plasma GnRH-like

modulate

testosterone results

420

gel Aca 202

terminus of the tide. They did

and

inhibitors.

Our GnRH-like

400

(mis)

possibility that these immunoreactive factors are artificially produced by enzymatic cleavage of label is unlikely. Incubation of radiolabeied

Hormone

Radioreceptor

displacement equilibrium

eluted

I0

/

I

COLLECTED

Chromatography

extracted

seminal

of extracted l-GnRH

with

I’l

0.

48h,oI4C

I

/ I

E 20

I

I,,

I’

(-50000)

I

PIos,,,o

!

A

10

40

I

(12.400)

C

(Mann peptide

seminal

and

of fluids Cowper’s

Mann-Hutwak,

hormones

plasma

semihuman

have

of

man.

been

These

374

SOKOL

include

follicle-stimulating

LH,

prolactin,

mone 1978; 1983). to

and

(Sheth Smith Inhibin,

et and

have

a role

in the

male, plasma

has of

of GnRH-like

trations markedly

in normal decreases

origin areas

hor-

secretion

also man

of pituitary

been and

FSH

identified in bull (Franchirnont

1978; Peek and Watkins, 1979; Scott 1980; Ramasharma et a!., 1984).

presence

are

(FSH),

al., 1975; Schoenfeld et al., Lugman, 1982; Pekary et al., a testicular substance postulated

in the seminal et al., Burger,

hormone thyrotropin-releasing

the

site and

of

in similar

their

production. activity

of

semen testes

the

The these

and The

concen-

and vasectomized the likelihood that

biologic

of future

factors

the

sites

factors

of

study. ACKNOWLEDGMENTS

Belsey,

M. A., Eliasson, R., Gallegos, A. J., Moghissi, K. S., Paulsen, C. A. and Prasad, M.R.N. (1980). World Health Organization Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Press Concern, Singapore. Bhasin, S. and Swerdloff, It. S. (1984). Testicular GnRH-like factors-characterization of biologic activity. Biochem. Biophys. Res. Commun. (in press). Bhasin, S., Heber, D., Peterson, M. and Swerdloff, R. S. (1983). Partial isolation and characterization of testicular GnRH-factors. Endocrinology 112: 1144-1146.

Bourne, G. A., Regiani, S., Payne, A. H. and Marshall, J. C. (1980). Testicular GnRH receptors. Characterization and localization on interstitial tissue. Clin. Endocrinol. Metab. 51:407-409. Clayton, R. N., Katikinen, M., Chan, V., Dufau, M. and Catt, K. J. (1980). Direct inhibition of testicular function by GnRH: mediation by specific GnRH receptors in interstitial cells. Proc. Nat!. Acad. Sci. USA 77:4459. Chan, S.Y.W. and Tang, L.C.H. (1983). Immunoreactive LHRH-like factors in human seminal plasma. Arch. Androl. 10: 29-32. DeLean, A., Munson, P. J. and Rodbard, D. (1978). Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am. J. Physiol. 235:97-102. Dutlow, C. M. and Millar, R. P. (1981). Rat testis immunoreactive LHRH differs structurally from hypothalamic LHRH. Biochem. Biophys. Res. 101:486-494. P., Demoulin,

A.,

1. 5. and of action of inhibin: perspectives in regulation of male infertility. Int. J. Androl. Suppl. 2:69-79. Heber, D. and Odell, W. D. (1978). Development of a GnRH RIA utilizing a superactive synthetic GnRH analog D (Lys)’ GnRH. Proc. Soc. Exp. Biol. Med. 158:643-646. Heber, D. and Odell, W. D. (1979). Estrogen modulation of pituitary LHRH receptor in the rat: in vivo and in vitro studies. Am. J. Physiol. 237: 136-141. Mann, T. and Mann-Hutwak, C. (1981). Male Reproductive Function and Semen. Springer-Verlag, Berlin, Germany, pp. 268. Nett, T. M. and Niswender, G. D. (1978). Gonadotropin-releasing hormone. In: Methods of Hormone M.

(1978).

Radioiminunoassay

(B.

Walton,

T., Nature

M.

and

Jaffe

mechanism

and

H.

R. Behr-

man, eds.). Academic Press, New York, pp. 5776. Nett, T. M., Akbar, A. M., Niswender, G. D., Helund, M. T. and White, W. F. (1973). Radioimmunoassay for GnRH in serum. J. Clin. Endocrinol. Metab. Peek,

36:880-885. J. C. and

Watkins,

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W. B. (1979).

in seminal plasma bulls. J. Reprod.

Gonadotropinfrom intact Fertil. 57:

A. E., Hershman, J. M. and Freidman, S. (1983). Human semen contains TRH, a TRHhomologous peptide and TRH binding substance. J. Androl. 4:399-407. Pharmacia Fine Chemicals. (1979). Affinity chromatography. In: Principles and Methods. Pharmacia Fine Chemicals, Uppala, Sweden, pp. Pekary,

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