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.
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(B.
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T., Nature
M.
and
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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,
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