Endocrinol.
Japon.
1977,
24 (6), 545-550
Further Studies on Gonadotropin-Inhibiting Substances Excreted in Human Urine MINORU
OTA,
NOBUKO
SATO
AND KIJURO
OBARA
Department of Biochemistry, Iwate Medical University of Medicine, Morioka, Iwate-ken 020, Japan
School
Synopsis Partial separation of human urinary substances which have properties to suppress the ovulation induced by PMS and HCG in mice was attempted by gel filtartion on Sephadex G-100 and ultrafiltration using the Amicon Diafio membranes UM-2 and UM-10. In addition to a thermostable inhibitor which has a molecular weight more than 10,000, the presence of a heat-labile inhibitor with a molecular weight less than 1,000 was newly demonstrated.
A substance, so called gonadotropininhibiting substance (GIS), which inhibits the stimulatory effects of HCG on mouse uterine weight (Safer et al., 1961) and suppresses the ovulation induced by PMS and/or HCG in mice (Dronkert et al., 1963 ; Ota et al., 1968a, 1968b) has been found in pubertal and adult human urine. The inhibitor can be extracted from urine specimens according to the tannic acid precipitation method used for the extraction of gonadotropins excreted in the urine. The inhibitor also can be obtained from the precipitate of acidified urine by a borate buffer extraction (Ota et al., 1967). In adult urine in which the activity of gonadotropin is predominant over that of GIS, the heat-stable inhibitor is demonstrable after the destruction of gonadotropin activity present in the extract by heating in a boiling water bath. The existence of GIS was also demonstrated in the urine of albino rats (Ota et al., 1970) and the pineal gland was postulated Received
April
15, 1977.
as a possible origin of this substance (Ota et al., 1971). Our recent studies provide evidence indicating the presence of two types of antiovulatory substances, a high molecular one and a low molecular one which is not thermostable, in the bovine pineal (Ota et al., 1975; Ota et al., 1976). In our previous study which dealt with chromatographic separation of GIS in the adult male urine by Sephadex G-100 column, an inhibition of ovulation was observed in the earlier emerged fractions and a weak inhibition was also found in the far later fractions (Ota and Dronkert, 1969). However, little investigation had been carried out on the far later fractions. In the present study, therefore, attention was given to a low molecular inhibitor and separation of these two inhibitors was attempted using gel filtration
and
ultramembrane
Materials
and
filtration.
Methods
Urine specimens were collected from healthy adult males during their working hours and pooled in a refrigerator without using preservatives for 1-4 days before they were processed.
546
OTA
Endocrinol. Japon. December 1977
et al. UM-10
residue
(Fraction Separation
of
Firstly
GIS
GIS
according
to
Johnsen
by
was the
(1958),
gel filtration
extracted
between
from
tannic
acid
which
is
the
pooled
the
extraction
precipitation used
for
urine
method
dissolving
gonadotropins.
with
ether
and
The final
dissolved
in
sodium
borate
buffer
(pH
obtained
was
heated
in
hour
to
the
destroy
pooled
acetic
acid
The
precipitate
tion.
The
twice,
8.6).
in
to
was was
ethanol
half
the
4.0
twice
and
with
centrifuga-
2%
ether
The
ing
ether,
to
Ota
the
et
al.
precipitate
was
of
(1967).
The
A
small
redissolved
borate
in
buffer.
Johnsen's
a
small
These
weights
extract,
the
heated
the
of
In
Ota's
extract
prepared
horn
20mm
6l
column
sodium
of
The
of
of
borate
sorption
After accord-
0.1M
e.g.,
of
sodium
the
Johnsen's
19-20
day
acidified
buffer
these
G-100, (pH
8.6).
extract,
fractions
was
assay
old
immature
female
constant
minimum
a
with
and
acidified
activity
borate
and
PMS
was
followed of
by
HCG
an
at
after
the
the
number
Each
injected
the
F1 mice.
ovulation,
intraperitoneally
1.25
second
ova
fraction
I.U.
per
the
third
injection,
of
mixed
mouse)
the
in
the
mice
I.U.
buffer
in
limit
of
the
number
standard
of
ova
deviation
divided
into
cording
to
an
indication
•vulation in
of
over
the
the
controls
presence
of
the
above
hr
(0.125
mice.
was
taken
pituitary
as
of.
GIS from to
was 10l
Ota
mg)
from
of
acidified
pooled
al.
(1967).
buffer
was
GIS.
Super-
indication
of
the
15min.
44.3mg.
material
of
sodium
The
The
weight
supernatant
ultramembrane filter
a
partitions
mixtures
molecular
of
solution
weight
nitrogen
pressure.
materials
below
ultrafiltration
The the
using MW
(MW)
substances
of the
above
104 MW
greater
range membrane than
with
the
The of
sodium
same
10-ml
buffer.
fractions I
absorption
lyophilized.
and
and
curve
The water
15min.
II,
ac-
(280nm).
residue and
Each
obtained
centrifuged
extract
at
was filtered
ultramembrane filter pressure.
to Thus,
UM-10
The
ultrafiltration
at
4•Ž
UM-10 filtrate
stances
above
between
104
using
the
UM-10
104MW
and at
a
was
Diafio
residue
and
on
(pH
8.6).
precipitate was
the
The
in
10ml
The
solution
membrane
contained
UM-02
was
hardly
UM-10 of
below 4•Ž
the under
added
Thus,
each
three
portions.
A
to
deionized
sub-
residue
or
UM-02
0.01M
did residue
sodium
obtained
was
obtained)
and
the
borate centrifuged
and
a
Sephadex
Fractions
103,
I
and
that
above
were F-1
104
and
and
solution
was
divided 104,
103
did
into F-I-B;
and
It was
contained
was column.
MW> MW>
F-II
super-
deionized
G-10
obtained.
mainly
the of
the
II
F-I-A; F-I-C;
F-II-C
volume
residue,
Thus,
and
course
small
through of
MW>
tion
effects
induced
the
mice
of
to which
1 I.U.
of same
the
at
the
PMS mice
this
F-II-A,
the
matter
materials
materials
F1
PMS
with
with
dose
human Another
fractions
and
HCG the
and
were
0.1 as
smaller
One
corresponding
the
was of the
mouse
to
As
HCG,
obtained
HCG
were
injected using
the of
a when
experiment
500ml
using
strain.
received to
ovula-
tested
sensitive
I.U. in
on were
ICR-SLC
ovulation
manner mice.
these
of
strain
minimum
in
the
of
with female
(cx129)
subjected
Thus,
lyophilized.
was
partly
constant
generally
UM-02 103.
103.
dissolved
23-day-old
precipitate
containing was
and
Inhibitory at
placed
UM-10 filtrate
a Diafio
min.
molecules.
(464.5
which
range,
eluted
deionized
Diafio
UM-02.
borate
centrifuged
was
UM-10,
and
ultraviolet
nitrogen
MW
according
lyophilized
was
the
subjected
of
obtained
(pH4.0)
solution
was,
0.01M
Fractions
for
F-II-B
0.1M
night
column
with
portions,
in
water
deviation
precipitate
urine
20ml
The
for
Diafio
retains
in
(pH8.6).
3,000rpm
104
The
dissolved
the
15
450•~25mm
two
was
(the
ultramembrane
extracted
et
was
by
over
the
gonadotropins.
GIS
for a
in
portion
104> Separation
on
collected
dissolved
was
animals,
standard
an
stand
A
below
control of
of
2,000rpm
were
was
under
and
HCG
mouse,
presence limit
0.1M
mixture
to
equilibrated
Each
natant was
The
determined.
of
the
of
I.U.
killed
three
per in
at
(pH8.6)
eluates
buffer decrease
aliquot
20ml
I.U.
0.125
were
into
lyophilized 101
in
allowed
placed
G-100
3,000rpm
p.m.,
Eighteen
was
0.375
injected
5
of
day.
oviducts
with
was
at
injection
on
The
meausing
(cx129)
intraperitoneally
3 p.m.
was
390.1mg,
the
(pH8.6). and
was
through of
were
another
dissolved
buffer
centrifuged
The
280nm.
fraction
hybrid
C
ab-
at
the
or
weight.
C
from
was
homogenized
borate
0.05M
for
in
and
(Fraction
B
dry
experiment, obtained
urine
Sephadex
a 900•~
ultraviolet
employed
inhibitory
obtain
on
eluted
measured
was
of
B
fraction
103 A,
of
small
respectively.
following
(407mg)
then,
was
placed The
This
MW
and
a
unheated
urine-each
urine-were
Sephadex
ovulation
surement
To
from
G-10 in
buffer.
Fractions
A,
deionized
dissolved
than
of
21.9mg,
the
supernatant the
Sephadex
measurement
Fractions
and
partially
of
material
of
extracts,
by buffer
acid
once.
volume
three
collected borate
was
borate smaller
aliquot
for
sodium was
respectively.
sodium
was
sodium
substances
8.2mg
acetic
lyophilized
B),
M
MW
substances
was
column material
0.01M
contained
served
days.
extracted
0.01
UM-02 filtrate
The
material removing
of
104
did
membrane
a 250•~25mm
C).
glacial
by
8.6).
volume
one
three
with
above
residue (Fraction
the
10ml
lyophilized.
extract for
for
collected
103 MW on
with
through
0.1M
Secondly,
pH
washed
washed
bath
UM-02
of
of
of
water
a refrigerator
formed precipitate
95%
A
acidified
stored
was
volume
gonadotropins.
was
and
small
a boiling
pituitary
urine
residue
a
substances
the
and
material
(pH urinary
and
104
Each of
contained
A)
the
the extract pooled
urine. aliquots
of
Fraction
I-A
(MW>
104)
and
Vol.
24.
No.
GONADOTROPIN-INHIBITOR
6
Fraction II-C (MW 104) as indicated by a low percentage of ovulation and significantly reduced number of ova per mouse as compared to those in the control. A weak inhibition was found in the mice given Fraction C (MW< 103), whereas no inhibition of ovulation was observed in the mice injected with Fraction B (104>MW> 103). As shown in Table 2, similar results were observed in the experiment using Fractions I-A, B and C, and Fractions IIA, B, and C, which were obtained by gel filtration of Sephadex G-100(as indicated
548
OTA Table
1.
1)
Each
mouse
2)
Each
mouse
3)
500ml of Data analyzed
4)
Data
1) Each 2) Each
Inhibitory effects of fractions of urinary extracts separated on ovulation induced with PMS and HCG in mice.
received , in male
analyzed
Table
Endocrinol. Japon. December 1977
et al.
adult by chi by
2.
mouse mouse
1 I .U.
addition
to
of
urine. square
t test
.
PMS,
PMS test *
followed
plus
.
P
Japon.
1977,
24 (6), 545-550
Further Studies on Gonadotropin-Inhibiting Substances Excreted in Human Urine MINORU
OTA,
NOBUKO
SATO
AND KIJURO
OBARA
Department of Biochemistry, Iwate Medical University of Medicine, Morioka, Iwate-ken 020, Japan
School
Synopsis Partial separation of human urinary substances which have properties to suppress the ovulation induced by PMS and HCG in mice was attempted by gel filtartion on Sephadex G-100 and ultrafiltration using the Amicon Diafio membranes UM-2 and UM-10. In addition to a thermostable inhibitor which has a molecular weight more than 10,000, the presence of a heat-labile inhibitor with a molecular weight less than 1,000 was newly demonstrated.
A substance, so called gonadotropininhibiting substance (GIS), which inhibits the stimulatory effects of HCG on mouse uterine weight (Safer et al., 1961) and suppresses the ovulation induced by PMS and/or HCG in mice (Dronkert et al., 1963 ; Ota et al., 1968a, 1968b) has been found in pubertal and adult human urine. The inhibitor can be extracted from urine specimens according to the tannic acid precipitation method used for the extraction of gonadotropins excreted in the urine. The inhibitor also can be obtained from the precipitate of acidified urine by a borate buffer extraction (Ota et al., 1967). In adult urine in which the activity of gonadotropin is predominant over that of GIS, the heat-stable inhibitor is demonstrable after the destruction of gonadotropin activity present in the extract by heating in a boiling water bath. The existence of GIS was also demonstrated in the urine of albino rats (Ota et al., 1970) and the pineal gland was postulated Received
April
15, 1977.
as a possible origin of this substance (Ota et al., 1971). Our recent studies provide evidence indicating the presence of two types of antiovulatory substances, a high molecular one and a low molecular one which is not thermostable, in the bovine pineal (Ota et al., 1975; Ota et al., 1976). In our previous study which dealt with chromatographic separation of GIS in the adult male urine by Sephadex G-100 column, an inhibition of ovulation was observed in the earlier emerged fractions and a weak inhibition was also found in the far later fractions (Ota and Dronkert, 1969). However, little investigation had been carried out on the far later fractions. In the present study, therefore, attention was given to a low molecular inhibitor and separation of these two inhibitors was attempted using gel filtration
and
ultramembrane
Materials
and
filtration.
Methods
Urine specimens were collected from healthy adult males during their working hours and pooled in a refrigerator without using preservatives for 1-4 days before they were processed.
546
OTA
Endocrinol. Japon. December 1977
et al. UM-10
residue
(Fraction Separation
of
Firstly
GIS
GIS
according
to
Johnsen
by
was the
(1958),
gel filtration
extracted
between
from
tannic
acid
which
is
the
pooled
the
extraction
precipitation used
for
urine
method
dissolving
gonadotropins.
with
ether
and
The final
dissolved
in
sodium
borate
buffer
(pH
obtained
was
heated
in
hour
to
the
destroy
pooled
acetic
acid
The
precipitate
tion.
The
twice,
8.6).
in
to
was was
ethanol
half
the
4.0
twice
and
with
centrifuga-
2%
ether
The
ing
ether,
to
Ota
the
et
al.
precipitate
was
of
(1967).
The
A
small
redissolved
borate
in
buffer.
Johnsen's
a
small
These
weights
extract,
the
heated
the
of
In
Ota's
extract
prepared
horn
20mm
6l
column
sodium
of
The
of
of
borate
sorption
After accord-
0.1M
e.g.,
of
sodium
the
Johnsen's
19-20
day
acidified
buffer
these
G-100, (pH
8.6).
extract,
fractions
was
assay
old
immature
female
constant
minimum
a
with
and
acidified
activity
borate
and
PMS
was
followed of
by
HCG
an
at
after
the
the
number
Each
injected
the
F1 mice.
ovulation,
intraperitoneally
1.25
second
ova
fraction
I.U.
per
the
third
injection,
of
mixed
mouse)
the
in
the
mice
I.U.
buffer
in
limit
of
the
number
standard
of
ova
deviation
divided
into
cording
to
an
indication
•vulation in
of
over
the
the
controls
presence
of
the
above
hr
(0.125
mice.
was
taken
pituitary
as
of.
GIS from to
was 10l
Ota
mg)
from
of
acidified
pooled
al.
(1967).
buffer
was
GIS.
Super-
indication
of
the
15min.
44.3mg.
material
of
sodium
The
The
weight
supernatant
ultramembrane filter
a
partitions
mixtures
molecular
of
solution
weight
nitrogen
pressure.
materials
below
ultrafiltration
The the
using MW
(MW)
substances
of the
above
104 MW
greater
range membrane than
with
the
The of
sodium
same
10-ml
buffer.
fractions I
absorption
lyophilized.
and
and
curve
The water
15min.
II,
ac-
(280nm).
residue and
Each
obtained
centrifuged
extract
at
was filtered
ultramembrane filter pressure.
to Thus,
UM-10
The
ultrafiltration
at
4•Ž
UM-10 filtrate
stances
above
between
104
using
the
UM-10
104MW
and at
a
was
Diafio
residue
and
on
(pH
8.6).
precipitate was
the
The
in
10ml
The
solution
membrane
contained
UM-02
was
hardly
UM-10 of
below 4•Ž
the under
added
Thus,
each
three
portions.
A
to
deionized
sub-
residue
or
UM-02
0.01M
did residue
sodium
obtained
was
obtained)
and
the
borate centrifuged
and
a
Sephadex
Fractions
103,
I
and
that
above
were F-1
104
and
and
solution
was
divided 104,
103
did
into F-I-B;
and
It was
contained
was column.
MW> MW>
F-II
super-
deionized
G-10
obtained.
mainly
the of
the
II
F-I-A; F-I-C;
F-II-C
volume
residue,
Thus,
and
course
small
through of
MW>
tion
effects
induced
the
mice
of
to which
1 I.U.
of same
the
at
the
PMS mice
this
F-II-A,
the
matter
materials
materials
F1
PMS
with
with
dose
human Another
fractions
and
HCG the
and
were
0.1 as
smaller
One
corresponding
the
was of the
mouse
to
As
HCG,
obtained
HCG
were
injected using
the of
a when
experiment
500ml
using
strain.
received to
ovula-
tested
sensitive
I.U. in
on were
ICR-SLC
ovulation
manner mice.
these
of
strain
minimum
in
the
of
with female
(cx129)
subjected
Thus,
lyophilized.
was
partly
constant
generally
UM-02 103.
103.
dissolved
23-day-old
precipitate
containing was
and
Inhibitory at
placed
UM-10 filtrate
a Diafio
min.
molecules.
(464.5
which
range,
eluted
deionized
Diafio
UM-02.
borate
centrifuged
was
UM-10,
and
ultraviolet
nitrogen
MW
according
lyophilized
was
the
subjected
of
obtained
(pH4.0)
solution
was,
0.01M
Fractions
for
F-II-B
0.1M
night
column
with
portions,
in
water
deviation
precipitate
urine
20ml
The
for
Diafio
retains
in
(pH8.6).
3,000rpm
104
The
dissolved
the
15
450•~25mm
two
was
(the
ultramembrane
extracted
et
was
by
over
the
gonadotropins.
GIS
for a
in
portion
104> Separation
on
collected
dissolved
was
animals,
standard
an
stand
A
below
control of
of
2,000rpm
were
was
under
and
HCG
mouse,
presence limit
0.1M
mixture
to
equilibrated
Each
natant was
The
determined.
of
the
of
I.U.
killed
three
per in
at
(pH8.6)
eluates
buffer decrease
aliquot
20ml
I.U.
0.125
were
into
lyophilized 101
in
allowed
placed
G-100
3,000rpm
p.m.,
Eighteen
was
0.375
injected
5
of
day.
oviducts
with
was
at
injection
on
The
meausing
(cx129)
intraperitoneally
3 p.m.
was
390.1mg,
the
(pH8.6). and
was
through of
were
another
dissolved
buffer
centrifuged
The
280nm.
fraction
hybrid
C
ab-
at
the
or
weight.
C
from
was
homogenized
borate
0.05M
for
in
and
(Fraction
B
dry
experiment, obtained
urine
Sephadex
a 900•~
ultraviolet
employed
inhibitory
obtain
on
eluted
measured
was
of
B
fraction
103 A,
of
small
respectively.
following
(407mg)
then,
was
placed The
This
MW
and
a
unheated
urine-each
urine-were
Sephadex
ovulation
surement
To
from
G-10 in
buffer.
Fractions
A,
deionized
dissolved
than
of
21.9mg,
the
supernatant the
Sephadex
measurement
Fractions
and
partially
of
material
of
extracts,
by buffer
acid
once.
volume
three
collected borate
was
borate smaller
aliquot
for
sodium was
respectively.
sodium
was
sodium
substances
8.2mg
acetic
lyophilized
B),
M
MW
substances
was
column material
0.01M
contained
served
days.
extracted
0.01
UM-02 filtrate
The
material removing
of
104
did
membrane
a 250•~25mm
C).
glacial
by
8.6).
volume
one
three
with
above
residue (Fraction
the
10ml
lyophilized.
extract for
for
collected
103 MW on
with
through
0.1M
Secondly,
pH
washed
washed
bath
UM-02
of
of
of
water
a refrigerator
formed precipitate
95%
A
acidified
stored
was
volume
gonadotropins.
was
and
small
a boiling
pituitary
urine
residue
a
substances
the
and
material
(pH urinary
and
104
Each of
contained
A)
the
the extract pooled
urine. aliquots
of
Fraction
I-A
(MW>
104)
and
Vol.
24.
No.
GONADOTROPIN-INHIBITOR
6
Fraction II-C (MW 104) as indicated by a low percentage of ovulation and significantly reduced number of ova per mouse as compared to those in the control. A weak inhibition was found in the mice given Fraction C (MW< 103), whereas no inhibition of ovulation was observed in the mice injected with Fraction B (104>MW> 103). As shown in Table 2, similar results were observed in the experiment using Fractions I-A, B and C, and Fractions IIA, B, and C, which were obtained by gel filtration of Sephadex G-100(as indicated
548
OTA Table
1.
1)
Each
mouse
2)
Each
mouse
3)
500ml of Data analyzed
4)
Data
1) Each 2) Each
Inhibitory effects of fractions of urinary extracts separated on ovulation induced with PMS and HCG in mice.
received , in male
analyzed
Table
Endocrinol. Japon. December 1977
et al.
adult by chi by
2.
mouse mouse
1 I .U.
addition
to
of
urine. square
t test
.
PMS,
PMS test *
followed
plus
.
P
