THOMAS. A. KOST. Department of Microbiology,. N. C. State. University,. Raleigh, ..... (Lowry et al.,. 1951). In addition,. Fraction. B was tested for the presence.
BIOLOGY
OF
REPRODUCTION
15,
Chromatographic
260-265
(1976)
Separation
of Puberty Male
JOHN
Mouse G.
of Mental
Research North S.
Health,
Carolina
FINLAYSON
Bureau of Biologics, and Drug Administration,
Food
Bethesda,
J.
from
Division,
Raleigh,
WALTER
Pheromone
VANDENBERGH
N. C. Department
J.
Accelerating Urine1
DOBROGOSZ,
Maryland
STEVEN
Department
S.
of
N.
State
C.
Raleigh,
DILLS
and
THOMAS
A.
KOST
Microbiology, University,
North
Carolina
ABSTRACT Dialysis and sequential ultrafiltration were used to separate urine from adult male mice into a number of fractions, all of which proved to be capable of accelerating the sexual maturation of female mice. Chromatography of the lowest molecular weight fraction on Sephadex G-15 yielded several fractions, only one of which was active. The active fraction was eluted at a position corresponding to a molecular weight of 860. It contained no detectable steroids but gave a strong positive test peptides.
for
Vandenbergh,
INTRODUCTION The is
rate
of
sexual
accelerated
(Vandenbergh, presence
other
on
the
increased of
pubertal adult
Bronson
within
male
(Bronson
and
produced
of by
by urine
a
The
puberty
exposure male
by
circulating four-fold
after
in to
females
(Vandenbergh, and
Wise,
Accepted May 11, 1976. Received March 16, 1976. ‘We thank Carol Bradley for bioassay procedure. This work Public Health Grant MH 16870 Institute of Mental Health to JGV.
can
Colby
or
al., the
1975). active
it of
Further component
to
iso-
accelerating is
associated male
urine
progress in
urine
in is
here.
AND
METHODS
Bioassay
be
The procedure for assaying the activity of urinary fractions depends upon the detection of uterine hypertrophy coincident with the onset of estrus cyclicity. The procedure has been described in detail previously (Vandenbergh et al., 1975). In brief, it
material 1969)
1972;
that
component et
of
steps for
to 1973;
bedding
indicated
MATERIALS
in
exposure Stetson,
responsible
protein
isolation
reported
1974).
early
(Cowley
h and
the
ef-
mediated since
the
(Vandenbergh
al.,
acceleratory
rise
4
the et
pheromone puberty
with
Preliminary
1974).
the
female
male
by
(Vandenhergh
apparently
Desjardins,
mice a
inhibited
hormone
females
female of
activity
luteinizing
Induction
male
is
hypophyseal
levels
soiled
and
1974). female
in
presence
females
Drickamer,
fect
an
the
1967) of
1972;
maturation
by
late
by
Procedure
employs female albino mice (Mus musculus) of the Swiss Webster strain born in the laboratory and reared under standardized conditions of food, water, caging, photoperiod, humidity, and litter size. Females are weaned and individually caged at 21 days of age. Beginning at 28 days of age and for 8 consecutive days thereafter, 0.05 ml of a control or test substance is applied directly to each female’s oro-nasal groove daily. At 36 days of age females are killed and their uteri are removed and weighed.
and
assistance with the was supported by from the National
260
PUBERTY
Dialysis
ACCELERATING
Procedure
this
In
Exp. I, 16 ml of urine was collected from 20 adult male stud mice by gentle massage of the bladder, pooled, sterilized by filtration through a Millipore Swinnex filter with a 0.45 m pore size, and divided into two equal fractions. One fraction was stored for 4 days at 5#{176}C and the other 8 ml was dialysed against 40 ml distilled water at 5#{176}C for 48 h with one change of water. Both the dialysate and retentate were freezedried and reconstituted to the original 8 ml quantity with distilled water. Thus, from the original urine collected we were able to test whole urine as well as the retentate and dialysate after dialysis and freezedrying.
Ultrafiltration
Procedure
In
Exp. II, 24 ml of urine was collected from 20 adult male stud mice and filtered as described above. The urine was further clarified by cen trifugation at 5000 X g for 10 mm at 4#{176}C prior to ultrafiltration using a RC2 Refrigerated Sorval Centrifuge with an SS1 Rotor. The supernatant fluid was filtered by means of an Amicon 202 stirred ultrafiltration cell equipped with Amicon series UM ultrafiltration membranes. The operating pressure for all filtration was 55 psi. Each retentate was washed with 10 ml distilled water. The urine was initially filtered through a UM1O membrane, general retentivity greater than 10,000 molecular weight. The filtrate was collected and filtered through a UM2 membrane, general resensitivity greater than 1,000 molecular weight. The filtrate was again collected and filtered through a UMO5 membrane, general retentivity greater than 500 molecular weight. The filtrate passing the UM1O membrane and each of the filtrates (including the wash) were freeze-dried and stored until tested. Prior to delivery to females each fraction was reconstituted with 6 ml distilled water to return it to the original concentration found in whole male urine.
Chromato
graphic
Procedure
In
Exp. III, 30 ml urine was collected from 20 adult male stud mice and processed as described under the ultrafiltration procedure. An aliquot of the UMO5 filtrate was retained for testing and the remainder was lyophilized and reconstituted to a volume of 3 ml with 0.1 M ammonium bicarbonate, pH 7.8. The sample was applied to a 75 X 1.5 cm column packed with G-15 Sephadex (Pharmacia Fine Chemicals) which had been equilibrated with 0.1 M ammonium bicarbonate. The column was developed at room temperature with
TABLE
PHEROMONE
1. Standard
compounds
used
to estimate
molecular
same
solution.
261
A
flow
rate
of
1.5
mI/mm
For
estimation
of the
molecular
weight
of
Fraction
B (Fig. 2) several standards were chromatographed. The void volume of the column, approximately 40 ml, was indicated by the elution volume of blue dextran (Pharmacia Fine Chemicals) and cytochrome c (Sigma Chemicals), which emerged at the same position. The standards (Table 1) together with Fraction B were passed through the column and the elution volumes were measured. The log, molecular weight was plotted vs. effluent volume to obtain a standard curve. The molecular weight of Fraction B was then estimated from the graph. Selected fractions from each of the procedures described above were analysed for total protein (Lowry et al., 1951). In addition, Fraction B was tested for the presence of steroids by the hot sulfuric acid reaction and by the 10 percent phosphomolybdic acid reaction in ethanol (Edwards, 1969). Fraction B was also subjected to two-dimensional paper chromatography by the following procedure: 25 MI of Fraction B was spotted on one corner of a 21 X 21 cm sheet of Whatman 31 ET paper and chromatographed in an ascending manner in two phases. In phase one, phenol saturated with H2 0 in the presence of 3 percent ammonia was used with a 4.5 h development time. The chromatograph was dried overnight and rinsed three times with ether. In phase two, n-butanol:HCOOH:H20 (100:30: 25) was used with a development time of 2 h. The chromatograph was then dried and sprayed with ninhydrin and aniline phosphate.
weight
of Fraction
B.
Molecular Compound
Cytochrome
c
Bacitracin NADH Glutathione
(reduced)
was
achieved by the use of a peristaltic pump. Fractions of 2 ml were collected and absorbance was measured at 280 nm using a Beckman DU spectrophotometer. On the basis of the elution pattern obtained (Fig. 1) fractions were combined into two pools (I and II) representing the major peaks and a third pool (III) consisting of material eluted later. The fractions were pooled, lyophilized, and reconstituted to half the original urine volume with distilled water. This doubling of presumed concentration was thought necessary to compensate for losses during the procedure. In Exp. IV, a similar quantity of urine was processed as described for Exp. Ill; however, only the fractions indicated on Fig. 2 were prepared for bioassay. Those tested were Fractions B, C, D (i.e., subdivisions of Fraction I, which proved active in Exp. Ill); Fraction E, which was eluted just after the latter; Fraction A, which comprised all fractions collected prior to Fraction B, including those representing the void volume; and a recombined fraction made up of equal volumes of Fractions A through E.
weight
Source
12,400 1,450 665 307
Schwarz-Mann Schwarz-Mann Sigma Chemical Sigma Chemical
Biochemicals Biochemicals
Company Company
VANDENBERGH
262 TABLE 2. Mean body weight rated by dialysis or membrane
and uterine filtration.
weight
ET
of female
AL.
mice
after
Bo dy wt.
Experiment
N
5
14 14 14 14
18.3 18.5 18.2 17.9
13 13 13 13
Fjltrate-UMO5 Water ANOVAb different
from
experiment
bwithmfl
water
analysis
Bioassay of plus the retentate
urine and
urine induced
I revealed hypertrophy
in
Exp. uterine
used
as
a
controls
of variance
weight induced
by
that
of to
filtration uterine
The
results
and dialysate fractions that the pheromone
of
a large and
being
assumption be expected
the
and
molecule, was
filtrate
is
9.31a 19.60 12.59a 3.39
molecular was then
the
volume, contained the other fractions in uterine weight major
10.89
8.45a 11.25 2.08
the i.e.,
fraction viz.,
were
before
and
tively) equal
and peak
(Vanto the
Fraction
D,
compared
after
the
and
Fig.
Fraction
I (A
indicated found
was
of by
in the
in-
original
(Filtrate-UMO
recombined
after
in Fraction eluted after
B, which the void
was the volume.
Using standards of (Table 1) chromatographed of
E, respec-
sample consisting A through E.
chromatographed
weight
eluted
and
material
molecular
tested.
material
as
weight
narrower
2) were
with
activity
uterine
noted
in
C, and
and a recombined volumes of Fractions
substance
in
repeated
was
(B,
These
creased
(Exp. I, Table bound to or
12,000
known this
5),
separation,
first
discrete
molecular with Fraction
fraction
was
weight B the estimated
molecular released.
the in its
If
I (Fig.
‘III’
25 11
molecular to about
1?
chromatogthe compo-
after
the
activity 1 and
Table
ii
void
(Table 3). None of significant changes with water. Once
containing
II
‘
24
membrane.
lowest those up
I
28
pheromone purest form
UMO5
eluted
activity induced compared
experiment
subfractions
filtrate (Fig. 1). Bioassay of (Exp. Ill) revealed that only first
data.
was
over
the
P
