BIOLOGY
OF
REPRODUCTION
15, 43 5-443
Uteroglobin
W.
D.
(1976)
Messenger
RNA:
S. L.
C. WOO
BULLOCK,
Department
of Cell
Biology,
Translation
and
Baylor
Houston,
in vitro’
B. W. O’MALLEY
College
Texas
of Medicine,
77030
ABSTRACT
The messenger
RNA (mRNA)
coding for the progesterone-induced
protein uteroglobin
has been
extracted
from
endometrial
tissue
of rabbits
in early pregnancy
and enriched
by binding
to oligodT-ceilulose.
After
translation
in a cell-free
system
derived
from wheat
germ, total mRNA
activity
was assessed
by measuring
the incorporation
of 35S-methionine
into TCA-precipitable
peptides
and
specific
mRNA
activity
by immunoprecipitation
with
specific
uteroglobin
antibodies
purified
by
affinity
chromatography.
Approximately
85 percent
of total
mRNA
activity
was recovered
after
dT-cellulose
chromatography,
10 percent
in the bound
fraction
and 75 percent
in the unbound
RNA,
suggesting
that the majority
of endometrial
mRNA
species
lacks poly A sequences
of longer
than about
20 residues.
No poly
A could
be detected
by
H-poly
U hybridization
in the unbound
fraction.
In contrast,
69 percent
of total mRNA
activity
was present
in dT-bound
RNA from
rabbit
liver.
The immunoprecipitable
cell-free
translation
products
of endometrial
dT-RNA
gave a single
peak
of radioactivity
on electrophoresis
in 15 percent
polyacrylamide
gels containing
sodium
dodecyl
sulfate.
The peak was completely
displaced
by the addition
of an excess of authentic
nonradioactive
uteroglobin
to the immunoprecipitation
reaction
and
was absent
from
products
of
translation
without
added endometrial
RNA.
The cell-free
product
migrated
more slowly than
authentic
uteroglobin,
suggesting
the synthesis
of a precursor
protein.
No uteroglobin
mRNA
could
be detected
in dT-bound
RNA
from
rabbit
liver. The proportion
of uteroglobin
mRNA
in endometrial dT-bound
RNA
reached
a peak on Day 4 of pregnancy
and declined
subsequently
to non’
pregnant levels on Day 8, a pattern
similar
to that of uteroglobin
secretion.
INTRODUCTION
the
Uteroglobin
rabbit
is a major
uterus
during
secretory
protein
the preimplantation
stage of pregnancy
(Beier,
of
its alleged
embryotrophic
protein
Daniel,
firm
was called
“blastokinin”
1967).
Recent
work
of
effects
in vitro
Despite
the
growth
function,
for
studies
of
The
protein
(Arthur
and
by
No
on
of
induced
1972)
by
specific
marker
for
chemical
action
of progesterone
malian
uterus
is available.
We
have
begun,
molecular
mechanism
one on the synthesis
guish
between
ac-
on
the
in
studies
on
of
of
progesterTo distin-
action
of uteroglobin.
at the
of uteroglobin
of
which
the
in.
reveals
The
changes
early
transcriptional,
we
the
results
Received
June
April
Presented
ety
for
the Study
in
14,
1976.
28, 1976.
part
at the 8th
of Reproduction,
July
Meeting,
22-25,
Sod-
were
435
in
and
of
the
basis
of uteroglobin
germ
for
the
the
endo-
its translation
from
synthesis
female
flushed
with
AND
New
Zealand
pentobarbital
wheat
germ,
a pro-uteroglobfor
mRNA
studies
of
during
METHODS
White
rabbits
were
sodium
and the uteri
pregnancy.
The uterine
horits
saline,
slit longitudinally
and the
anesthetized
with
excised on Day 4 or 6 of
1975.
wheat
pregnancy.
Adult,
Annual
the
in a
sys-
mRNA
and its
evidence
mRNA
rabbits
provide
in levels
in the
derived
MATERIALS
Accepted
uteroglobin
rabbits,
present
uteroglobin
of pregnant
in a system
vitro
of
mRNA
vitro system.
In this
paper,
metrium
condi-
for
in oocytes
of Xen opus laevis.
Levey
(1976)
also have reported
the trans-
presence
biomam-
therefore,
an action
in
Willen,
the
level, specifbe quantita-
specific
protein.
have reported
re-
(1975)
the extraction
progesterone-stimulated
lation
is
corresponding
Rungger
translation
and Daniel
model
and
the
and
cently
from
progesterone
(Bullock
into
Beato
and its production
estradiol
tem
blastocyst
hormone
or translational
(mRNA)
must
RNA
ted in vivo
under
different
hormonal
tions.
This, in turn,
requires
a procedure
extraction
of mRNA
and its translation
cell-free,
heterologous
protein-synthesizing
basis
the
1976).
its biological
a useful
mechanism
is
other
Beier,
about
provides
Daniel,
modulated
On the
properties,
uteroglobin
the
of
(Krishnan
and
has failed
to con-
(Maurer
and
uncertainty
uteroglobin
tion.
1974).
1968).
posttranscriptional
ic messenger
BULLOCK
436
surface
endometrium
scraped
off by blunt
dissection
on a towel
placed
over ice. The tissue
was frozen
immediately
in liquid
N2 and stored
at -100#{176}C until
extraction.
In some experiments
the whole uterus was
frozen in liquid N3 immediately
after excision.
Extraction
of mRNA
The frozen
tissue
was weighed,
broken
into small
pieces and homogenized
in a Waring
blendor
in 5 volumes (vlw)
of a buffer
containing
sodium
dodecyl
sulfate (SDS)
and 5 volumes
of redistilled
phenol.
Various extraction
conditions
were employed.
Whole
uterine
tissue was extracted
initially
at room
temperature
in 0.5
percent
(w/v)
SDS,
0.025
M
Na3 EDTA
and 0.07 5 M NaCI,
pH 8.0,
and buffersaturated
phenol,
pH 8.0 (Rosen
et al., 1975).
Whole
uterus
was also extracted
at room
temperature
in 0.5
percent
(w/v)
SDS, 0.01 M NaOAc,
pH 5.2, and buffer-saturated
phenol.
In both
cases the extract
was
allowed
to stand
on ice for 30 min and centrifuged
at
5000
g for 10 mm. The aqueous
phase
and protein
interphase
were re-extracted
with
an equal volume
of
the
phenol
solution.
Following
centrifugation,
the
aqueous
phase
was removed
carefully
and brought
to
0.25
M in NaCI.
In the case of pH-8 extraction,
the
aqueous
solution
was overlayered
with
an equal
volume of cold 95 percent
ethanol
(EtOH)
and the DNA
was removed
by spoolin&
This step was omitted
from
the pH-5.2
procedure.
In later experiments,
whole
uterine
tissue or endometrial
tissue
was extracted
at 0#{176}C
in a pH-9 bufferphenol
mixture,
similar
to that described
by Brawerman (1973).
The frozen
tissue was homogenized
in a
Waring
blendor
with
5 volumes
of 0.1 M Tris-HCI,
pH
9, 2 mM Na2 EDTA,
0.5 percent
SDS and 5 volumes
of
cold watersaturated
phenol,
adjusted
to pH 9 with
10
N NaOH.
After
centrifugation,
the aqueous
phase was
removed
and the phenol
plus protein
interphase
Yeextracted
with
an equal
volume
of cold buffer.
The
aqueous
phases were combined
and extracted
3 times
with 2.5 volumes
of the phenol
solution,
until
free of
protein.
In all three
extraction
procedures,
the aqueous
phase was made 0.25 M in NaCI and total
nucleic
acid
precipitated
at -20#{176}C for 16 h by the addition
of 2
volumes
of cold
95 percent
EtOH.
After
centrifugation
at 5000 g for 15 mm the pellet
was vacuum-dried
and dissolved
in 0.01 M NaOAc,
pH 5.2. The ethanol
precipitation
was repeated
and the final
pellet
dissolved
in a minimum
cuantity
of deionized
water,
frozen
and stored
at -20
C.
The poly A-containing
RNA was fractionated
from
total
nucleic
acid extracts
by chromatography
over a
column
of oligo-dT-cellulose
(Collaborative
Research,
T3),
by a method
similar
to that of Aviv
and Leder
(1972).
The total
extract
was diluted
to 1 mg/mi
with
0.5 M KCI-0.01
M Tris-HC1,
pH 7.6, and applied
at a
flow
rate of 0.5 ml per mm to a column
of 5 g of
oligo-dT-cellulose,
previously
equilibrated
at
room
temperature
with
the same
buffer.
The effluent
was
monitored
at 254 nm and elution
with
the salt buffer
continued
until
the large peak of unbound
material
was excluded.
The poly A-containing
RNA
was eluted
from
the column
with
0.01
M Tris-HCI,
pH 7.6, and
the dT-bound
and flow-through
fractions
were pooled
separately.
with
NaCI
The bound
and RNA
was
fraction
was
precipitated
brought
from
to 0.25 M
both
frac-
ET AL.
tions
with
cold
95 percent
EtOH.
The peUets were
washed
with
EtOH,
dried,
dissolved
in water
and the
concentration
of RNA
determined
by reading
optical
density
at 260 nm,
assuming
25 A36 0unit
for a 1-cm
light path equals
1 mg/mi.
As a control
tissue,
rabbit
liver
from
Day
6 of
pregnancy
was also extracted
by the pH-9 procedure.
3H-poly
U Hybridization
The
total
(FT)
poly
extract
fractions
A content
of each of the endometrial
(TE),
dT-bound
(dT)
and flow-through
was assayed
by hybridization
with
‘H-
poly
U (Gillespie
et al., 1972).
Samples
were prepared
in a volume
of 100 Ml containing
5 to 50 zg RNA and
0.14
MCi of ‘H-poly
U (sp.
act.
26.3
mCi/mmol,
Schwartz-Mann)
in 8 mM Tris-HCI,
pH 7.2, 36 mM
sodium
citrate,
0.36 M NaCI, containing
50 percent
formamide.
Standards
of 27
to 212
ng poly
A
(Schwartz-Mann)
were prepared
in a similar
mansler.
From
each
sample,
25-izl
and
50-gil
volumes
were
sealed
in capillary
tubes
and heated
at 60#{176}Cfor
10
min, followed
by hybridization
at 36#{176}C
overnight.
The
samples
were then incubated
at 30#{176}C
for 2 h in 0.01 M
Tris-HCI,
pH 7.2, 0.5 M NaCI, 0.01 M MgCI2,
containing 5 gg/ml
of pancreatic
sibonuclease
A (Worthington)
and 20 Mg/mI
of deoxyribonuclease
1 (Worthington).
The hybrids
were precipitated
with
cold 10 percent trichloracetic
acid (TCA),
collected
on Milhipore
filters
and the radioactivity
counted
in Spectrafluor
(Amersham-Searle).
From
the poly A standard
curve,
the amount
of poly A in each endometrial
RNA
fraction
was calculated
and expressed
as a percentage
of
the amount
of RNA
added to the reaction.
Translation
Assay
mRNA
activity
in the TE, dT and FT fractions
was
aasessed by translation
in vitro
in a cell-free,
heterologous protein-synthesizing
system
derived
from
wheat
germ (Roberts
and Patesson,
1973).
The ingredients
of
the
assay
were
the
same
as described
previously
(Rosen
et al., 1975),
except
that ‘5S-methionine
(sp.
act. 275 to 340 Ci/mmol)
was used as the radioactive
precursor
and non-radioactive
methionine
(1 MM) was
induded.
Assays
were
performed
normally
in a total
volume
of 100 p1, incubated
at 25#{176}C
for 2 h. At the
end of incubation,
the samples
were
centrifuged
at
100,000
g for 60 mm, to remove
ribosomes.
Total
mRNA
activity
was determined
in a l5ijl
portion
of the postribosomal
supernatant
by precipitation
with
TCA.
From
a dose-response
curve
of TCAprecipitable
radioactivity
versus
pg of RNA,
the
specific
activity
(cpm/izg
RNA)
of the mRNA
was calculated
from
the linear
portion
of the curve. Multiplication
of the specific
activity
by the mass of RNA
allowed
estimation
of recovery
of mRNA
activity
in the
different
fractions
and calculation
of the enrichment
of mRNA
by
comparison
with
the
total
extract.
Uteroglobin
mRNA
activity
was measured
by immunoprecipitation
using
75 Ml of the supernatant,
as described
below.
The fraction
of total
TCA-precipitable
radioactivity
represented
by
immunoprecipitable
radioactivity
is thus
a measure
of concentration
of
utcroglobin
mRNA
relative
to total mRNA.
Purification
of Uteroglobin
Uteri
of
flushed
with
rabbits
on Day
5 ml of isotonic
5 or 6 of pregnancy
saline;
the flushinge
were
were
TRANSLATION
pooled,
OF UTEROGLOBIN
dialysed
against
deionized
water
at 4#{176}C
and
fractionation
was achieved
by gel
filtration
over
a 2 X 20-cm
column
of Ultrogel
54
(LKB)
in 0.01
M Tris-HCI,
pH 7.6, at 4#{176}C.
The fraction
containing
uteroglobin
was applied
to a column
of DEAE-cellulose
(Whatman,
DE 52) and eluted
at
4#{176}C
with
a gradient
of 0 to 0.1 M KCI in 0.01 M TrisHC1, pH 7.6. Uteroglobin
eluted
at 0.03
M KC1 and
gave a single
band
on electrophoresis
in 15 percent
polyacrylamide
gels
containing
0.1
percent
SDS
(Laemmli,
1970)
and on electrophoresis
in 4 percent
polyacrylamide
gels in 0.35
M jl-alanine
buffer,
pH
4.3.
lyophiized.
Protein
Immunoprecipitation
Antibodies
against
uteroglobin
were purified
from
a goat anti-uteroglobin
(anti-UG)
antiserum
by binding
to a matrix
of uteroglobin
cross-linked
with
glutaraldehyde
(Avrameas
and Ternynck,
1969).
The purified
antibodies
were
eluted
with
0.1 M glycine-HCI,
pH
2.8, and dialysed
against
0.01 M Tris-HC1,
pH 7.6. The
specificity
of the antibody
preparation
was tested
by
immunoelectrophoresis
on
1 percent
agar
against
uteroglobin
and whole
uterine
fluid proteins.
Immunoprecipitation
of the products
of the translation
assay
was performed
by mixing
75 p1 of the
post-ribosomal
supernatant
with
75 p1 of a buffer
consisting
of 2.8 percent
sodium
deoxycholate,
1.6 percent
Triton
X-100
and 1 mM sodium
phosphate-15
mM
NaCI,
pH 7.5.
A double-antibody
precipitation
was performed,
using
rabbit
anti-goat
IgG antiserum
(Antibodies,
Inc.)
as the second
antibody.
Optimum
conditions
were
determined
by incubation
with
different
amounts
of first
and second
antibody,
for various times,
at 25#{176}C
or 4#{176}C.
The specificity
of immunoprecipitation
was tested
by substituting
normal
goat serum
for the first antibody
and
by
double-antibody
precipitation
in the
presence
of 20 pg of standard
uteroglobin.
A further
test
of specificity
was performed
by translation
of
purified
ovalbumin
mRNA
(Woo
et al., 1975)
and
immunoprecipitation
of the translation
products
in
the
uteroglobin
double-antibody
system.
Following
incubation,
0.25 ml of 0.15 M NaC1 and
0.25 ml of a cold “wash”
buffer,
consisting
of 10 mM
sodium
phosphate,
pH 7.5, 0.15
M NaCI,
2 percent
Triton
X-100
(v/v)
and
10 mM
non-radioactive
Lmethionine,
were
added
and the immunoprecipitate
was collected
by centrifugation
at 10,000
g for 10
TABLE
different
1. Yield
methods,
of poly-A
containing
with
a comparison
RNA
to hen
Tissue
and
extraction
method
RNA
Whole
uterus,
pH 8
Whole
uterus,
pH 5.2
Hen oviduct,
pH 5.2
Whole
uterus,
pH 9
Endomecrium,
pH 9
Rabbit
liver, pH 9
Filter-bound
d’F-bound
dT-bound
dT-bound
dT-bound
dT-bound
fraction
min.
of
mRNA
The
437
precipitate
was
washed
3 times
with
0.5
ml
cold
“wash”
buffer
and collected
by filtration
on a
Millipore
filter.
The filters were dried
under
an infrared light
and the radioactivity
counted
by liquid scintillation
in Spectra±luor
(Amersham-Searle).
Product
Analysis
The identity
of the immunoprecipitated
products
of translation
was examined
further
by electrophoresis
in 15 percent
polyacrylamide
gels
containing
0.1
percent
SDS,
using
the method
of Laemmli
(1970).
The precipitate
was dissolved
in 80 p1 of a buffer
consisting
of 1 percent
SDS, 10 mM dithiothreitol
(DTT)
and 10 mM sodium
phosphate,
pH 7.5, and 20 p1 of
standard
uteroglobin
(1 mg/mI)
were
added.
After
heating
at 90#{176}C
for 5 mm, the sample
was cooled
and
50 p1 of 40 percent
(wlv)
sucrose
containing
0.1 percent
bromophenol
blue
were
added.
Electrophoresis
was performed
at a constant
current
of 2 mA per gel
for 3 to 4 h at room
temperature.
The gels were cut
off at the dye front
and fixed
and stained
in 0.8 percent
Amido
Schwartz
dye in methanol
:acetic
acid:
water
(45:10:45,
by vol.)
for 16 h. After
destaining
in methanol:acetic
acid:water,
the gels were cut into
1-mm
slices.
Each slice was dissolved
in 0.5 ml of 15
percent
H3O3
(v/v)
at 60#{176}Cfor 16 h and the radioactivity
counted
in 5 ml of a liquid
scintillation
cocktail (ACS,
Amersham-Searle).
Parallel
gels of rabbit
serum
albumin
(MW
68,000),
ovalbumin
(44,000),
soya
bean trypsin
inhibitor
(22,700)
and cytochrome
c(13,370)
were run as molecular
weight
standards.
RESULTS
Table
taining
1 shows
RNA
procedures,
mRNA
the
recovery
resulting
from
represented
by
activity
bound
to
in
one
case,
nitrocellulose
et
a!.,
1972).
Extraction
NaC1-containing
containing
both
ing
buffer
RNA
bound
over
and mRNA activity
oviduct
and rabbit
and
fraction.
material
the
at
TE.
in rabbit
liver.
pH
That
uterus
5.2
and
and
total
or,
was
(Rosenfeld
8.0
uterus
or
gave
the
in
low
not
endometrium
of
A-contain-
activity
only
in
NaOAcyields
poly
specific
enriched
this
mass
whole
pH
in
The
was
the
A-con-
extraction
filters
at
activity
poiy
oligo-dT-cellulose
of
buffer
mass
of
different
of
2 to
a fault
the
3-fold
of
extracted
RNA
mass
yield
(96)
mRNA
activity
yield
(%)
mRNA
enrichment
(fold)
1.2
1.2
1.2
0.8
1.0
1.9
2.3
3.8
26.9
7.0
10.3
68.6
2.0
3.1
24.5
9.1
10.8
34.4
the
by
BULLOCK
438
procedure
and
is shown
the
mRNA
by
25-fold
extracted
by
Extraction
HCI
of
buffer
(1973)
at
to
RNA,
dT
the
increase
increase
of
to
DNA
whole
by
procedure.
the
and
obtained
starting
mRNA
extracted
metnum
by
In
contrast
tracted
ity
on
pH-9
to
pH-8
liver
percent
of
of
the
uterine
data
of
1.9
total
chromatography.
was
mRNA
activity
mRNA
and
The
FT
background
added
ed
each
plateau
at
activity
about
TE
2).
total
mass
able
mRNA
fraction,
Because
bound,
contain
length
column.
results
which
Calculation
in
poly
1
A
percent
bulk
at
the
of
of
3H-poly
U
no
a
least
en-
tion,
but
with
50
of
the
of
FT
were
labeled
on
each
dT
and
of
FT
uteroglobin
affinity
single
the
has
products
of
fractions.
antibody
The
the
was
results
on
pg
for
by
1251
by
Fig.
2
solution
22.5
conditions
determined
available
in
of
the
to
permit
precipitawere
obtained
against
PUF
2.5
pg
protein.
immunoprecipitation
incubation
the
pregnant
preparation.
quantitative
antibody
and
5-day
demonstrates
of
lactoperoxidase
uteroglobin,
meth-
sufficient
A
been
the dT
by the
in
3),
the
TABLE
2. Total
mRNA
activity
of rabbit
endometrium
after
separation
of total
RNA
extract
(TE)
on
oligo-dT-cellulose
into
bound
(dT)
and flow-through
(FT) fractions.
FT
carried
translation
specificity
is shown
line
12
does
(Table
preparation
chromatography
precipitin
10
antibody
by
fraction.
Immunoprecipitation
the
titer
p1 of
with
and
(PUF)
material
uteroglobin
The
percent
of
of
the
translat-
of
poly
Insufficient
estimation
75
hybridization
detectable
8
uteroglobin
flushings
monospecificity
of
the
pure
uterine
to
endometnum
sequences
sis against
the
in
(>20
residues)
to be retained
by
This interpretation
is supported
revealed
6
FIG.
1. Dose-response
curves of total mRNA
activity of endometrial
RNA
translated
in a 50-p1 wheatgerm assay. TE = total
extract;
dT
dT-bound
RNA;
FT = flow-through
RNA (not bound
to dT-cellulose).
whole
a 10.3-fold
present
that
of
of
compared
the
4
of TE,
reaches
portion
was
mRNA
1).
total
subtract-
RNA.
only
activity
dT-
absence
been
linear
RNA
2
pg RNA
in
of
fraction
revealed
dT
was
total
of
the
suggesting
the
not
the
the
has
dT
0
endometrium.
in
pg
curves
of
(Table
of
4
from
dose-response
richment
6-day
The
of
(Table
curves
ct/mm)
value.
specific
TE
0
activ-
in a 50-/11 assay
of
(60,000
percent
The
the
obtained
RNA
from
to
FT
ex-
34-fold
dose-response
fractions
It
endo-
mRNA
enriched
obtained
activity
dT
with
RNA
yielded
compared
re-
obtained
rabbit
liver
RNA
the
procedure.
dT-cellulose
1 shows
in activity
percent
endometrium,
69
ap-
tissue
10
bound
Figure
we
increase
thus
E
extraction
pregnant
the
was
problem,
extraction
rabbit
and
DNA
to
were
from
the
mass
All
en-
the
the
further
paper
from
total
undoubtedly
when
fraction
large
fractions
to endometrial
this
in
TE
this
activity.
ported
extrac-
procedure
dT
in the
very
overcome
a small
the
and
yield
the
had
in
A-rich
yield
previous
of
RNA
spooling
pH-9
in
total
of
To
plied
all
and
losses
removed
the
Tris-
poly
mass
observed
in
uterus
countered
cold
activity
We
400
Brawerman
of
in
in
binding
oviduct
in
by
yield
compared
amounts
yield
uterus
the
of
hen
method.
claimed
9,
procedures.
from
pH-5.2
a 3-fold
fraction
level
of
whole
pH
5-fold
tion
high
increase
gave
nearly
the
enrichment
ET AL.
out
of
TE,
of
the
purified
in
immunoelectrophore-
Fig.
by
2.
A
RNA
fraction
TE
dT
FT
mg
Specific
activity
cpm/pg
Activity
96
52.2
0.5
34.2
14011
151372
16011
100
10.3
74.9
TRANSLATION
TABLE
3. Poly A content
of endometrial
OF
UTEROGLOBIN
RNA fractions
RNA
3H-poly
RNA
fraction
added
pg
hybridized
cpm
TE
dT
FT
58
10
57
4369
16
TE
Total
=
ND
extract;dT
dT-bound;
=
FT
Flow
=
439
by 3H-poly
U
U hybridization.
Poly
A
equival ent
pg
0.02
0.26
ND
Poly A
96
0.06
5.2
.
...
through.
Not detectable.
1972),
with 2 p1 of the
solution
and various
amounts
of anti-lgG
(second
at 4#{176}Cor 2 h at 25#{176}Cin
Figure
of time
or
at 4#{176}C
with
at
determined
403
od (Hubbard
and Cohn,
purified
anti-UG
antibody
tions.
mRNA
3 shows
that
there
25#{176}Cwith
the
second
translation
products
RNA,
using
different
antibody,
15
bodies
and
amounts
shows
that
was little
antibody
the
percent
20
was
of
were obtained.
The
cipitable
radioactivity
about
for 16 h
combina-
temperature.
Precipitation
the first antibody
followed
chosen
for convenience.
When
this procedure
second
antibody)
different
p1 of
chosen
for
omission
has
applied
been
to
the
results
shown
anti-UG
which
routine
of the
Fig.
were
assays.
specific
at
antithe
Figure
5
anti-UG
antibodies
or the addition
of 20 pg of nonradioactive
uteroglobin
reduced
the radioactivity
in
the precipitate
to 2.5 percent
and 1.4 percent
respectively.
I mmunoprecipitation
of translation
products
of dT-bound
liver RNA
gave results
which
were
indistinguishable
from
nonspecific
trapping.
The
identity
of
the
immunoprecipitable
radioactivity
with
the
data
obtained
phoresis
(Fig.
6).
uteroglobin
is confirmed
by analytical
Day 4 endometrial
gel
normal
goat
serum
the specific
anti-UG
peak was completely
of excess
(6A).
a similar
was
in place
(6B)
by the
uteroglobin
of
and the
addition
(6C).
The
of radioactivity
migrated
more
slowly
authentic
uteroglobin,
indicating
the cell-
free
translation
than
relative
Fig.
7.
curve
to
gave
values
uteroglobin,
of
11,300
15,600
for the
Figure
8 shows
be
larger
in size
weight
versus
mobility
is shown
in
of the linear
regression
4 replicates
of
may
of molecular
electrophoretic
Interpolation
fitted
and
product
uteroglobin.
A standard
the
for
standard
authentic
proteins
uteroglobin
cell-free
product.
that the specific
expressed
mRNA
as a percentage
for
of
the
8
0
6
st A
by
electrodT-RNA
Negligible
mobility
used
antibodies
displaced
nonradioactive
(6D),
of trans-
peak
than
4
of immunoprea maximum
4 p1 of
anti-IgG,
in
dTand
of radioactivity
the products
lation
without
added
RNA
radioactivity
was present
at
when
16 h
by 2 h
4-day
endometrial
amounts
of first
percentage
reached
with
effect
for
gave a single
large peak
which
was
absent
from
-4
2nd Ab.
#{149}2h
6 h
o 16h
2h
16 h
E
2
0
I
I
0
20
I
40
60
2nd
FIG. 2. Immunoelectrophoresis
uteroglobin
antibodies
against
whole pregnant uterine flushings
(18,000
of purified
anti(UG) and
(PUF).
uteroglobin
100
II
120
140
160
ANTIBODY-A
3. Immunoprecipitation
of I 2luoglobin
cpm) incubated
for 2 h at 25#{176}C
or 16 h at
4#{176}C
in different
combinations
with 2 p1 of first antibody (purified
anti-UG)
and various
amounts of second antibody
(anti-lgG).
FIG.
+
I
80
BULLOCK
440
ET AL.
20
‘5
I0
EIE
a
5A
5
2ndAb
0-
a
I
0
I
I
I
2
3
A
st ANTIBODY
FIG.
4.
Percent
translation
products
various
by
2
body
amounts
of
antibody
first
h at 25#{176}Cwith
(anti-IgG).
total
(mean
various
mRNA
activity,
± S.E.)
in
metrium
pregnancy
1.4
immunoprecipitable
incubated
for 16 h
to
percent
of
on
Day
fold
increase
in the
mRNA
in dT-bound
a
8. Thus,
there
proportion
RNA
on
0.
wheat-germ
C
C
4#{176}Cwith
at
0
followed
second
rose from
0.9
non-pregnant
10.2
± 1.3 percent
on
and declined
subsequently
C
4
(anti-UG)
amounts
the
I
±
Day
to
was
anti-
1.4%
endo4 of
1.9 ±
an 11-
of uteroglobin
Day 4 of preg-
0
Slice
N:i:iiber
radioactivity
in wheatgerm translation
products
on electrophoresis
in 15%
polyacrylamide-SDS
gels by the method
of Laemmli
(1970). A; blank (no added RNA), anti-UG antibodies
plus anti-IgG.
B; dT-bound
endometrial
RNA, normal
goat serum
plus anti-lgG.
C; dT-RNA,
anti-UG
plus
anti-IgG
plus 20 pg nonradioactive
uteroglobin.
D; dT
RNA,
anti-UG
plus anti-IgG.
The arrows mark the
position of authentic
uteroglobin.
FIG.
20
+
15
0
0
6.
Immunoprecipitable
x
10. 10
nancy.
ill’-’
.0<
of
<0
I-
Analysis
fitting
of
variance,
constants
that
the value
ferent
(P<0.01)
on
using
(Snedecor,
the
method
revealed
1956),
Day 4 was significantly
from
the values
on Days
dif0, 2
and 8.
5
DISCUSSION
T1
I’
translation
antibodies
goat serum
that
uteroglobin
the
can
specific
be quanti-
Ab1
tated
in the normal
endometrium
of early pregnancy.
Several
interesting
findings
about
the
Ab2
Ah2
Al)2
translatable
immunoprecipitable
wheat-germ
products
incubated
with
purified
anti-UG
(Ab1)
and anti-IgG
(Ab2), or with normal
(NGS) and Ab2, or with Ab1 plus Ab2 in
of
demonstrate
for
NG S
5. Percent
the presence
(UG).
results
coding
Ab1
UG
FIG.
The
mRNA
20 pg
nonradioactive
uteroglobin
In
many
mRNA
in the
contrast
to
other
eukaryotic
percent
of
mRNA
unbound
was bound
translatable
the
total
material
mRNA.
hen
uterus
oviduct,
tissues,
activity
also
emerged.
rabbit
liver
and
only
about
10
of the
endometrial
to oligo-dT-cellulose
contained
the majority
Histone
mRNA
and the
of the
is known
TRANSLATION
OF UTEROGLOBIN
0.6
Produd
0.5
Cyt.
Trypsin
mRNA
sensitive
to 0.01
of
cell-free
the
0.4
In X
will be reported
subsequently.
specificity
of translation
of
Ovalbumin
Albumin
to
and
the
10
20
30
40
50
I
I
presence
I
I_I
00708090100
MW x l0
was
the
denaturing
of radioactivity
very low.
In
study
of
in the
of
a single
of
peak
when
of radio-
conditions.
displaced
the
gel
radio-
prepara-
be completely
to
immuno-
electrophoresis
Nonspecific
trap-
in the immunoprecipitate
the presence
of non-immune
or an excess
of nonradioactive
the immunoprecipitate
contained
2 percent
Similar
products
were
dTendo-
uteroglobin
binding
antibody
subjected
ping
was
1 to
I
by
a monospecific
under
serum
globin,
0.1
I
mRNA
is indicated
precipitate
I
non-dT-bound
activity,
which
could
by authentic
uteroglobin,
0.3
0.2
products
metrium
The
tion
1.144-0.245
-
A. A detailed
translation
and
activity
V
pg of poly
bound
mRNA
InhibItor
441
of the
TCA-precipitable
results
were
found
of translation
of
precipitated
uteroonly
with
counts.
when
the
ovalbumin
uteroglobin
cell-free
mRNA
antibodies.
FIG. 7. Molecular
weight
determinations
on 15
percent
polyacrylamide-SDS
gels. Cyt.
c = Cytochrome c; UG = uteroglobin;
Product = immunoprecipitable
cell-free
translation
product
of endometrial
mRNA.
Vertical lines show standard error of the mean
(n=4).
uteroglobin
mRNA
activity
in the dT fraction
of endometrial
RNA
accounted
for 10 to 20
to
and 8) is due to
RNA
preparations
lack
polyadenylation
Greenberg
and
(Adesnik
Perry,
poly A-minus
mRNA
sea urchin
embryo
1972)
et al.,
and
servation
poly
of greater
was
A could
by
3H-poly
than
20
substantiated
not
nonhistone
be detected
U hybridization,
Hartley,
an unusually
lacks poly A
and
residues.
by
the
This
finding
percent
obthat
in the
FT extract
under
conditions
8
U
.0
‘C
U
6
‘C
I-
4
DAY OF PREGNANCY
the
activity
the
detected
on
specific
Day
total
mRNA
would
of the
cannot
thus
While
was
rabbit
detected
TE
or
10
to
frac-
the
of
the
liver,
no uteroglobin
in this RNA fraction.
than
cell-free
uteroglobin
cent
mate
polyacrylamide-SDS
of molecular
weight
dT
of
uteroglobin
1 to
2 percent
mRNA
activity,
from
nonspecific
69 percent
present
in
of
percent
and
amount
FT
since
about
activity,
total
endometrial
be distinguished
ping.
activity
the
surprising,
only
mRNA
4
for aver(Fig. 4, 5
among
the three
Little
uteroglobin
in
is not
represented
which
trap-
the total
dT-fraction
mRNA
mRNA
from
could
product
migrated
on electrophoresis
be
more slowly
in 15 per-
gels. An accurate
estiis difficult
to obtain
in this system,
because
the molecular
uteroglobin
lies at the extreme
of
weight
of
the linear
range
value
of
the
protein
standards.
approximately
previous
1975).
FIG.
8. Changes
in proportion
of uteroglobin
mRNA
activity
in dT-bound
endometrial
ENA during
early pregnancy.
Vertical
lines show ± S.E. of mean
(d.f. = 7).
trapping,
differences
involved.
result
fraction
with
0
mRNA
be
This
mated,
2 _1
the
could
tions.
The
10
a
of
for
pregnancy.
The variation
in the values
age percentage
of uteroglobin
mRNA
12
SE
correction
mRNA
been described
in the
(Nemer
et al., 1974)
and
has
spinach
chloroplast
(Wheeler
1975).
Our
results
indicate
that
large proportion
of uterine
mRNA
sequences
1972;
After
It
observations
is
clear,
The
11,000,
however,
migrates
ahead
of cytochrome
free product
migrates
behind
is
(Beato
and
that
this
Rungger,
uteroglobin
c and the
standard.
protein
synthesized
in vitro
appears
molecular
weight
about
4000
greater
native
protein,
suggesting
the synthesis
uteroglobin.
esti-
consistent
to
cellThe
have
than the
of a pro-
a
442
BULLOCK
Beato
and
Rungger
duct
in
vitro
globin,
perhaps
cessing
cell-free
ilar
results
have
gen
of
the
fraction
in
the
of wheat
germ
been
reported
for
et
1975)
(Kemper
whether
precursor
Levey
et
and
Daniel
proto
germ.
Sim-
placental
It
lacto-
hor-
is not
known
is derived
from
a recent
publication,
reported
the
product
which
size data were
rapidly-labelled
endometrium
4 to 5 days of pregnancy
Levey
and Daniel
(1976).
was
Our
transla-
during
polysomal
the first
also reported
data show
by
that
the specific
mRNA
for uteroglobin
accounts
for
an increasing
proportion
of the poly
A-rich
endometrial
mRNA
during
this period,
reaching
a peak on Day 4 of pregnancy.
The pattern
of
change
in
uteroglobin
pattern
of
secretion
mRNA
of
is similar
uteroglobin
of uteroglobin
be reported
mRNA
subsequently.
to the
during
pregnancy
and reflects
the changing
status
of the animal.
Studies
of the
induction
globin
will
and
early
hormonal
kinetics
of
of utero-
ACKNOWLEDGMENTS
from PHS grant NIH RO-1 HD
09378-1.
We thank Mrs. Sara Dinyari and Ms. Rebecca
Russell for technical
assistance. We are grateful to Dr.
Henning Beier for advance notice of his work with Dr.
Maurer.
Supported
a
wheat
germ
showed
an
co-migrated
not given.
by funds
REFERENCES
Adesnik,
M. and Darnell,
J. E. (1972).
Biogenesis and
characterization
of histone
messenger
RNA
in
HeLa cells. J. MoI. Biol. 67, 397406.
Arthur,
A. T. and Daniel, J. C., Jr. (1972).
Progesterone regulation
of blastokinin
production
and maintenance
of rabbit
blastocysts
transferred
into uteri
of castrate
recipients.
Fertil.
Steril.
23, 115-122.
Aviv,
H. and Leder, P. (1972).
Purification
of biologically active globin
messenger
RNA
by chromatography
on oligothymidylic
acid
cellulose.
Proc.
NatI. Aced.
Sci., USA 69, 1408-1412.
Avrameas,
S. and Ternynck,
T. (1969).
The
crosslinking
of proteins
with
glutaraldehyde
and its use
for the preparation
of immunoadsorbents.
Immunochemistry
6, 53-66.
Beato,
M. and Rungger,
D. (1975).
Translation
of the
messenger
RNA
for rabbit
uteroglobin
in Xenopus
oocytes.
FEBS Letters
59, 305-309.
Beier, H. M. (1968).
Biochemisch-entwicklungs
phy-
untersuchungen
am Proteinmiieu
fur
die Blastozystenentwicklung
des Kaninchens
(Oryctolagus
cuniculus).
Zool. Jahrb.
Anat.
85, 72-190.
Boime,
I., Boguslawski,
S. and Caine, J. (1975).
The
translation
of a human
placental
lactogen
mRNA
fraction
system
parathyroid
mRNA
in the
These
authors
in
greater
compared
vivo
(1976)
but
increase
from
rabbit
1974).
In
immunoprecipitable
with uteroglobin,
An
and
in
uteroglobin
system.
utero-
translation
human
al.,
uteroglobin
molecule.
tion
of
cell-free
RNA
(S-30)
wheat
al.,
of the
oocyte
siologische
a pro-
with
intact
(Boime
mone
obtained
co-migrated
a reflection
ability
the
(1975)
which
ET AL.
in
heterologous
cell-free
systems:
The
synthesis
of a possible
precursor.
Biochem.
Biophys.
Res. Commun.
62, 103-109.
Brawerman,
C. (1973).
The
isolation
of RNA
from
mammalian
cells.
In:
Methods
in Cell
Biology,
Prescott,
D. M. (ed).
Academic
Press, N.Y.
Vol.
7, pp. 1-22.
Bullock,
D. W. and Willen,
C. F. (1974).
Regulation
of
a specific
uterine
protein
by estrogen
and progesterone
in ovariectomized
rabbits.
Proc.
Soc. Exp.
Biol. Med. 146, 294-298.
Gillespie,
D., Marshall,
S. and Gallo,
R. C. (1972).
RNA
of RNA
tumor
viruses
contains
poly A.
Nature
(Lond.),
New Biol. 236, 227-231.
Greenberg,
J. R. and Perry,
R. P. (1972).
Relative
occurrence
of
polyadenylic
acid
sequences
in
messenger
and heterogeneous
nuclear
RNA of L
cells
as determined
by
poly
U-hydroxylapatite
chromatography.
J. Mol. Biol. 72, 91-98.
Hubbard,
A. L. and Cohn,
Z. A. (1972).
The enzymatic iodination
of the red cell membrane.
J. Cell
Biol. 55, 390-405.
Kemper,
B., Habener,
J. F., Mulligan,
R. C., Potts,
J. T., Jr. and Rich, A. (1974).
Preproparathyroid
hormone:
A direct
translation
product
of parathyroid
messenger
RNA.
Proc.
NatI.
Acad.
Sci. USA
71, 373 1-3735.
Krishnan,
R. S. and Daniel,
J. C., Jr. (1967). “Blastokinin”:
Inducer
and regulator
of blastocyst
development
in the rabbit
uterus.
Science
158, 490492.
Laemmli,
U. K. (1970).
Cleavage of structural
proteins
during
the assembly
of the head of bacteriophage
T4. Nature
Lond.
227, 680-688.
Levey, I. L. and Daniel,
J. C., Jr. (1976).
Isolation
and
translation
of blastokinin
mRNA.
Biol.
Reprod.
14, 194-201.
Maurer,
R.
R.
and
Beier,
H.
M.
(1976).
Uterine
pro-
teins
and development
of pre-implantation
embryos
of the rabbit.
J. Reprod. Fertil. (In press).
Nemer,
M., Graham,
M. and Dubroff,
L. M. (1974).
Co-existence
of nonhistone
messenger
RNA species lacking and containing
polyadenylic
acid in sea
urchin
embryos.
J. Mol. Biol. 89, 435454.
Roberts,
B. W. and Paterson,
B. M. (1973).
Efficient
translation
of tobacco
mosaic
virus
RNA
and rabbit globin
9S RNA
in a cell-free
system
from commercial
wheat
germ.
Proc.
Nail
Aced.
Sci.,
USA
70, 2330-2334.
Rosen,
J. M., Woo, S. L. C., Holder,
J. W., Means, A.
R. and O’Malley,
B. W. (1975).
Preparation
and
preliminary
characterization
of purified
ovalbumin
messenger
RNA
from
the hen oviduct.
Biochemistry
14, 69-78.
Rosenfeld,
G. C., Comstock,
J. P., Means,
A. R. and
O’Malley,
B. W. (1972).
Estrogen
induced
synthe-
sis of ovalbumin
messenger
RNA
and its translation
in a cell-free
system.
Biochem.
Biophys.
Res.
Commun.
47, 387-392.
Snedecor,
G. (1956).
Statistical
Methods.
Iowa State
College
Press, pp. 388-391.
Wheeler,
A. M. and
Hartley,
M. R. (1975).
Major
mRNA
species
from
spinach
chloroplasts
do not
TRANSLATION
contain
Woo,
poly
(A).
S. L. C., Rosen,
son,
D.
L.,
Choi,
Nature,
J. M.,
V. C.,
OF
Lond.
257, 66-67.
Liarakos,
C. D., RobberBusch,
II., Means,
A. R.,
UTEROGLOBIN
and
mRNA
O’Malley,
443
B. W.
(1975).
Physical
characterization
of purified
ovalbumin
ger RNA. J. Biol. Chem.
250, 7027-7039.
cal
and
chemimessen-