BIOLOGY
OF
REPRODUCTION
17,
749-759
(1977)
Temporal Changes in Proteins of Oviduct Fluids During the Preimplantation Period E. B. TUCKER Division
and
G. A. SCHULTZ
of Medical Faculty
of
University
Calgary,
Biochemistry, Medicine, of
Alberta,
and Uterine in the Rabbit
Calgary,
Canada
T2N
1N4
ABSTRACT A study of the polypeptides which contribute to the environment surrounding the rabbit preimplantation embryo was undertaken. At various times during the first 6 days of pregnancy the lumen of the oviducts and uteri of normally mated and superovulated does were flushed with saline, and the polypeptides present in these flushings were separated in a two-dimensional (isoelectric focusing, sodium dodecyl sulfate) electrophoresis system. Information was obtained regarding similarities and differences in polypeptide patterns between the stages and between the tissues. The major protein in the oviduct is albumin whereas in the uterus at later preimplantation stages it is uteroglobin. During the preimplantation period the polypeptide environment in the oviduct remains virtually the same, while in the uterus there is a disappearance of albumin and a marked increase in uteroglobin. lmmunoelectrophoresis demonstrated that of the more than 50 polypeptides resolved in two-dimensional electrophoresis of oviduct fluid, at least 5 are identical to serum proteins (albumin, lgG, transferrin, and possibly haptoglobin and n-2-macroglobulin). Uterine fluid at Day 1 to 2 postcoitum (p.c.) also contains these 5 serum proteins. By Day 3 p.c., the uterine fluid contains little protein which reacts with antisera directed toward rabbit serum proteins. Some differences were noted in polypeptide patterns of uterine flushes during early preimplantation stages of superovulated does versus those from normal matings. Comparable differences were not observed in oviduct fluids. INTRODUCTION
The
biochemical
composition
and uterine fluids of development parameter luminal
in fluids
passage
of
ronment sides and
spermatozoa in
and
specific
which derives
patterns
uterine
fluids for the
and
during may
well
ova
globin Daniel,
the
of
Arthur
These for the retime-
have
stages
proteins isoelectric rell, 1975)
concerned
with
oviduct They
temporal 1969;
the developof studies on
tide ine
and uterine have been changes Beier,
poral uterine
whether proteins are derived by endometrial secretion or transudation of serum (Fiegelson and Kay, 1972; Beier, 1974; Shapiro et al., 1971; Dies, 1973) and the relationship between
Accepted Received
July April
high developed.
by
two
components lumen fluids
period
14, 1977. 25, 1977.
749
by
present
method
Willen,
unrelated
communi-
parameters weight the
in oviducal and during in rabbits.
the The
of
(O’Farpolypepand utersix day polypep-
of both normally ovulated does were compared. The or absence also present
immunological
of in
oviduct serum
procedures.
have allowed us to verify clarify previously reported
addition information
to
teins
reproductive
contributing regarding
new temporal tract
the
of separating
methods cases to
in
and 1970;
and
molecular to reexamine
present before
occurrence proteins
noted
In the
and used
utero-
(Krishnan et al.,
techniques for of polypeptides
sensitive
the point was
protein of
Bullock
resolution separation
highly
tide patterns superovulated
1974),
tract
induction
1972;
Daniel,
preimplantation in
the
and
the
conse-
important
reproductive production 1968; Urzua
been
cation
oviduct
and
blastokinin 1967; Beier
1974). Recently, two-dimensional
envi-
embryo Any
or
have
composition of been published. (Kirchner,
stages important
preimplantation
the protein fluid have composition
particularly,
and
composition
synchronization of embryo. A number
general
status
patterns,
process. a medium
the developing nourishment. in the
of development quences ment of
during preimplantation in mammals is an
the reproductive serve both as
hormonal oviducal
of
and
and was These
and
more changes
fluids
and tem-
in some results in detailed in pro-
during
the
TUCKER
750
preimplantation
period
and
Oviduct
Flushings
AND
Preparation
of
METHODS Uterine
SCHULTZ
NaCI,
in rabbits.
MATERIALS Collection
AND
and
Dutch belted rabbits were used throughout the work. A complete series of normally ovulated animals and a complete series of superovulated animals was studied. The latter were induded because of relevance to the many studies on preimplantation embryos which have depended heavily on the use of hormonal superovulation to increase embryo yields. For the normal series, animals were mated on Day 0 and groups of at least three animals sacrificed at daily intervals throughout the 6 day preimplantation period prior to removal of uteri and oviducts. Animals induced to ovulate without mating were given 2.5 mg of luteinizing hormone (LH) in an ear vein and sacrificed 24 h later. Superovulation was performed as described by Manes (1969). Does were brought into the same cycle by a single injection of 25 IU human chorionic gonadotrophin. Nineteen days later they were given 0.3 mg of follicle stimulating hormone subcutaneously twice daily for three days. On the fourth day they were mated and injected with 2.5 mg of LH into an ear vein or ovulated with LH administration alone without mating. As for the normal series, coitus was taken as Day 0 and groups of at least three animals were sacrificed at daily intervals for recovery of oviducts and uteri. The flushing obtained by passing saline through the respective reproductive organ was cleaned of debris by centrifugation, dialyzed against water using No. 3 Spectraphor membranes (Spectrum Med. Ind. Inc., Los Angeles) and lyophilized. All procedures prior to lyophiization were performed at 0#{176}C. Protein concentration of samples was determined by the Biuret method (Layne, 1957).
0.025
Using System
the
Lyophiized samples were dissolved in lysis buffer (9.5 M Urea, 2 percent (w/v) Nonidet P-40, 2 percent Ampholines pH 3.5 to 10, and 5 percent -mercaptoethanol) and frozen at -70#{176}C until use. Proteins were separated according to isoelectric point by isoelectric focusing in tube gels in the presence of pH 3.5-10 ampholines in the first dimension, followed by molecular weight separation by sodium dodecyl sulfate (SDS) electrophoresis on 10-15 percent exponential gradient acrylamide slab gels in the second dimension (O’Farrell, 1975). Forty g of protein was used in all cases. Gels were stained for 2 h in 50 percent methanol, 9 percent acetic acid, 3 percent glycerol and dried on a Hoefer gel slab drier. A mixture of marker proteins was applied to a small piece of cellulose acetate membrane and was placed beside the first dimension gel on top of the second dimension gel for molecular weight calibration of the second dimension. Immunological
Procedures
agar plates for double immunodiffusion (Ouchterlony) studies were obtained from Hyland Laboratories, Inc. Lyophiized oviduct and uterine fluid samples were dissolved in 0.15 M Goat
anti-rabbit
serum
and
buffer,
pH
7.2
at a concentration
RESULTS
Identification
the
of
Albumin following
albumin sample marked min
Separation of Polypeptides Two Dimensional (2-D)
M Tris
of 50 mg/mi. Rabbit serum (60 mg/mI) was used without dilution. Double immunodiffusion studies were performed at room temperature for 24 h using gels containing 2 percent agar, 7.5 percent glycine, 1 percent NaCl, and 0.1 percent sodium aside at a pH of 7.2. Five MI samples of oviduct, uterine, or serum protein were applied to peripheral wells and S til goat anti-rabbit serum to the center well. lmmunoelectrophoresis was performed using the Model R-103 accessory to the Beckman Microzone electrophoresis apparatus. Samples containing 25 ug protein in 0.5 M’ were applied to embossed membranes and subjected to electrophoresis in 0.0375 ionic strength barbiturate buffer, pH 8.6, for 45 min at 150 volts. After electrophoresis, 20 MI of goat antiserum to total rabbit serum proteins (A-RSP) was put into each trough and immunodiffusion was allowed to proceed for 24 h under paraffm oil. Membranes were then washed with saline to remove unprecipitated protein and were stained with Ponceau S dye to visualize precipitin lines. Standard cellulose acetate electrophoresis was performed using 0.07 5 ionic strength barbiturate buffer pH 8.6 for 30 mm at 250 volts. Membranes were fixed and stained with Ponceau S dye to visualize proteins.
flush the
Proteins
of uterine manner.
(Sigma protein albumin is well
blood marked
Major
flushing When
known
to
serum (Kozma albumin on oviduct
flush
protein
of
min is present rabbits and in
be
the
(see
protein prominent
all three. This peptide three spots which are albumin. proteins
them,
“transferrin” has and
iron bound tentatively serum
protein
identically
to
That
albu-
sec-
of blood serum is or oviduct flush
other and
than albumin is present in as a series of are more basic
immunoelectrophoresis
allowed in
p.c. by
Immunological
appears larger and
The
serum
of
the spot of uterine
in uterine flushes of 1 day oviduct flushes was verified
is another is relatively
has
with spot albu-
protein and
serum.
tion). When the 2-D pattern matched with that of uterine
system
major
migrates blood
immunoelectrophoresis
90,000, point,
Uterus
was identified in purified rabbit
et al., 1967) the 2-D pattern
and
than
the
Chemical Co.) was mixed it migrated with the in Fig. 1. Furthermore,
major
there which
in
the uterine has
identification flushes
of and
a molecular
one
weight
5
of of
a slightly more basic isoelectric exists in several forms, the apo and forms (Sober, 1970). Thus identified this polypeptide transferrin.
we have as the
UTERINE
OVIDUCT
ISOELECTRIC
PROTEINS
751
FOCUSING
(0 ‘Ii
r m C, -4
0 0 “I 0)
(0
FIG. 1. Two-dimensional gel electrophoresis separation of rabbit uterine flush proteins. In the first dimension, proteins were separated by isoelectric focusing on 110mm cylindrical gels. In the second dimension, proteins were separated according to molecular weight on SDS exponential gradient (10-15 percent acrylamide) polyacrylamide slab gels. Forty g protein was used in each separation. A, Estrus; B, 2 day p.c.; C, 3 day p.c.; D, 4 day p.c.; E, 5 day p.c.; F, 6 day p.c. Albumin (AIb), uteroglobin (Ug), and transferrin (Tr) are identified.
The
been
molecular reported
15,000 14,000-16,000 tein is the uterine and is Daniels,
weight
as (Murray major
flushes induced 1972).
(Bullock
there which
is
postcoitus
uteroglobin
et
has
et al., 1972)
al., 1977). of 4, 5, and
cells of the endometrium it has been isolated and
and trans-
al.,
1976).
In our
2-D
uterine
flush
and
system
which
can
uteroglobin
which
the
procedure
we
have
found
polypeptide similar to et
and
supports
spot
At with
Swank the
and
on
al. (1977) their
molecular
under
same
molecular
be
as
1. Using of
daltons. determined
This
is by
uteroglobin
identical, subis a noticeable weight
from
composition
that
but
modification “acidic”
we to
form.
both do
has
of uteroglobin nancy (Arthur
in
been the et
1972a,
the authentic different The
et al.
spots
are
as
the subunit It may be
which
yet
(1977)
and to the These
uteroglobin know
what
has occurred in the related to hormone
suggested
as a function
uterus during al., 1972; El
b; Beato
by the (1971),
according al. (1976).
not
of
is indistinguishable
Nieto
we purified Bullock et
indicate
subunits
It
basis
Weltman
also by
the
compositions and
reported
of uteroglobin procedure of
Daniel,
acid
point.
On
uteroglobin” and are not detectably
acid that
amino
Marchalonis
this “acidic uteroglobin amino
isoelectric
inducible.
and
Baier,
early Banna
pregand
1975).
this
conditions
that
of
binding days
acidic
(1971),
weight
denaturing
conclusion
of two similar, if not Day 4 and 5 p.c. there the
Fig.
Munkres
to be 6,700-7,000 the value of 8,000
Nieto consists units.
is so labeled
of
a more of
method
results
polypeptide the 6 day
induced when does are injected subcutaneously with 4 mg progesterone for 5 consecutive (Fig. 3). Thus we identified this protein
but
is progesterone
comparison
This pro6 day p.c. 1974) and and
molecular weight major protein in
globin also
1976), and
(Kirchner, 1969; Beier, by progesterone (Arthur This protein is produced
et
a low is the
of
(Bullock et al.,
(Neito protein
secreted by the the mRNA for lated
11,400
as utero-
Temporal
Changes
of
Nonsuperovulated
When unmated
40
j.zg of
does
polypeptides slab gel (Fig. overloaded
Uterine
Rabbit
Proteins (2-D
uterine
flush
protein
is electrophoresed, could 1A).
with
in
Electrophoresis)
58
be identified on If the first dimension
protein
up
to
88
from distinct the
polypeptides
2-D gel is
TUCKER
752
AND
ISOELECTRIC
pH
SCHULTZ
FOCUSING
Ij
FIG. 2. Two-dimensional gel electrophoresis separation of hormonally treated rabbit’s uterine and oviduct flush. Superovulation was performed with FSH and LB as described by Manes, 1969. Electrophoretic methods described in Fig. 1. A, uterine flush of unstimulated doe ovulated with LH; B, superovulated 1 day p.c. uterine flush; C, superovulated 2 day p.c. uterine flush; D, superovulated 3 day p.c. uterine flush; E, normal 1 day p.c. oviduct flush; F, superovulated 1 day p.c. oviduct flush. Albumin (AIb), uteroglobin (Ug), and transferrin (Tr) are identified.
may
be
found
Of the molecular
24
molecular
weight
between Albumin
30 and makes
and
uteroglobin
The
protein
flush
small
50,000; up the
protein
takes
present
to
the
of
2 days p.c. but there (note
place
of proteins and greater
50 and
in the
with (note
at at
1C). least Day
By this 4 new 2 p.c.
arrow)
30-50,000
p.c.
flush
17
which
is more
there
1A) these of a
3 p.c. pattern
disappearance
weight a slight
of 50,000 appearance
is an appearance which polypeptides
concentrated (note “acidic uteroglobin”
were
which
have
(note
ring).
The
marked
time were except
change
becomes
uteroglobin (Fig.
as
very
the
faint
is the protein iF). Furthermore,
of
and highest at this
there is a reappearance of proteins which present at Days 2 and 3 (note arrows) for the high molecular weight proteins.
Albumin
and
transferrin
do not
doe
not
stimulated
with
was
ing
Hormone
was
injected
(FSH), with
ovulation, to
was
Luteinizing the
Day
uterine
3 p.c.
of
reappear. Follicle
not
If the Stimulat-
mated,
but
Hormone flush
pattern
normal
only
(LH)
to
looks
mating
(Fig.
2A). of
arrow). By is becoming
an isoelectric
is another
authentic concentration
similar
absent become
(Fig. 1D) and there is a complete of proteins of molecular weight and
albumin
uteroglobin”
cause
has the same molecubut is more acidic
than
there
“acidic
acidic
Day
basic
“acidic uteroglobin” is the major protein of the 5 day p.c. uterine flush while the other polypeptides appear identical to those of Day 4 p.c. (Fig. 1E). At Day 6 p.c., the time of implantation,
uterine (Fig.
no loss of appearance
is a complete a molecular ring) and
polypeptides and other
relatively more Day 4 p.c. the concentrated disappearance
1 day
there is is a slight
time
a a
amounts.
(Fig. 1B). However, by change in the polypeptide as there
more
have 32 have
70,000;
small
oestrus
of the polypeptide which lar weight as uteroglobin (Fig.
results). 7
and 2 below 30,000. highest concentration
is pattern
than albumin a remarkable
(unpublished
polypeptides 70,000 or larger,
of between
is identical
and at proteins
h p.c.
58 distinct weight of
point
Temporal
Changes
Superovulated There
of Uterine
Rabbits are
superovulated uterine flush predominance
detectable
(2-D
Proteins
in
Electrophoresis)
differences
between
the
rabbit flush and the normal proteins. At Day 1 p.c. there is a of polypeptides with a molecular
UTERINE
weight
of
50-70,000
concentration
and
of
Fig.
2B
with
p.c.
superovulated
day
p.c.
which
uterine p.c. normal
Fig.
flush
a decrease
albumin
day p.c. superovulated what like the 2 day
the
the
the
flush
looks
like
The
3, 4,
the
31
a molecular
3
6 day
proteins 50,000.
same
polypeptides
wise
oviducts
superovulated
the ine
by far oviduct,
normal flush
does, contains
uteroglobin
at
to Day become present.
The
With
Day
2 p.c. does
6 day
p.c.
uterine
which
uterof
is maintained
superovulated
and
have
identical
makes
up
the
trations. day
polypeptides is only normal
the
major-
of uterine fluids and slightly present during
uteroestrus.
mating
the
serum
proteins
and transferrin begins to disappear so that by Day 3 p.c. they are almost absent and by Day 4 p.c. they are not detectable. When rabbits are superovulated (FSH followed by LH) or are just injected
with
LH,
slightly present p.c. Uteroglobin concentrations mating
there
form major
by Day amount
1 day
p.c.
is
normally
during
When
is a high p.c. and
clearly
proteins
and
are
are
absent
present
oestrus
and
rabbits
only 2 days
in
with
increase
4 and 5 p.c. By of protein in the
trace
normal
in an acidic
Day 6 p.c. the uterine flush is
are
1
shown
by
of
conventional
cellulose
accompanied uteroglobin tify
by band.
this
band
inducible
by
significant
Changes
Oviducts
(2-D
When could
40 first be
and in
(Fig. of
because
it
is not
present
fluid
from
uterine
in non-
Proteins
in
oviduct
dimension distinguished
flush gel (Fig.
protein about 2E).
is applied 68 Of
there
relative
concen-
noticeable a
on
number
of
concentrated
in
3
the
polypeprelation
to
presented).
of Proteins
in Uteri
position markedly
of oviduct during the
fluid does preimplantation
and
2-D electrothat the comnot
change period
while the uterine protein different in late (Day compared to early (Day Immunological techniques
patterns are markedly 3 to 6 p.c.) stages 1 to 2 p.c.) stages. have been utilized to
monitor any modulations and oviduct fluids which
in proteins are common
to
verify
When sion was a
results
from
the technique used, uterine
lesser
degree lines
2-D
Day
2
with
identity
serum. However, of preimplantation
uterine
with
of uterine to serum
electrophoresis.
of double immunodiffuproteins from Day 1 and
A-RSP
and
3
p.c.
show
reactions
with
fluids from later show little or no
(Fig.
4)
in concentration or loss shared in common between serum. Oviduct fluid proteins
stages of the preimplantation A-RSP to form precipitin reactions of identity with lmmunoelectrophoresis
These
peptides these
although
indicating
a
of
these uterine from all
period react with lines which give rabbit serum (Fig. 4). was used to resolve
precisely the antigenic and oviduct fluids shared
components with serum.
of In
rabbits from normal matings, there are at least five serum proteins in uterine fluid at 2 day p.c. which react with A-RSP (NO series, Fig. 5).
Electrophoresis)
zg
their
with the indicated
more uterine
3).
the up
present
obtained technique has
decrease proteins fluid and is
LH
as makes
that
always
in
where
Changes
cross-reaction
increase in the above, we iden-
uteroglobin
progesterone
rabbits
Temporal
the
as
amounts
pregnant
to
a concomitant As indicated
been
given
(Immunological)
Evidence
rabbit stages
acetate electrophoresis of Day 1 to 6 p.c. uterine fluid (Fig. 3). There is a marked decrease in staining intensity of proteins migrating similar to serum proteins after 1 day p.c.
are
more
(not
precipitin
uteroglobin
is significant
phoretic
to concentration
the Like-
have
or just
polypeptides Albumin
is particularly
oviduct
Oviducts
have
oviduct.
which LH)
a change
become
and
superovulated
concentration of uteroglobin this is maintained throughout
preimplantation. The increased is
serum
is a noticeable
uteroglobin. there day
these
be
Temporal
albumin
oviduct
rabbits
It
This
albumin
p.c.
the same (Fig. 2F).
30 and percent greater
greatest amount of the protein in the and a spot migrating as uteroglobin is
to
p.c.
70,000;
Thus 58 weight
oestrus
and
polypeptides
appears
poly-
day
as the from
than
25 between
30,000. a molecular
1-6
present.
tides
albumin
the
greater
70,000;
(FSH
same
6 day
The
to have oviduct
always
the acidic uteroglobin although it is always
flush
summarize,
ity of globin
superovulated does’ major concentration
a
6 p.c. Never predominant
p.c. normal peptides. To
the
appear normal
and
3 below have
than
p.c. superovulated uterine flushes are similar to the 3 day normal except that they have a higher concentration of uteroglobin (Fig. 2D). Unlike
weight
50 and
the
of
normal
753
between
50,000;
1 day
5 and
PROTEINS
have 1
flush look someflush (compare
Progressively
2C).
in
makes
1B).
(Fig.
OVIDUCT
9
protein
are
albumin, migrating
immunoglobulin to
the
same
G (IgG), position
a as
TUCKER
754
AND
SCHULTZ
5:: 4 3 -
#{149}i.
p g FIG. 3. Cellulose acetate electrophoresis of rabbit uterine proteins. Proteins from uterine fluid of normally mated rabbits from day I to 6 p.c. (samples 1-6, respectively) were subjected to electrophoresis along with rabbit serum (S). Uterine fluid proteins from a progesterone stimulated doe (P) and from a nonpregnant doe (C) run on a separate electrophoresis strip are included to show the location of the uteroglobin (Ug) band. The location of serum albumin is indicated by (Alb).
transferrin, a protein or ceruloplasm, and which bourg,
may 1975).
be
migrating a fifth
like minor
haptoglobin component
alpha-2-macroglobulin Monospecific
prepared certainty.
(Arquemsera
have
not
been
LB fewer
24
to identify In rabbits h previously, serum
proteins,
the latter induced the
components to ovulate
uterine with
lgG
fluid
with with contains
predominant.
UTERINE
OVIDUCT
OVIDUCT
UTERUS
PROTEINS
lated that
755
animals. The in superovulated
transferrin
are
lost
than in uterine animals (Fig. 5). results ences
in
Oviduct which react early stage demonstrate phoretic
FIG. 4. Double immunodiffusion of oviduct and uterus fluid proteins. The centre well contained goat antiserum to total rabbit serum proteins (A-RSP) in each plate. Serum (S) and proteins derived from oviduct or uterus of normally mated animals from 1-6 days p.c. (wells 1-6, respectively) were placed peripherally.
pattern. of serum
period. same
In addition, whether the
IgG
transferrin fluids.
and
are After
a
protein
present Day 2
in p.c.
migrating
day there
p.c. is a
1
like
ovulated
As in the munoelectrophoresis between rabbits. from
superovulated
pare
SO
series
albumin is Additionally, induced mated
with not
(for
day LH
NO
yield p.c.)
induced
the
does
and
ovulation
and
LH)
as the matings
2 day (com-
5) although
concentration. 24 h after
LH
same
pattern
as the
are
clearly
differ-
in nonsuperovu-
LH
losses
or
in common
preimplantation
1 the pattern is the have been mated or (Fig.
from
ovulated patterns
there
Also,
5).
derived
normally
to
broaden rabbit
agrees (1969)
the
oviduct
the
same,
fluid
super-
does. have
our embryo
fluid
proteins
Only
been
pre-
knowledge development
stages proteins
of
protein
the and to
as
IgG,
of
while
in
remain
in
the
uterine
stages. to
by
estrus well
The
contain
serum
-macroglobulin)
the endometrium 3-globulins).
We
and 1 to 2 day as all preimplantation
(e.g. have p.c.
oviduct fluid contain five serum they are: albumin, IgG, a protein the
same
migrating and
occur
albumin,
results that
patterns
reported
secreted uteroglobin, that
of
protein
preimplantation
is
fluid
previous (1974)
changes
the
(e.g.
the Beier
the
marked
fluid
illustrated uterine
with and
during
uterine
plasmin,
a high does,
the
at Day animals
ovulated
study
Kirchner
(FSH
Fig.
major
see Schultz and Tucker, 1977) it is to study the in tOo embryo environnamely the oviduct and uterine fluids.
migrating
series,
no shared
review
normally mated at 1 day p.c.
five components from normal
in such nonmated
ovulation (1
from
animals
same fluid
are
DISCUSSION
ment,
studies, imdifferences
and proteins
the
necessary
uterine marked
fluids.
electrophoretic demonstrates
superovulated Uterine fluid
contain the p.c. uterine
ent
2-D
oviduct
by
sented.
and proteins prealbumin,
and
previously
in fluids
versus
(see arrows in 4 day p.c. uterus, Fig. 5). This latter protein is clearly distinct from one of the major components migrating like transferrin in fluid
mated
noted technique.
with
normally
This
uterine
h earlier
proteins
difference
decrease in the amount and number of proteins which cross-react with A-RSP as suggested earlier by the double immunodiffusion studies. From Day 3 p.c. onward, only two proteins in uterine fluid seem to be shared in common with serum. These appear to be IgG and a protein migrating in a similar position to haptoglobin
early
24
throughout
ovulated no
2 p.c.,
is and
from normally immunoelectrophoretic
There
these
In order preimplantation Albumin,
Day
difference albumin
consistent with differand normally ovulated
proteins
with
the
by
fluid These
of
fluid proteins from stages 1 to 6 p.c. with A-RSP are similar to those of (1 to 2 day p.c.) uterus and they a very constant immunoelectro-
additions
simply
point rabbits,
are completely superovulated
rabbit uterine 2-D electrophoretic
was
final
position
like
a fifth
as transferrin,
haptoglobin
component
a-2-macroglobulin. Beier (1974)
that
and transferrin low albumin. results from
and that at Furthermore 2-D electrophoresis,
or which
Our results support at estrus there is high Day 4-6 because and
a
cerulomay
be
those of albumin there is of our immuno-
electrophoresis it must be stressed that albumin and transferrin are negligible quite rapidly after coitus or administration of superovulating hor-
TUCKER
756
mones,
whereas
remain
during
could
not
other the
correlate
the
proteins
noted
results
because
of
for
the
acetate are
of secretory globin from at
uterus
least
Beier of
of
(1974)
these used. the
both
50
of
postalwith
our
parame-
and of
an increase
percent
We
by
SCHULTZ
tion
we
forms
cellulose
uterine
polypeptides
with
(around
the
(Kirchner,
of uteroat Day 6
protein 1969).
in
7,000)
points. these
The forms reported the
the
In addi-
tion,
there
that
is,
identical but
by “acidic
of
be
Nieto
different
et
al.
two weights
isoelectric
these
As is not
a the
described by Beier to be a modified
although modification bound forms
both from
(1977). likely
postalbumin it appears
two
observe
molecular
with
phosphorylation, one
may we
uteroglobin”
subunit of uteroglobin yet know what this ly
that
amino acid composition of are not detectably different
same as uterine (1974). Rather,
in concentration that
noted
uteroglobin,
result,
fluid
have
of
that 2-D
proteins, particularly Day 1 to 6 p.c., and
is uteroglobin
IgG)
polypeptides
studies
with
and
appropriate
of
electrophoresis
consistent
p.c.
by
techniques
results
(e.g. period.
prealbumin
lack
comparison
different The
proteins
preimplantation
bumin ters
serum
AND
we do not as (e.g., acetylahormone)
is
present
is. Onin
large
UTERUS OVIDUCT
NO
SO
I H
-___ .%-
1
2
S
:LJi,: 3
-“-
4
cj 5
FIG. 5. lmmunoelectrophoresis contained goat antiserum to total after LH induced ovulation without samples are from non-superovulated superovulated (SO) animals up to series is presented. Immunoglobulin
4lII
_J-
of preimplantation period oviduct and uterine fluids of the rabbit. Troughs rabbit serum proteins (A-RSP). Samples are as follows: LH, obtained 24 h mating; 5, rabbit serum; 1-6, 1-6 days postcoitum respectively. Oviduct animals. Uterus samples include both normally ovulated (NO) and Day 3 p.c. No differences were observed past Day 3 p.c. and only the NO G (IgG), Transferrin (Tr) and serum albumin (Aib) are identified.
UTERINE
concentrations
after
does
lated
or treated
with
progesterone
have
form
is present
in
days forms
p.c. are
large
been
OVIDUCT
Superovulation crease the number
superovu-
but
the
other
concentrations
4 to
in the unsuperovulated never found in oviduct
5
does. Two or in uterus
where uteroglobin is in low concentrations (i.e. in oestrus or early pregnancy). The changing pattern of these uterine fluid proteins likely reflects
the
selective
transport
of
serum
pro-
teins as well as the secretion of uterine specific proteins during early embryonic development. The
oviduct
contains selective
fluid
proteins transudation
secretion et al.,
of 1971;
1972).
However
like
which
oviduct Dies, some
the in
relative concentration the oviduct fluids.
of
in
small
our
of the Urzua
uteroglobin in the oviduct
the preimplantation (1972) reported found
we
in
stages. transferrin far
as to
was absent found it with
present at all times
Dies in low during
Fiegelson and and prealbumin
greater
quantity
Kay to
relative
to
albumin in oviduct fluid rather than serum while Dies (1973) found these proteins to be in proportions comparable with those in serum. By comparing the stain density of transferrin with albumin of both serum and oviduct fluid on the 2-D and immunoelectrophoretic systems we agree with Dies (1973) that they are in comparable In
proportions.
all
the
presented,
samples
contained important resolving 3.5
to
2-D
electrophoretic of
oviduct
uterine
fluid
40 ig of protein in each case. It is to consider, however, that we are polypeptides only in the IpH range of 10.
The
polypeptides cluded in the
presence
or new
outside this first dimension
range and
appearance would would
of be exnot be
seen on our gels. Hence, some caution must be taken regarding quantitative aspects of the gel as more or less protein may be retained in the procedure material the This
range
of physical
is unlikely
but may changes fluids.
depending upon in each preparation to
the which
parameters alter
contribute to in polypeptide
our our
amount of lies outside for
major
having composition
There used
pattern (Kirchner,
of
profile the
are reports that the pregnancy the 1969)
(Beier
tion
and
Kiihnel,
in
phoresis temporal
separation.
and
quantity
to infemale
when superovulaspecific protein
1973).
albumin
differ
used by the
uterus is not fully or deviates from
prealbumin,
developed the normal For
example,
postalbumin
from
the
frac-
normal
and
24-3 0 h early. Likewise electrophoresis, cellulose and immunoelectro-
that when does are changes in uterine
superovulated protein pattern
the is
advanced by at least 24 h. In fact, the rapid disappearance of albumin (the protein in the largest concentration) is remarkable and so far unexplained. The two have
dimensional
composition very complex. been stages.
electrophoretic
presented of
show
oviduct More than
resolved in each Our knowledge
activity
of
very
these
determining
roles
the
at early concerning
of
is
the
step
these
first
fluids
still in
in embry-
fact
be cultured
can
is have
components
the reproductive of the components.
embryos
fluids
preimplantation the biological
onic development and is a detailed analysis that
protein
and uterine 50 polypeptides
However, the
profiles
that
protein
incomplete.
process The
in vitro
from
the fertilized egg stage to blastocysts (Whitten, 1971; Seidel et al., 1976) and beyond (Hsu al., 1974) argues that there are no polypeptides with specific ing capacities
growth promoting in these fluids.
At
there is clearly a synchrony development and reproductive response
separations and
is
we
information
agree
is
as to
and
serum proteins et al. (1970)
From
patterns
by and
(Shapiro and Kay,
fluid
rabbit. tion
is commonly of ova released
13-glycoprotein appears we have noted by 2-D acetate electrophoresis
uterus
exists
oviduct
amounts.
2-D
(1973) that concentrations
be
the
blastokinin (uteroglobin) oviduct while Dies (1973)
present
the
uncertainty
composition
from
of
specific proteins 1973; Fiegelson
the
reported in the
that
have arisen both of serum proteins
757
PROTEINS
bryos the low
to
postovulatory
artificially reproductive survival
synchrony substances tion stages
factors
to assign
out
of
Rabbit synchrony than with
emwith
24 h show those in
(Chang, 1950). Growth inhibitory have been reported in preimplantaof mouse uteri (McLaren, 1973).
are
Williams, 1974). as we have reported us
between embryo tract changes in
tract by more rates compared
Carbonic anhydrase leucine aminopeptidase sent and regulated tion
or differentiatthe same time,
stimuli.
placed
et
some
also
and are precapacita-
1955)
1974) Sperm
present
(McRorie
and
High resolution here might
analyses ultimately
such allow
of
functional
continued
factors research
to
specific
missed
allow the elucidation of what subtle influence each component contributes to a proper environment for early development.
other these
and
these
observations of
proteins
(Lutwak-Mann, (Beier, hormonally.
may
TUCKER
758
AND
laboratory
ACKNOWLEDGMENTS
This
work was supported by operating grant MA-4854 from the Medical Research Council Canada. We wish to thank Ms. E. W. Garbutt and R. Bray for valuable technical assistance. We also to thank Dr. E. H. Peters for doing the amino composition analyses.
No. of Mr. wish acid
REFERENCES Arquembourg,
theory,
P. C.
methods,
(1975).
SCHULTZ
Immunoelectrophoresis:
identifications, interpretations. Kargea Publishing Co., BasIc,
2nd. edition, S. Switzerland. 104 pp. Arthur, A. T., Cowan, B. D. and Daniel, Jr., J. C. (1972). Steroid binding to blastokinin. Fert. Steril. 23, 85-92. Arthur, A. T. and Daniel, Jr., J. C. (1972). Progesterone regulation of blastokinin production and maintenance of rabbit blastocysts transferred into uteri of castrate recipients. Fert. Steril. 23, 115-122. Beato, M. and Baier, R. (1975). Binding of progesterone to the proteins of the uterine luminal fluid. Biochim. Biophys. Acta. 392, 346-356. Beier, H. M. (1968). Uteroglobin: a hormone-sensitive endometrial protein involved in blastocyst development. Biochim. Biophys. Acta 160, 289-291. Beier, H. M. (1974). Oviducal and uterine fluids. J. Reprod. Fert. 37, 221-237. Beier, H. M. and KihneI, W. (1973). Pseudo-pregnancy in the rabbit after stimulation by human chorionic gonadotropin. Hormone Res. 4, 1-27. Bullock, D. W. and Willen, G. F. (1974). Regulation of a specific uterine protein by estrogen and progesterone in ovariectomized rabbits. Proc. Soc. Exp. Biol. Med. 146, 294-298. Bullock, D. W., Woo, S. L. C. and O’Malley, B. W. (1976). Uteroglobin Messenger RNA: Translation in vitro. Biol. Reprod. 15, 435-443. chang, M. C. (1950). Development and fate of transferred rabbit ova or blastocyst in relation to ovulation time of recipients. J. Exp. Zool. 114, 197-225. Dies, R. (1973). Influence of hormones on the protein pattern and development of embryos in the genital tract of the rabbit (Oryctolagus cuniculus). Zool. Jahrb. Abt. AlIg. Zool. Physiol. Tiere 77, 360-441. El Banns, A. A. and Daniel, Jr., J. C. (1972a). Stimulation of rabbit blastocysts in vitro by progesterone and uterine proteins in combination. Fert. Steril. 23, 101-104. El-Banna, A. A. and Daniel, Jr., J. C. (1972b). The effect of protein fractions from uterine fluids on embryo growth and uptake of nucleic acid and protein precursors. Fert. Steril. 23, 105-114. Fiegelson, M. and Kay, E. (1972). Protein patterns of rabbit oviducal fluid. Biol. Reprod. 6, 244-252. Hsu, Y., Baskar, J., Stevens, L. C. and Rash, J. E. (1974). Development in vitro of mouse embryos from the two-cell Stage to the early somite stage. J. Embryol. Exp. Morph. 31, 23 5-245. Kirchner, C. (1969). Untersuchungen an uterusspezifischen glykoproteinen wiihrend der friihen graviditat des kaninchens Oryctolagus cuniculus. Wilhelm Roux Arch. Entro Mech. 164, 97-13 3. Kozma, C. K., Pelas, A. and Salvador, R. A. (1967). Electrophoretic determination of serum proteins in
animals.
J.
Amer.
Vet.
Med.
Ass.
151,
865-869. Krishnan, R. and Daniel, Jr., J. C. (1967). “BlastoIdnin”: inducer and regulator of blastocyst development in the rabbit uterus. Science, 158, 490492. Layne, E. (1957). Spectrophotometric and turbidimetric methods for measuring proteins. In S. Colowick and N. Kaplan, eds. Methods in Enzymology, vol. III, Academic Press, N.Y., N.Y. pp. 450-451. Lutwak-Mann, C. (1955). Carbonic anhydrase in the
female
reproductive
tract.
Occurrence,
distribution
and hormonal dependence. J. Endoer. 13, 26-38. Manes, C. (1969). Nucleic acid synthesis in preimplantation rabbit embryos. I. Quantitative aspects, relationship to early morphogenesis and protein synthesis. J. Exp. Zool. 172, 303-310. Marchalonis, J. J. and Weltman, J. K. (1971). Relatedness among proteins: a new method of estimation and its application to immunoglobulins. Comp. Biochem. Physiol. 38B, 609-625. McRorie, R. A. and Williams, W. L (1974). Biochemistry of mammalian fertilization. Ann. Rev. Biochem. 43, 777-803. Murray, F. A., McGaughey, R. W. and Yarus, M. J. (1972). Blastokinin: its size and shape and an indication of the existence of sub-units. Fert. Steril. 23, 69-80. Neito, A., Ponstingi, H. and Beato, M. (1977). Purification and quaternary structure of the hoemonally induced protein uteroglobin. Arch. Biochem. Biophys. 180, 82-92. O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 40074021. Schultz, G. A. and Tucker, E. B. (1977). Protein synthesis and gene expression in preimplantation rabbit embryos. In Development in Mammals, M. Johnson, ed. Vol. 1, Elsevier PubI. Co., Amsterdam. pp. 69-97. Seidel, G. F., Bowen, R. A. and Kane, M. T. (1976). In vitro fertilization, culture and transfer of rabbit ova. Fert. Steed. 27, 861-870. Shapiro, S. S., Jentsch, J. P. and Yard, A. 5. (1971). Protein composition of rabbit oviducal fluid. J. Reprod. Pert. 24,403-408. Sober, H. A. (1970). Handbook of Biochemistry selected data for Molecular Biology. 2nd edition. CRC Press Publishing Co., Cleveland, Ohio, USA. p. c-28. Swank, R. T. and Munkres, K. D. (1971). Molecular weight analyses of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 39,462477. Urzua, M. A., Stambaugh, R., Flickinger, G. and Mastroianni, L. (1970). Uterine and oviduct fluid protein patterns in the rabbit before and after ovulation. Fert. Steed. 21, 860-865. Whitten, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos in vitro. Advan. BioSci. 6, 129-1 39. RECOMMENDED Beier, H. Reprod. Sherman,
M. (1974). Oviducal Fert. 37, 221-237. M. I. and Saloman,
REVIEWS and D.
uterine 5.
fluids. (1975).
J. The
UTERINE
OVIDUCT
relationship between the early mouse embryo and its environment. In The Developmental Biology of Reproduction, 33rd Symposium of the Society for
PROTEINS
Developmental Papaconstantinou, 277-309.
759
Biology, eds.
C. L. Academic
Markert Press,
and J. N.Y. pp.
OF
REPRODUCTION
17,
749-759
(1977)
Temporal Changes in Proteins of Oviduct Fluids During the Preimplantation Period E. B. TUCKER Division
and
G. A. SCHULTZ
of Medical Faculty
of
University
Calgary,
Biochemistry, Medicine, of
Alberta,
and Uterine in the Rabbit
Calgary,
Canada
T2N
1N4
ABSTRACT A study of the polypeptides which contribute to the environment surrounding the rabbit preimplantation embryo was undertaken. At various times during the first 6 days of pregnancy the lumen of the oviducts and uteri of normally mated and superovulated does were flushed with saline, and the polypeptides present in these flushings were separated in a two-dimensional (isoelectric focusing, sodium dodecyl sulfate) electrophoresis system. Information was obtained regarding similarities and differences in polypeptide patterns between the stages and between the tissues. The major protein in the oviduct is albumin whereas in the uterus at later preimplantation stages it is uteroglobin. During the preimplantation period the polypeptide environment in the oviduct remains virtually the same, while in the uterus there is a disappearance of albumin and a marked increase in uteroglobin. lmmunoelectrophoresis demonstrated that of the more than 50 polypeptides resolved in two-dimensional electrophoresis of oviduct fluid, at least 5 are identical to serum proteins (albumin, lgG, transferrin, and possibly haptoglobin and n-2-macroglobulin). Uterine fluid at Day 1 to 2 postcoitum (p.c.) also contains these 5 serum proteins. By Day 3 p.c., the uterine fluid contains little protein which reacts with antisera directed toward rabbit serum proteins. Some differences were noted in polypeptide patterns of uterine flushes during early preimplantation stages of superovulated does versus those from normal matings. Comparable differences were not observed in oviduct fluids. INTRODUCTION
The
biochemical
composition
and uterine fluids of development parameter luminal
in fluids
passage
of
ronment sides and
spermatozoa in
and
specific
which derives
patterns
uterine
fluids for the
and
during may
well
ova
globin Daniel,
the
of
Arthur
These for the retime-
have
stages
proteins isoelectric rell, 1975)
concerned
with
oviduct They
temporal 1969;
the developof studies on
tide ine
and uterine have been changes Beier,
poral uterine
whether proteins are derived by endometrial secretion or transudation of serum (Fiegelson and Kay, 1972; Beier, 1974; Shapiro et al., 1971; Dies, 1973) and the relationship between
Accepted Received
July April
high developed.
by
two
components lumen fluids
period
14, 1977. 25, 1977.
749
by
present
method
Willen,
unrelated
communi-
parameters weight the
in oviducal and during in rabbits.
the The
of
(O’Farpolypepand utersix day polypep-
of both normally ovulated does were compared. The or absence also present
immunological
of in
oviduct serum
procedures.
have allowed us to verify clarify previously reported
addition information
to
teins
reproductive
contributing regarding
new temporal tract
the
of separating
methods cases to
in
and 1970;
and
molecular to reexamine
present before
occurrence proteins
noted
In the
and used
utero-
(Krishnan et al.,
techniques for of polypeptides
sensitive
the point was
protein of
Bullock
resolution separation
highly
tide patterns superovulated
1974),
tract
induction
1972;
Daniel,
preimplantation in
the
and
the
conse-
important
reproductive production 1968; Urzua
been
cation
oviduct
and
blastokinin 1967; Beier
1974). Recently, two-dimensional
envi-
embryo Any
or
have
composition of been published. (Kirchner,
stages important
preimplantation
the protein fluid have composition
particularly,
and
composition
synchronization of embryo. A number
general
status
patterns,
process. a medium
the developing nourishment. in the
of development quences ment of
during preimplantation in mammals is an
the reproductive serve both as
hormonal oviducal
of
and
and was These
and
more changes
fluids
and tem-
in some results in detailed in pro-
during
the
TUCKER
750
preimplantation
period
and
Oviduct
Flushings
AND
Preparation
of
METHODS Uterine
SCHULTZ
NaCI,
in rabbits.
MATERIALS Collection
AND
and
Dutch belted rabbits were used throughout the work. A complete series of normally ovulated animals and a complete series of superovulated animals was studied. The latter were induded because of relevance to the many studies on preimplantation embryos which have depended heavily on the use of hormonal superovulation to increase embryo yields. For the normal series, animals were mated on Day 0 and groups of at least three animals sacrificed at daily intervals throughout the 6 day preimplantation period prior to removal of uteri and oviducts. Animals induced to ovulate without mating were given 2.5 mg of luteinizing hormone (LH) in an ear vein and sacrificed 24 h later. Superovulation was performed as described by Manes (1969). Does were brought into the same cycle by a single injection of 25 IU human chorionic gonadotrophin. Nineteen days later they were given 0.3 mg of follicle stimulating hormone subcutaneously twice daily for three days. On the fourth day they were mated and injected with 2.5 mg of LH into an ear vein or ovulated with LH administration alone without mating. As for the normal series, coitus was taken as Day 0 and groups of at least three animals were sacrificed at daily intervals for recovery of oviducts and uteri. The flushing obtained by passing saline through the respective reproductive organ was cleaned of debris by centrifugation, dialyzed against water using No. 3 Spectraphor membranes (Spectrum Med. Ind. Inc., Los Angeles) and lyophilized. All procedures prior to lyophiization were performed at 0#{176}C. Protein concentration of samples was determined by the Biuret method (Layne, 1957).
0.025
Using System
the
Lyophiized samples were dissolved in lysis buffer (9.5 M Urea, 2 percent (w/v) Nonidet P-40, 2 percent Ampholines pH 3.5 to 10, and 5 percent -mercaptoethanol) and frozen at -70#{176}C until use. Proteins were separated according to isoelectric point by isoelectric focusing in tube gels in the presence of pH 3.5-10 ampholines in the first dimension, followed by molecular weight separation by sodium dodecyl sulfate (SDS) electrophoresis on 10-15 percent exponential gradient acrylamide slab gels in the second dimension (O’Farrell, 1975). Forty g of protein was used in all cases. Gels were stained for 2 h in 50 percent methanol, 9 percent acetic acid, 3 percent glycerol and dried on a Hoefer gel slab drier. A mixture of marker proteins was applied to a small piece of cellulose acetate membrane and was placed beside the first dimension gel on top of the second dimension gel for molecular weight calibration of the second dimension. Immunological
Procedures
agar plates for double immunodiffusion (Ouchterlony) studies were obtained from Hyland Laboratories, Inc. Lyophiized oviduct and uterine fluid samples were dissolved in 0.15 M Goat
anti-rabbit
serum
and
buffer,
pH
7.2
at a concentration
RESULTS
Identification
the
of
Albumin following
albumin sample marked min
Separation of Polypeptides Two Dimensional (2-D)
M Tris
of 50 mg/mi. Rabbit serum (60 mg/mI) was used without dilution. Double immunodiffusion studies were performed at room temperature for 24 h using gels containing 2 percent agar, 7.5 percent glycine, 1 percent NaCl, and 0.1 percent sodium aside at a pH of 7.2. Five MI samples of oviduct, uterine, or serum protein were applied to peripheral wells and S til goat anti-rabbit serum to the center well. lmmunoelectrophoresis was performed using the Model R-103 accessory to the Beckman Microzone electrophoresis apparatus. Samples containing 25 ug protein in 0.5 M’ were applied to embossed membranes and subjected to electrophoresis in 0.0375 ionic strength barbiturate buffer, pH 8.6, for 45 min at 150 volts. After electrophoresis, 20 MI of goat antiserum to total rabbit serum proteins (A-RSP) was put into each trough and immunodiffusion was allowed to proceed for 24 h under paraffm oil. Membranes were then washed with saline to remove unprecipitated protein and were stained with Ponceau S dye to visualize precipitin lines. Standard cellulose acetate electrophoresis was performed using 0.07 5 ionic strength barbiturate buffer pH 8.6 for 30 mm at 250 volts. Membranes were fixed and stained with Ponceau S dye to visualize proteins.
flush the
Proteins
of uterine manner.
(Sigma protein albumin is well
blood marked
Major
flushing When
known
to
serum (Kozma albumin on oviduct
flush
protein
of
min is present rabbits and in
be
the
(see
protein prominent
all three. This peptide three spots which are albumin. proteins
them,
“transferrin” has and
iron bound tentatively serum
protein
identically
to
That
albu-
sec-
of blood serum is or oviduct flush
other and
than albumin is present in as a series of are more basic
immunoelectrophoresis
allowed in
p.c. by
Immunological
appears larger and
The
serum
of
the spot of uterine
in uterine flushes of 1 day oviduct flushes was verified
is another is relatively
has
with spot albu-
protein and
serum.
tion). When the 2-D pattern matched with that of uterine
system
major
migrates blood
immunoelectrophoresis
90,000, point,
Uterus
was identified in purified rabbit
et al., 1967) the 2-D pattern
and
than
the
Chemical Co.) was mixed it migrated with the in Fig. 1. Furthermore,
major
there which
in
the uterine has
identification flushes
of and
a molecular
one
weight
5
of of
a slightly more basic isoelectric exists in several forms, the apo and forms (Sober, 1970). Thus identified this polypeptide transferrin.
we have as the
UTERINE
OVIDUCT
ISOELECTRIC
PROTEINS
751
FOCUSING
(0 ‘Ii
r m C, -4
0 0 “I 0)
(0
FIG. 1. Two-dimensional gel electrophoresis separation of rabbit uterine flush proteins. In the first dimension, proteins were separated by isoelectric focusing on 110mm cylindrical gels. In the second dimension, proteins were separated according to molecular weight on SDS exponential gradient (10-15 percent acrylamide) polyacrylamide slab gels. Forty g protein was used in each separation. A, Estrus; B, 2 day p.c.; C, 3 day p.c.; D, 4 day p.c.; E, 5 day p.c.; F, 6 day p.c. Albumin (AIb), uteroglobin (Ug), and transferrin (Tr) are identified.
The
been
molecular reported
15,000 14,000-16,000 tein is the uterine and is Daniels,
weight
as (Murray major
flushes induced 1972).
(Bullock
there which
is
postcoitus
uteroglobin
et
has
et al., 1972)
al., 1977). of 4, 5, and
cells of the endometrium it has been isolated and
and trans-
al.,
1976).
In our
2-D
uterine
flush
and
system
which
can
uteroglobin
which
the
procedure
we
have
found
polypeptide similar to et
and
supports
spot
At with
Swank the
and
on
al. (1977) their
molecular
under
same
molecular
be
as
1. Using of
daltons. determined
This
is by
uteroglobin
identical, subis a noticeable weight
from
composition
that
but
modification “acidic”
we to
form.
both do
has
of uteroglobin nancy (Arthur
in
been the et
1972a,
the authentic different The
et al.
spots
are
as
the subunit It may be
which
yet
(1977)
and to the These
uteroglobin know
what
has occurred in the related to hormone
suggested
as a function
uterus during al., 1972; El
b; Beato
by the (1971),
according al. (1976).
not
of
is indistinguishable
Nieto
we purified Bullock et
indicate
subunits
It
basis
Weltman
also by
the
compositions and
reported
of uteroglobin procedure of
Daniel,
acid
point.
On
uteroglobin” and are not detectably
acid that
amino
Marchalonis
this “acidic uteroglobin amino
isoelectric
inducible.
and
Baier,
early Banna
pregand
1975).
this
conditions
that
of
binding days
acidic
(1971),
weight
denaturing
conclusion
of two similar, if not Day 4 and 5 p.c. there the
Fig.
Munkres
to be 6,700-7,000 the value of 8,000
Nieto consists units.
is so labeled
of
a more of
method
results
polypeptide the 6 day
induced when does are injected subcutaneously with 4 mg progesterone for 5 consecutive (Fig. 3). Thus we identified this protein
but
is progesterone
comparison
This pro6 day p.c. 1974) and and
molecular weight major protein in
globin also
1976), and
(Kirchner, 1969; Beier, by progesterone (Arthur This protein is produced
et
a low is the
of
(Bullock et al.,
(Neito protein
secreted by the the mRNA for lated
11,400
as utero-
Temporal
Changes
of
Nonsuperovulated
When unmated
40
j.zg of
does
polypeptides slab gel (Fig. overloaded
Uterine
Rabbit
Proteins (2-D
uterine
flush
protein
is electrophoresed, could 1A).
with
in
Electrophoresis)
58
be identified on If the first dimension
protein
up
to
88
from distinct the
polypeptides
2-D gel is
TUCKER
752
AND
ISOELECTRIC
pH
SCHULTZ
FOCUSING
Ij
FIG. 2. Two-dimensional gel electrophoresis separation of hormonally treated rabbit’s uterine and oviduct flush. Superovulation was performed with FSH and LB as described by Manes, 1969. Electrophoretic methods described in Fig. 1. A, uterine flush of unstimulated doe ovulated with LH; B, superovulated 1 day p.c. uterine flush; C, superovulated 2 day p.c. uterine flush; D, superovulated 3 day p.c. uterine flush; E, normal 1 day p.c. oviduct flush; F, superovulated 1 day p.c. oviduct flush. Albumin (AIb), uteroglobin (Ug), and transferrin (Tr) are identified.
may
be
found
Of the molecular
24
molecular
weight
between Albumin
30 and makes
and
uteroglobin
The
protein
flush
small
50,000; up the
protein
takes
present
to
the
of
2 days p.c. but there (note
place
of proteins and greater
50 and
in the
with (note
at at
1C). least Day
By this 4 new 2 p.c.
arrow)
30-50,000
p.c.
flush
17
which
is more
there
1A) these of a
3 p.c. pattern
disappearance
weight a slight
of 50,000 appearance
is an appearance which polypeptides
concentrated (note “acidic uteroglobin”
were
which
have
(note
ring).
The
marked
time were except
change
becomes
uteroglobin (Fig.
as
very
the
faint
is the protein iF). Furthermore,
of
and highest at this
there is a reappearance of proteins which present at Days 2 and 3 (note arrows) for the high molecular weight proteins.
Albumin
and
transferrin
do not
doe
not
stimulated
with
was
ing
Hormone
was
injected
(FSH), with
ovulation, to
was
Luteinizing the
Day
uterine
3 p.c.
of
reappear. Follicle
not
If the Stimulat-
mated,
but
Hormone flush
pattern
normal
only
(LH)
to
looks
mating
(Fig.
2A). of
arrow). By is becoming
an isoelectric
is another
authentic concentration
similar
absent become
(Fig. 1D) and there is a complete of proteins of molecular weight and
albumin
uteroglobin”
cause
has the same molecubut is more acidic
than
there
“acidic
acidic
Day
basic
“acidic uteroglobin” is the major protein of the 5 day p.c. uterine flush while the other polypeptides appear identical to those of Day 4 p.c. (Fig. 1E). At Day 6 p.c., the time of implantation,
uterine (Fig.
no loss of appearance
is a complete a molecular ring) and
polypeptides and other
relatively more Day 4 p.c. the concentrated disappearance
1 day
there is is a slight
time
a a
amounts.
(Fig. 1B). However, by change in the polypeptide as there
more
have 32 have
70,000;
small
oestrus
of the polypeptide which lar weight as uteroglobin (Fig.
results). 7
and 2 below 30,000. highest concentration
is pattern
than albumin a remarkable
(unpublished
polypeptides 70,000 or larger,
of between
is identical
and at proteins
h p.c.
58 distinct weight of
point
Temporal
Changes
Superovulated There
of Uterine
Rabbits are
superovulated uterine flush predominance
detectable
(2-D
Proteins
in
Electrophoresis)
differences
between
the
rabbit flush and the normal proteins. At Day 1 p.c. there is a of polypeptides with a molecular
UTERINE
weight
of
50-70,000
concentration
and
of
Fig.
2B
with
p.c.
superovulated
day
p.c.
which
uterine p.c. normal
Fig.
flush
a decrease
albumin
day p.c. superovulated what like the 2 day
the
the
the
flush
looks
like
The
3, 4,
the
31
a molecular
3
6 day
proteins 50,000.
same
polypeptides
wise
oviducts
superovulated
the ine
by far oviduct,
normal flush
does, contains
uteroglobin
at
to Day become present.
The
With
Day
2 p.c. does
6 day
p.c.
uterine
which
uterof
is maintained
superovulated
and
have
identical
makes
up
the
trations. day
polypeptides is only normal
the
major-
of uterine fluids and slightly present during
uteroestrus.
mating
the
serum
proteins
and transferrin begins to disappear so that by Day 3 p.c. they are almost absent and by Day 4 p.c. they are not detectable. When rabbits are superovulated (FSH followed by LH) or are just injected
with
LH,
slightly present p.c. Uteroglobin concentrations mating
there
form major
by Day amount
1 day
p.c.
is
normally
during
When
is a high p.c. and
clearly
proteins
and
are
are
absent
present
oestrus
and
rabbits
only 2 days
in
with
increase
4 and 5 p.c. By of protein in the
trace
normal
in an acidic
Day 6 p.c. the uterine flush is
are
1
shown
by
of
conventional
cellulose
accompanied uteroglobin tify
by band.
this
band
inducible
by
significant
Changes
Oviducts
(2-D
When could
40 first be
and in
(Fig. of
because
it
is not
present
fluid
from
uterine
in non-
Proteins
in
oviduct
dimension distinguished
flush gel (Fig.
protein about 2E).
is applied 68 Of
there
relative
concen-
noticeable a
on
number
of
concentrated
in
3
the
polypeprelation
to
presented).
of Proteins
in Uteri
position markedly
of oviduct during the
fluid does preimplantation
and
2-D electrothat the comnot
change period
while the uterine protein different in late (Day compared to early (Day Immunological techniques
patterns are markedly 3 to 6 p.c.) stages 1 to 2 p.c.) stages. have been utilized to
monitor any modulations and oviduct fluids which
in proteins are common
to
verify
When sion was a
results
from
the technique used, uterine
lesser
degree lines
2-D
Day
2
with
identity
serum. However, of preimplantation
uterine
with
of uterine to serum
electrophoresis.
of double immunodiffuproteins from Day 1 and
A-RSP
and
3
p.c.
show
reactions
with
fluids from later show little or no
(Fig.
4)
in concentration or loss shared in common between serum. Oviduct fluid proteins
stages of the preimplantation A-RSP to form precipitin reactions of identity with lmmunoelectrophoresis
These
peptides these
although
indicating
a
of
these uterine from all
period react with lines which give rabbit serum (Fig. 4). was used to resolve
precisely the antigenic and oviduct fluids shared
components with serum.
of In
rabbits from normal matings, there are at least five serum proteins in uterine fluid at 2 day p.c. which react with A-RSP (NO series, Fig. 5).
Electrophoresis)
zg
their
with the indicated
more uterine
3).
the up
present
obtained technique has
decrease proteins fluid and is
LH
as makes
that
always
in
where
Changes
cross-reaction
increase in the above, we iden-
uteroglobin
progesterone
rabbits
Temporal
the
as
amounts
pregnant
to
a concomitant As indicated
been
given
(Immunological)
Evidence
rabbit stages
acetate electrophoresis of Day 1 to 6 p.c. uterine fluid (Fig. 3). There is a marked decrease in staining intensity of proteins migrating similar to serum proteins after 1 day p.c.
are
more
(not
precipitin
uteroglobin
is significant
phoretic
to concentration
the Like-
have
or just
polypeptides Albumin
is particularly
oviduct
Oviducts
have
oviduct.
which LH)
a change
become
and
superovulated
concentration of uteroglobin this is maintained throughout
preimplantation. The increased is
serum
is a noticeable
uteroglobin. there day
these
be
Temporal
albumin
oviduct
rabbits
It
This
albumin
p.c.
the same (Fig. 2F).
30 and percent greater
greatest amount of the protein in the and a spot migrating as uteroglobin is
to
p.c.
70,000;
Thus 58 weight
oestrus
and
polypeptides
appears
poly-
day
as the from
than
25 between
30,000. a molecular
1-6
present.
tides
albumin
the
greater
70,000;
(FSH
same
6 day
The
to have oviduct
always
the acidic uteroglobin although it is always
flush
summarize,
ity of globin
superovulated does’ major concentration
a
6 p.c. Never predominant
p.c. normal peptides. To
the
appear normal
and
3 below have
than
p.c. superovulated uterine flushes are similar to the 3 day normal except that they have a higher concentration of uteroglobin (Fig. 2D). Unlike
weight
50 and
the
of
normal
753
between
50,000;
1 day
5 and
PROTEINS
have 1
flush look someflush (compare
Progressively
2C).
in
makes
1B).
(Fig.
OVIDUCT
9
protein
are
albumin, migrating
immunoglobulin to
the
same
G (IgG), position
a as
TUCKER
754
AND
SCHULTZ
5:: 4 3 -
#{149}i.
p g FIG. 3. Cellulose acetate electrophoresis of rabbit uterine proteins. Proteins from uterine fluid of normally mated rabbits from day I to 6 p.c. (samples 1-6, respectively) were subjected to electrophoresis along with rabbit serum (S). Uterine fluid proteins from a progesterone stimulated doe (P) and from a nonpregnant doe (C) run on a separate electrophoresis strip are included to show the location of the uteroglobin (Ug) band. The location of serum albumin is indicated by (Alb).
transferrin, a protein or ceruloplasm, and which bourg,
may 1975).
be
migrating a fifth
like minor
haptoglobin component
alpha-2-macroglobulin Monospecific
prepared certainty.
(Arquemsera
have
not
been
LB fewer
24
to identify In rabbits h previously, serum
proteins,
the latter induced the
components to ovulate
uterine with
lgG
fluid
with with contains
predominant.
UTERINE
OVIDUCT
OVIDUCT
UTERUS
PROTEINS
lated that
755
animals. The in superovulated
transferrin
are
lost
than in uterine animals (Fig. 5). results ences
in
Oviduct which react early stage demonstrate phoretic
FIG. 4. Double immunodiffusion of oviduct and uterus fluid proteins. The centre well contained goat antiserum to total rabbit serum proteins (A-RSP) in each plate. Serum (S) and proteins derived from oviduct or uterus of normally mated animals from 1-6 days p.c. (wells 1-6, respectively) were placed peripherally.
pattern. of serum
period. same
In addition, whether the
IgG
transferrin fluids.
and
are After
a
protein
present Day 2
in p.c.
migrating
day there
p.c. is a
1
like
ovulated
As in the munoelectrophoresis between rabbits. from
superovulated
pare
SO
series
albumin is Additionally, induced mated
with not
(for
day LH
NO
yield p.c.)
induced
the
does
and
ovulation
and
LH)
as the matings
2 day (com-
5) although
concentration. 24 h after
LH
same
pattern
as the
are
clearly
differ-
in nonsuperovu-
LH
losses
or
in common
preimplantation
1 the pattern is the have been mated or (Fig.
from
ovulated patterns
there
Also,
5).
derived
normally
to
broaden rabbit
agrees (1969)
the
oviduct
the
same,
fluid
super-
does. have
our embryo
fluid
proteins
Only
been
pre-
knowledge development
stages proteins
of
protein
the and to
as
IgG,
of
while
in
remain
in
the
uterine
stages. to
by
estrus well
The
contain
serum
-macroglobulin)
the endometrium 3-globulins).
We
and 1 to 2 day as all preimplantation
(e.g. have p.c.
oviduct fluid contain five serum they are: albumin, IgG, a protein the
same
migrating and
occur
albumin,
results that
patterns
reported
secreted uteroglobin, that
of
protein
preimplantation
is
fluid
previous (1974)
changes
the
(e.g.
the Beier
the
marked
fluid
illustrated uterine
with and
during
uterine
plasmin,
a high does,
the
at Day animals
ovulated
study
Kirchner
(FSH
Fig.
major
see Schultz and Tucker, 1977) it is to study the in tOo embryo environnamely the oviduct and uterine fluids.
migrating
series,
no shared
review
normally mated at 1 day p.c.
five components from normal
in such nonmated
ovulation (1
from
animals
same fluid
are
DISCUSSION
ment,
studies, imdifferences
and proteins
the
necessary
uterine marked
fluids.
electrophoretic demonstrates
superovulated Uterine fluid
contain the p.c. uterine
ent
2-D
oviduct
by
sented.
and proteins prealbumin,
and
previously
in fluids
versus
(see arrows in 4 day p.c. uterus, Fig. 5). This latter protein is clearly distinct from one of the major components migrating like transferrin in fluid
mated
noted technique.
with
normally
This
uterine
h earlier
proteins
difference
decrease in the amount and number of proteins which cross-react with A-RSP as suggested earlier by the double immunodiffusion studies. From Day 3 p.c. onward, only two proteins in uterine fluid seem to be shared in common with serum. These appear to be IgG and a protein migrating in a similar position to haptoglobin
early
24
throughout
ovulated no
2 p.c.,
is and
from normally immunoelectrophoretic
There
these
In order preimplantation Albumin,
Day
difference albumin
consistent with differand normally ovulated
proteins
with
the
by
fluid These
of
fluid proteins from stages 1 to 6 p.c. with A-RSP are similar to those of (1 to 2 day p.c.) uterus and they a very constant immunoelectro-
additions
simply
point rabbits,
are completely superovulated
rabbit uterine 2-D electrophoretic
was
final
position
like
a fifth
as transferrin,
haptoglobin
component
a-2-macroglobulin. Beier (1974)
that
and transferrin low albumin. results from
and that at Furthermore 2-D electrophoresis,
or which
Our results support at estrus there is high Day 4-6 because and
a
cerulomay
be
those of albumin there is of our immuno-
electrophoresis it must be stressed that albumin and transferrin are negligible quite rapidly after coitus or administration of superovulating hor-
TUCKER
756
mones,
whereas
remain
during
could
not
other the
correlate
the
proteins
noted
results
because
of
for
the
acetate are
of secretory globin from at
uterus
least
Beier of
of
(1974)
these used. the
both
50
of
postalwith
our
parame-
and of
an increase
percent
We
by
SCHULTZ
tion
we
forms
cellulose
uterine
polypeptides
with
(around
the
(Kirchner,
of uteroat Day 6
protein 1969).
in
7,000)
points. these
The forms reported the
the
In addi-
tion,
there
that
is,
identical but
by “acidic
of
be
Nieto
different
et
al.
two weights
isoelectric
these
As is not
a the
described by Beier to be a modified
although modification bound forms
both from
(1977). likely
postalbumin it appears
two
observe
molecular
with
phosphorylation, one
may we
uteroglobin”
subunit of uteroglobin yet know what this ly
that
amino acid composition of are not detectably different
same as uterine (1974). Rather,
in concentration that
noted
uteroglobin,
result,
fluid
have
of
that 2-D
proteins, particularly Day 1 to 6 p.c., and
is uteroglobin
IgG)
polypeptides
studies
with
and
appropriate
of
electrophoresis
consistent
p.c.
by
techniques
results
(e.g. period.
prealbumin
lack
comparison
different The
proteins
preimplantation
bumin ters
serum
AND
we do not as (e.g., acetylahormone)
is
present
is. Onin
large
UTERUS OVIDUCT
NO
SO
I H
-___ .%-
1
2
S
:LJi,: 3
-“-
4
cj 5
FIG. 5. lmmunoelectrophoresis contained goat antiserum to total after LH induced ovulation without samples are from non-superovulated superovulated (SO) animals up to series is presented. Immunoglobulin
4lII
_J-
of preimplantation period oviduct and uterine fluids of the rabbit. Troughs rabbit serum proteins (A-RSP). Samples are as follows: LH, obtained 24 h mating; 5, rabbit serum; 1-6, 1-6 days postcoitum respectively. Oviduct animals. Uterus samples include both normally ovulated (NO) and Day 3 p.c. No differences were observed past Day 3 p.c. and only the NO G (IgG), Transferrin (Tr) and serum albumin (Aib) are identified.
UTERINE
concentrations
after
does
lated
or treated
with
progesterone
have
form
is present
in
days forms
p.c. are
large
been
OVIDUCT
Superovulation crease the number
superovu-
but
the
other
concentrations
4 to
in the unsuperovulated never found in oviduct
5
does. Two or in uterus
where uteroglobin is in low concentrations (i.e. in oestrus or early pregnancy). The changing pattern of these uterine fluid proteins likely reflects
the
selective
transport
of
serum
pro-
teins as well as the secretion of uterine specific proteins during early embryonic development. The
oviduct
contains selective
fluid
proteins transudation
secretion et al.,
of 1971;
1972).
However
like
which
oviduct Dies, some
the in
relative concentration the oviduct fluids.
of
in
small
our
of the Urzua
uteroglobin in the oviduct
the preimplantation (1972) reported found
we
in
stages. transferrin far
as to
was absent found it with
present at all times
Dies in low during
Fiegelson and and prealbumin
greater
quantity
Kay to
relative
to
albumin in oviduct fluid rather than serum while Dies (1973) found these proteins to be in proportions comparable with those in serum. By comparing the stain density of transferrin with albumin of both serum and oviduct fluid on the 2-D and immunoelectrophoretic systems we agree with Dies (1973) that they are in comparable In
proportions.
all
the
presented,
samples
contained important resolving 3.5
to
2-D
electrophoretic of
oviduct
uterine
fluid
40 ig of protein in each case. It is to consider, however, that we are polypeptides only in the IpH range of 10.
The
polypeptides cluded in the
presence
or new
outside this first dimension
range and
appearance would would
of be exnot be
seen on our gels. Hence, some caution must be taken regarding quantitative aspects of the gel as more or less protein may be retained in the procedure material the This
range
of physical
is unlikely
but may changes fluids.
depending upon in each preparation to
the which
parameters alter
contribute to in polypeptide
our our
amount of lies outside for
major
having composition
There used
pattern (Kirchner,
of
profile the
are reports that the pregnancy the 1969)
(Beier
tion
and
Kiihnel,
in
phoresis temporal
separation.
and
quantity
to infemale
when superovulaspecific protein
1973).
albumin
differ
used by the
uterus is not fully or deviates from
prealbumin,
developed the normal For
example,
postalbumin
from
the
frac-
normal
and
24-3 0 h early. Likewise electrophoresis, cellulose and immunoelectro-
that when does are changes in uterine
superovulated protein pattern
the is
advanced by at least 24 h. In fact, the rapid disappearance of albumin (the protein in the largest concentration) is remarkable and so far unexplained. The two have
dimensional
composition very complex. been stages.
electrophoretic
presented of
show
oviduct More than
resolved in each Our knowledge
activity
of
very
these
determining
roles
the
at early concerning
of
is
the
step
these
first
fluids
still in
in embry-
fact
be cultured
can
is have
components
the reproductive of the components.
embryos
fluids
preimplantation the biological
onic development and is a detailed analysis that
protein
and uterine 50 polypeptides
However, the
profiles
that
protein
incomplete.
process The
in vitro
from
the fertilized egg stage to blastocysts (Whitten, 1971; Seidel et al., 1976) and beyond (Hsu al., 1974) argues that there are no polypeptides with specific ing capacities
growth promoting in these fluids.
At
there is clearly a synchrony development and reproductive response
separations and
is
we
information
agree
is
as to
and
serum proteins et al. (1970)
From
patterns
by and
(Shapiro and Kay,
fluid
rabbit. tion
is commonly of ova released
13-glycoprotein appears we have noted by 2-D acetate electrophoresis
uterus
exists
oviduct
amounts.
2-D
(1973) that concentrations
be
the
blastokinin (uteroglobin) oviduct while Dies (1973)
present
the
uncertainty
composition
from
of
specific proteins 1973; Fiegelson
the
reported in the
that
have arisen both of serum proteins
757
PROTEINS
bryos the low
to
postovulatory
artificially reproductive survival
synchrony substances tion stages
factors
to assign
out
of
Rabbit synchrony than with
emwith
24 h show those in
(Chang, 1950). Growth inhibitory have been reported in preimplantaof mouse uteri (McLaren, 1973).
are
Williams, 1974). as we have reported us
between embryo tract changes in
tract by more rates compared
Carbonic anhydrase leucine aminopeptidase sent and regulated tion
or differentiatthe same time,
stimuli.
placed
et
some
also
and are precapacita-
1955)
1974) Sperm
present
(McRorie
and
High resolution here might
analyses ultimately
such allow
of
functional
continued
factors research
to
specific
missed
allow the elucidation of what subtle influence each component contributes to a proper environment for early development.
other these
and
these
observations of
proteins
(Lutwak-Mann, (Beier, hormonally.
may
TUCKER
758
AND
laboratory
ACKNOWLEDGMENTS
This
work was supported by operating grant MA-4854 from the Medical Research Council Canada. We wish to thank Ms. E. W. Garbutt and R. Bray for valuable technical assistance. We also to thank Dr. E. H. Peters for doing the amino composition analyses.
No. of Mr. wish acid
REFERENCES Arquembourg,
theory,
P. C.
methods,
(1975).
SCHULTZ
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