receptors for prostaglandins. (PG). F2 and. E2 were quantified in ovine corpora ... of reproductive status or day post-estrus, concentrations of luteal receptors for.
BIOLOGY
OF REPRODUCTION
Receptors
47,
984-991
(1992)
for Prostaglandins
F2a and E2 in Ovine Recognition
Gj.
Animal
WIEPZ,
WILTBANK,3
M.C.
Reproduction
and
NETF,
G.D.
NISWENDER,
Department
Laboratoiy,
Collins,
Fort
Lutea
during
Maternal
of Pregnancy1
T.M.
Biotechnology
Corpora
and
H.R
of Pbysiology,
Colorado
SAWYER2
Colorado
State
Universiiy
80523
ABSTRACT Plasma pregnant
membrane and
Regardless those
was apparent
than
in nonpregnant >
0.05;
2.8
only ewes
±
ewes
concentrations
is not
resistant
the
to the
maternal
concentrations
at which ±
fmol/mg)
2.7
luteolytic
±
for
actions for
for
and
and
tic ruminants terone from
0.2
PGE2
until
is not
due
Day
corpus
to regress approximately 16 days post-estrus if embryonic tissue is present in the uterus
the
uterus
causes
the
understood,
embryo
prevents
maintenance
receptors higher
(p ±
corpora
lutes
in
that
in the
is
structure
not
completely
and
function
[3,7,8]. and
may
teum
to PGF2,.
PGF2,.
which between
ewes
administered of by
gree
of
are
luteum secretion
to the
luteolytic
of PGE2
from
uterus. in
the
of the
than lowest
nonpregnant
ewes
ewes
were
similar obtained
lutea
fmol/mg)
post-estrus.
nonpregnant
ewes
luteum
corpus
for
on
to PGF
or an increase
PGF,
nonpregnant
ewe
pulsatile
actually
of
luteal
involved
serum
be higher
than
terized by distinct pulses beginning on Day 12-13 post-estrus, reaching maximum frequency and amplitude by Day 15-16 [2,3, 6,7]. In contrast, secretion of PGF2,. from the gravid uterus is characterized by fewer pulses of lower am-
[16, 17], [18-20].
[9] and
in
‘Supported by NIH Grant HD11590 and a grant from the Colorado Agricultural Experiment Station. GJ.W. (predoctoral trainee) and M.C.W. (postdoctoral trainee) were supported by NIH Grant HD07031. 2Correspondence: Dr. Heywood R. Sawyer, Animal Reproduction and Biotechnology I2boratory, Department of Physiology, Colorado State University, Fort Collins, CO 80523. FAX: (303) 491-7569. 3Current address: Dept of Dairy Science, University of Wisconsin, Madison, WI
13 and to
are
nonpregnant
ewe.
16 [9-11].
PGF2a
selu-
Doses
of
in pregnant ineffective Since
is correlated
the
if de-
with
present, it has been suggested factors from the embryo and/or
of evidence indicate that PGE2 of the corpus luteum during
administration corpus luteum
as well
from
as luteolysis
resistance
during
hypothesized
early
the
that reuterus
of the
corpus
is greater of pregnancy
of PGE2 has spontaneous
induced
is involved early pregin the [13-
been shown regression
by exogenous
PGF2.,
secretion, both be responsible
in vivo for the
luteum
to PGF2,.
that
oc-
pregnancy.
to a hormone receptors that
is dependent
located decreased
pus luteum to PGF2,. during nancy was due to decreased or, alternatively, an increase 984
pregnant
PGE2 stimulates progesterone in vitro [21]. Thus, PGE2 may
Sensitivity
53706.
in the
of the changes in PGF2a sensitivity of the corpus
in the
Days
is re-
[10, 12].
of functional we
of PGF2,. concentrations
Secretion of PGE2 from the uterus ewe during maternal recognition
increased curs
occur
resistance
15]. Additionally, to protect the
is charac-
secretion
mean
sufficient to induce luteolysis Days 8 and 12 post-estrus are
number of embryos lease of additional
nancy. pregnant
of PGF2,.
non-
in pregnant
corpora
0.4
from
ewe,
between
2) resistance
Secretion
in
±
obtained
also
are
of PGF2,.,
the
and
higher
than
Although
PGF2,.,
corpus
and
in pregnant
0.2 vs. 2.0
in the pregnant
action
3) increased
0.05)
greater
pregnant
of receptors
Several lines in the maintenance
of the
100000
AA
mean
and
SEM
±
GTP--y-S)
PGF2,. 13H1-PGE2
(nM)
by various
AA.*
PG
9C
and
El AL.
(n
± ± ±
338.1
±
±
186.7 4.2 1413.6
±
1754.7
±
>100000
2 replicate
=
7.7 12.7 6.3
±
experiments).
from
their
membrane bated
stream of nitrogen and appropriate concentration. suspended
with
in 50 l
0.1 pmol
of[3H]-PG
of assay (25
buffer
p.l) with
in assay of plasma were
either
incu-
0.1 nmol
of PG (25 p.1; nonspecific binding) or 25 p.1 of assay buffer (final assay volume of 10 p.1). After incubation all tubes were immediately placed in an ice bath (5-30 mm) to inhibit dissociation
of the
of the unbound accomplished
hormone-receptor
hormone by filtration
complex.
from that through
bound glass
Separation
to receptor fiber filter
addition
of 100
that had been presoaked in a 0.6% solution of polyethylenimmne in deionized H2O [27]. After filtration, each filter was washed twice with 3 ml of buffer (10 mM Tris-HCI; I mM
CaC12;
1 mM
MgCl2;
individual sample vials containing
4
present
sample
in
each
areas ml
0.02%
were placed of scintillation was
LS5000CE liquid scintillation Specificity of the receptors ing the ability of PGF2,,, PGFm, and AA (0.1-10 PGE2
or
[3H]PGF2a
plus
increasing
percentage
000
measured
milligram
was incubated h at 22#{176}C) or of the
of labeled
6.0)
with
dried;
a Beckman by measur-
PGA2, PGFIU, PGJ2, binding of [3H]-
equivalents
of stan-
as described above with [3H]-PGE2 (16 h at 4#{176}C)
different
hormone
and
in 7-ml scintillation fluid. Radioactivity
PGD2, PGEI, nM) to inhibit
PGE2,
doses
pH
counter. was determined
Twenty
dard pool membrane 1 nM [3H]PGF2a (2
NaN3;
that
prostaglandins. bound
was
The subjected
to a logit (response)-log (dose) transformation and fit with a simple regression equation. The concentration that inhibited [3H]-PG binding by 50% (IC50) was calculated using the corresponding linear regression equations for all unlabeled PGs (Table 1). The rank order of inhibition by the unlabeled PG tested was similar for both [3H]-PG5, but considerably greater concentrations of competing PGs were required
to inhibit
binding
of [3H]-PGF2,.
than
for
[3H]-PGE2.
30
mm
separated
from
As shown
in Figure
of
0.05)
by GMP
GTP-’y-S
(0.01,0.1,
1, 10, 100,
1000
receptor
of GTP
evidence receptors.
complex
by
(GTP-y-S)
is indicative
nucleotide
binding
receptor
also
some
PGE2
PGE2 in displacing 2.4 times as effective
[3H]-PGF2,. from its receptor, but in displacing [3H]-PGE2 binding.
only
To further ascertain whether PGF2,. and PGE2 were ing to different receptors, an additional experiment performed. The ability of guanosine nucleotides (GMP,
bindwas GTP,
[28],
analysis [30]. Analysis was from competition (increasing hormone,
(increasing
concentrations
15-mg ure
equivalents 3, only
for PGE2
high
and
pool
can
per
milligram
was
3.46
cific
1cd and
fmol
for
PGE2
demonstrated
the
the corpora
included
tissue
lutea
15-mg the
equivalents
sample
and
assays
one
constants
(Kd)
12.4
± 0.6 and of receptors
pool
fmol
membrane
for
PGF2,..
for
PGE2
described
of the
receptor
for
PGF2,.
was
determined.
receptor
and
The and
above
(Fig. 4). To and standards
assayed
PGE2
and
present
for standard
PGF,.
that the parallel,
at 12.5in triplicate
receptors.
Parallelism
indicates
curve
that
of in-
membrane 111.0-888.0
and
curve
conin in-
consisted
confirm were
in duplicate
receptors
which the
A standard
assay
spePGF2,.
or satamount
assays, PGE2
5- or in Fig-
detected,
of receptor from the standard fmol receptor for PGE2 and
was for
22.2
experiments
validity
in each
sample
equivalents
were
standard
Scat-
with
As shown
of receptors
of receptors
dividual was
nM)
whether determined by competition assays and were independent of the
of tissue. After completing centration
and
concentration
did not change uration binding
saturation
0.1-6
dissociation
in the
PGE2
by
on data obof unlabeled
and PGF2,. were The concentration
equivalent
Although receptors
and
sites
The
that
it should be noted from their G pro-
performed amounts nM)
binding
for the receptors for PGE2 6.4 ± 0.3 nM, respectively.
conclude that
membrane.
for PGF2C.
analog to a guanine
determined
of [3H]-PG,
affinity
one
These
of GTP [29]. for PGF2,. and
were
0.1-1000 of pooled
at the
(1 mM).
evidence
chard tained
homologous
dis-
only
to a G protein.
plasma
and
[3H]-PGF2,. bind of a hormone-
one
to a G protein, not dissociate
(p
p.M),
contrast,
coupling
in the
fmol receptor for PGF2,. ing curves for samples
than
In 0.05)
