Receptors for Prostaglandins F2a and E2 in Ovine Corpora Lutea ...

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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)