BIOLOGY

OF

REPRODUCTION

21,

The Effect

187-191

(1979)

of Prostaglandin

and Progesterone

F2a on Ovarian

Concentrations

W. B. WEHRENBERG,2

J.

D.

Wisconsin

Regional

in Cyclic

DIERSCHKE3

Primate

1223 Madison,

Blood

Flow

Guinea

Pigs1

and

Research

R. C. WOLF

Center,

Capitol Court, Wisconsin 53706 ABSTRACT

Prostaglandin in ovarian blood

F2u (PGF20) may provoke luteolysis flow. To test this hypothesis, 15 m

in guinea and 25Mm

pigs indirectly radiolabeled

by causing changes microspheres were

injected into guinea pigs before and after treatment with saline or a dose of PGF,a which resulted in a significant decrease in plasma progesterone concentrations. The percent cardiac output to the ovaries and the percent arteriole-venule shunting within the ovaries estimated by the number of microspheres in this tissue were unchanged 6 h after treatment, yet plasma progesterone concentratiOns were significantly reduced in the PGF,a treated animals. These results, although confirming the luteolytic action of PGF,0 in guinea pigs, suggest that its mode of action does not require the intervention of hemodynamic adjustments. INTRODUCTION Poyser which is

(1976) indicates

has that

luteolytic

in

demonstrates of

guinea

stimulates

tissue

progesterone

suppresses

it

vation

and

vasoconstrictor (1970)

initiates

luteolysis

to

blood

We

et

fact

al.,

in

tissue

of

necessary

inducing

incu-

shunting

tissue,

undertaken

potent

significantly

terone

concentrations

percent

led

ovary

PGF2a

of

to

in

the

of ovarian

1978).

This

guinea

percent blood

observation

flow

March

Received

November

29,

prior

causes

an

a decline

in

(Wehren-

led

us

to

Lauderdale, was defined

tration

‘Research supported by Grants RR-00167, HD and I T32 HD07007-01 from NIH and by Foundation Grant 630-0505B. Publication No. 18-041 of the Wisconsin Regional Primate Research Center. ‘Trainee of the Endocrinology-Reproductive Physiology Program. Present address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, 630 West 168 08737 Ford

Capitol

Court,

Madison,

in the

within

plasma

the

progesterone.

AND

METHODS

Adult female guinea pigs (Cavia porcellus) of heterogeneous stock raised at the Wisconsin Regional Primate Research Center and weighing 700-900 g were utilized in this study. The estrous cycle was monitored daily by vaginal smears with Day 0 corresponding to a vaginal cytology of squamous and cornified epithelial cells.

there

arteriole-

1979.

Donald Research

proges-

increase

shunting

a

to

6, 1978.

Street, New York, NY 10032. ‘Reprint requests: Dr. Wisconsin Regional Primate

dose plasma

A preliminary experiment identify a luteolytic dose of PGF,0 Accepted

a

As

study was of PGF2a

reducing

pig, of

regression.

decreases

MATERIALS that,

if

this

ultimately

the present

arteriole-venule

prior

and

determine

which

blood

depriving

(CL)

than

PGF2

of

thereby

luteum

of

shunting

nutrients

corpus

to

action

the

test of this hypothesis,

obser-

a

luteolytic by

vitro

1968)

by

reported

luteolysis

al.,

luteal

that

indirectly

isa significant increase in the et

al.,

hypothesize

recently

spontaneous

berg

is

mediated

flow.

have

venule

from

This

PGF2a et

vivo

(PGF2a) PGF,a

away

rather

1968).

that

(Ducharme

Pharriss

ovarian

PGF2a

synthesis

(Pharriss the

be

vivo, in

in

with

the

may

Although

action

that

evidence

F2a

pigs. this

luteal

the

prostaglandin

clearly

bation

hypothesize

reviewed

Upjohn Co., by a significant

was conducted (provided by

Dr.

to J.

Ml). Luteolysis in plasma concenof progesterone. Four animals were assigned to each of the 4 treatment groups which a) saline, b) 1 mg PGF2n, c) 3 mg PGF,0, or

randomly received: d) 5 mg PGF,0. Blood puncture into heparinized 10 of the cycle while

Kalamazoo, decline

samples

anesthesia (methoxyflurane, North Chicago, sample, animals dose of PGF20

were

syringes the animals

taken

by

cardiac

at 0900 h on Day were under light

Pentrane, Abbott

Lab.,

IL). Immediately following the blood were injected i.p. with their assigned or saline. Subsequent blood samples were drawn at 12, 24, 48, 72 and 96 h postinjection. Blood samples were centrifuged within 15 mm of collection. Plasma was collected and stored at -20#{176}C

J. Dierschke, Center, 1223

WI 53706.

187

WEHRENBERG

188

until assayed for progesterone. Plasma progesterone concentrations were determined by radioimmunoassay after purification of extracts on Sephadex LH-20 columns (Clark et al., 1978). A second experiment was conducted to evaluate the arteriole-venule shunting within the ovary before and after treatment with a luteolytic dose of PGF,0. Seven animals were assigned to each of 2 treatment groups saline and 3 mg PGF,0. Between 1500-1700 h on Day 9 of the cycle, the animals were anesthetized with methoxyflurane. The tip of a polyethylene catheter (PE 50, Clay Adams, New York, NY) was introduced into the left common carotid artery, advanced to the left ventricle (as detected by blood pressure recording) and secured in place. The opposite end of the catheter was then passed s.c. to the nape of the animal’s neck and exteriorized. The next day at 0900 h, a 0.1 ml suspension of radiolabeled microspheres (3M Co., St. Paul, MN, see below) was injected into the animal via the catheter and was followed with a 0.5 ml flush of saline. A 1.5 ml blood sample was then drawn from the catheter into a heparinized syringe and this in turn was followed by an i.p. injection of 3 mg PGF,0 or saline. Based on the preliminary experiment, we knew plasma progesterone would be decreased within 12 h following treatment with

3

mg

PGF20.

As

changes

in

ovarian

hemody-

namics were hypothesized to occur prior to the progesterone decline, we decided to evaluate ovarian hemodynamics for changes at 6 h after treatment. Therefore, at 1500 h on the same day, a second suspension of radiolabeled microspheres was injected followed by a saline flush. Immediately after this a second blood sample was drawn. The animals were then sacrificed and the ovaries removed. The number of microspheres trapped was quantitated by assaying the tissue in a 3-channel gamma counter. Data reduction was accomplished as described previously (Buss et al., 1975; Rankin and Schneider, 1975). The first microsphere suspension consisted of “.120,00O 15 m microspheres (labeled with ‘“I, and 73,000 25 Mm microspheres (labeled with ‘7Co). The second microsphere suspension consisted of “.‘165.000 15 m microspheres (labeled with Cr) and 184,000 25 m microspheres (labeled with 40Sc). The different numbers of microspheres were injected to establish equal counting rates for each isotope in the tissue, thereby minimizing the effect of channel overlap (Chaichareon et al., 1976). In all instances the minimum number of microspheres for I isotope trapped in the ovaries was 120. The percent cardiac output (% CO.) to the ovaries was calculated for the different microspheres as follows: % C.O. = (number of spheres in ovary #{247} number of spheres injected) X 100. The injected quantity was determined by assaying the radioactivity of the microsphere suspensions before and after administration. The % C.O. calculated by using 25 m microspheres was defined as “total” blood flow to the ovaries and the % C.O. calculated by using 15 m microspheres was defined as “functional” blood flow (Wehrenberg et al., 1978). The difference between functional and total blood flow was considered to represent that blood flow which traversed the ovary via arteriole-venule shunts. The percent shunting was calculated as follows: % shunting 11 - (functional blood flow #{247}total blood flow)l X 100. Although this experiment was designed to eluci-

ET

date luteal

AL.

changes function,

of

ovarian we know

blood flow that excision

with regard of CL from

to the

ovaries results in loss of microspheres from the tissue (Wehrenberg et al., 1978) and therefore CL and stroma were left intact. llowever, it is clear from other data (Novy, 1972; Niswender et al., 1976) that changes in blood flow to the entire ovary primarily reflect changes within the luteal tissue. Progesterone data were subjected to a two-way analysis of variance for repeated observations in the same animal (Winer, 1962). This method separates the inherent between-animal variation from treatment effects. Significant differences between means were detected by Duncan’s new multiple range test (Bruning and Kintz, 1968). The % CO. and % arteriolevenule shunting were subjected to arcsine transformation and then handled identically to the progesterone data.

RESULTS Treatment

of

experiment Day

guinea

with

10

of

the

that

a single

PGF2a

was

effective

0.01)

(45%

PGF2a

clearly

injection in

3 or

concentrations within

12

throughout

the

h.

received

PGF2a

the

3 mg

of

progesterone

resulted

gradual

were

h

96

of

Plasma

progesterone

used

ovarian

6

of

treated results

to

and

blood treatment

PGF2a

groups

on

showed the

concen-

whereas of 28%

than

3%

there

was

in animals

confirming

the

experiment. flow

treated

was

PGF2a

less

thus

6 h posttreatment flow

in guinea of

decline PGF2a,

blood

and

over

experiment)

preliminary

ovarian

saline

such

increased

saline

of by

concentrations

changed with

the

decline followed

posttreatment

3 rug

from

Total the

h

(P0.05)

between

demonstrated the

percent

within

the

the

of

ovaries

decline

total

ovarian

adequate

to

PGF2

TABLE PGF,0

luteolysis

2. Percent

arteriole-venule

on

Day

10 of the

luteolysis,

flow

was

CL by

istered reducing

in ovaries

of

flow

3

dose

clusion

mg

and

further in

bNo

=

yet

were

the

in

admin-

significantly

concentrations. support ovarian

a single

our

blood

injection

(i.p.)

con-

flow

of

do

3 mg

postinjection 6

0

an

flow

animals

cycle.a

Hours

Percent shuntingb 95% Confidence Upper limit Lower limit

plasma

blood

the

PGF2a,

progesterone

following

shunting though

effective

plasma

pigs

with

functional

of

changes

results

declined.

after

was

The

demonstrate

even

observations that

guinea

clearly

unchanged

with

These

If

mechanism

shunting

estrous

treated

even presum-

blood

change

arteriole-venule

treatment

also

proges-

function. a

experiment in

this

concomitant

concentrations

Total were

prior

in plasma

of

blood

occurred

or

in

following

progesterone.

change

progesterone

expected

shunting

to,

circulating

PGF2

luteolysis, be

Furthermore, prior

present

such

shunting

pigs

maintain

induces

the

occurring would

arteriole-venule

in

after

al.,

increase

arteriole-venule

is indicative

which

though

a significant

of guinea

spontaneous

terone,

ably

that

et

naturally increase

occur

no (Wehrenberg

to

administration.

would

expected.

DISCUSSION

to

differences

which

declines

in

were

means.

guinea the

PGF20

group.

treatment means. CExpreSsed as percent treatment

6

Saline

Saline

PGF20

Saline

PGF,n

8.5

9.7

3.2

4.5

11.4 6.0

14.5 5.8

7.6 0.6

11.1 0.7

interval

7/treatment differences

group. (P>0.05)

were

noted

between

treatment

means.

WEHRENBERG

190

not

occur

prior

to

concentrations Four on

possible

these

natural its

action of

possibility.

al.,

1974)

that

our

results

rats

and

in

(Nett

et

dict

this

guinea

al.,

1976;

O’Shea

explanation

for However,

(Behrman

et

which

in

in

which

between

possibility

exists, be

difficult

redistribution tissue,

may

The

be

blood

ovary In

rabbits

of

of

CL

perfusion

been

effective

dose ‘\‘3

consistent dosage

with

their

is also

ovary

stromal possibility

CL in

weight

guinea

and pigs

has

Chaichareon that

PGF2

PGF2a

the

this

vivo

reported of

mg

significant

and

on

in

1977) admin-

pigs.

documented. (1976)

a

and

of this

1973) in

in

the

reducing

(single

our in

and minimum CL

injection,

report, effective

as

for the

Folly,

within

guinea

PGF2a

of

well

and

flow

secretion

total

determined

Therefore, in

effect

progesterone

Ginther

regression.

which

decreased

evaluating

The

luteal

in

resulted

increased.

is worth

for

Cook,

blood

such stromal

an evaluation

and

perfusion

ovary, to

(Varga

PGF2

redistribution

was

permit

and

flow

were

bitches (Novy

istration with

not

do

possibility. and

flow

this

Fourth,

the

blood

experiments

functional whole

luteal

the

occurs

tested

substantiate.

responsible

present

it While

been

flow within

of

of mecha-

as

not

to

in blood

changes

10

same

12-14.

has

Third,

Day the

luteolysis

it

itself

flow.

on

Days

reflect

system

by

regulate

naturally

may

PGF2a

mediated

by

could

vascular blood

by be

in

estimated

effluent,

ovarian

not

1971),

was

and

induced

nisms

flow

not

experiments

McCracken,

data

reducing

observed

feedback

al.

(1976)

in

proges-

an

ineffective

The

recovery

following

PGF2a

system

before

et decline

prostaglandin.

of

progesconcen-

of

may

a

involve

a

progesterone

on

hormone.

REFERENCES

function. is

former

1971;

to

may

CL

fimbria

addition

cycle

in regulating

venous

the

luteolysis

contra-

discrepancies

luteinizing

plasma

concentrations dose

pigs

ewes

ovarian

these

al.,

in

and

that

blood of

changes

results

1970)

of

on

guinea

to

following

dose

in

Leader transient

progesterone

negative

in

also

PGF2a

increase

those

concentrations

noneffective

However,

1977)

the

al.,

ovarian

collection

et

HilIer,

suggest

and

is involved

and

than humans,

similar

cycle

an

treat-

have

1 mg

decline by

In

luteolytic of

initial

PGF2a

(1976)

estrous

higher

a

terone

(Behrman

other

al.,

trations

reported

is available

an

progesterone

1 rug with

the

followed

and

involved

case.

and et

in

after

surprised

in

Busch

treatment 9 of

in

terone

flow

rats

the

pigs

possibility

flow

is

(Pharriss

apparent.

this

this

resulted

treatment.

be

(Bruce

8 and

this

Evidence

rabbits

suggests

rabbits

not

Days

h

and

that

were

increase

96 Mellin

against

blood may

1971),

and

as

luteolysin

ovarian

function.

1971),

the

which

argues

ment.

we

a significant

reported

therefore mimic

However,

observe

concentrations

the

However,

natural

(McCracken,

placed be

evidence

and

shunting CL

ewes

blood

the

Second,

regulating

from

and to

the as

arteriole-venule

pigs

luteolysin.

is substantial

be not

expected

natural

PGF2a pigs

An

be

progesterone. to

may

in guinea not

the

supports

progesterone

can

PGF2a

discussed,

guinea

in

PGF2a.

by

First,

would

previously

et

decline

interpretations

data. luteolysin

action

in

a

induced

AL.

ET

weight i.m.); indicate plasma

Behrman, H., Yoshinaga, K. and Greep, R. (1971). Extraluteal effects of prostaglandins. Ann. N.Y. Acad. Sci. 180, 426-43 5. Bruce, N. W. and Hillier, K. (1974). The effect of prostaglandin F2 on ovarian blood flow and corpora lutea regression in the rabbit. Nature 249, 176-177. Bruning, J. L. and Kintz, B. L. (1968). Computational Handbook of Statistics. Scott, Foresman and Co., Atlanta, GA. Buss, D. D., Bisgard, G. E., Rawlings, C. A. and Rankin, J HG. (1975). Uteroplacental blood flow during alkalosis in the sheep. Am. J. Physiol. 228, 1497-1500. Chaichareon, D. P. and Ginther, 0. J. (1976). Effects of uterus and prostaglandin F2a on corpora lutea in mongolian gerbils and guinea pigs. Am. J. Vet. Res. 37, 573-578. Chaichareon, D. P., Rankin, J. H. and Ginther, 0. J. (1976). Factors which affect the relative contributions of ovarian and uterine arteries to the blood supply of reproductive organs in guinea pigs. Biol. Reprod. 15, 281-290. Clark, J. R., Dierschke, D. J. and Wolf, R. C. (1978). Hormonal regulation of ovarian folliculogenesis in rhesus monkeys: I. Concentrations of serum luteinizing hormone and progesterone during

laparoscopy ment during

and

patterns of follicular develop-

successive Reprod. 18, 779-783. Ducharme, D. W., Weeks, J. G. (1968). Studies on

hypertensive

effect of

menstrual

cycles.

Biol.

R. and Montgomery, the mechanism

of

prostaglandin

F2u.

R. the

J.

Pharmacol. Exp. Therap. 160, 1-10. Leader, A., Bygdeman, M., Eneroth, P., Martin, J. N. and Wiqvist, N. (1976). The effect of infusions with two analogues of prostaglandin F20 on corpus luteum function. In: Advances in Prostaglandin and Thromboxane Research. Vol. 2. (Samuelsson, B. and Paoletti, R., eds.). Raven Press, New York, NY. pp. 679-686. McCracken, J. (1971). Prostaglandin F20 and corpus

PROSTAGLANDIN

luteum

regression.

Ann.

F20

N.Y.

AND

Acad.

OVARIAN

Sci.

180,

456572. Mellin, T. M. and Busch, R. D. (1976). Corpus luteum function in the guinea pig: Effect of PGF20 and inhibitors of prostaglandin and progesterone biosynthesis. Theriogenology 6, 533-551. Nett, T. M., McClellan, M. C. and Niswender, G. D. (1976). Effects of prostaglandins on the ovine corpus luteum, Blood flow, secretion of progesterone and morphology. Biol. Reprod. 15, 66-78. Niswender, G. D., Reimers, T. J., Diekman, M. A. and Nett, T. M. (1976). Blood flow: A mediator of ovarian function. Biol. Reprod. 14, 64-81. Novy, M. J. (1972). Distribution of ovarian blood flow in rabbits as measured by radioactive microspheres. Biol. Reprod. 7, 105-106. Novy, M. J. and Cook, M. J. (1973). Redistribution of blood flow by prostaglandin F,0 in the rabbit ovary. Am. J. Obstet. Gyn. 117, 381-385. O’Shea, J. D., Nightingale, M. G. and Chamley, W. A. (1977). Changes in small blood vessels during cyclical luteal regression in sheep. Biol. Reprod. 17, 162-177. Pharriss, B. B. (1970). The possible vascular regulation of luteal function. Perspect. Biol. Med. 13, 434-444.

BLOOD

FLOW

IN CYCLIC

GUINEA

PIGS

191

(1968).

L. J. and Gutknecht, Biological interactions between

glandins

and

Pharriss,

B. B., Wyngarden,

luteotropins

in the

rat.

In:

G. D.

prostaGonado-

tropins. Altos,

(Rosemberg. E., ed). Geron-X, Inc., Los CA. pp. 121-129. Pharriss, B. B., Cornette, J. C. and Gutknecht, G. D. (1970). Vascular control of luteal steroidogenesis. J. Reprod. Fertil., Suppl. 10, 97-103. N. L. (1976). Prostaglandin F20 is the uterine luteolytic hormone in the guinea pig: The evidence reviewed. In: Advances in Prostaglandin and Thromboxane Research. Vol. 2. (Samuelsson, B. and Paoletti, R., eds.). Raven Press. New York, NY pp. 633-643. Rankin, J.H.G. and Schneider, J. M. (1975). Effect of surgical stress on the distribution of placental blood flows. Resp. Physiol. 24, 373-383. Varga, B. and Folly G. (1977). Effects of prostaglandins on ovarian blood flow in the bitch. J. Reprod. Fertil. 51, 315-319. Wehrenberg, W. B., Dierschke, D. J., Rankin, JUG. and Wolf, R. C. (1978). Variations in “functional” blood flow as related to corpus luteum activity in cyclic guinea pigs. Biol. Reprod. 19, 380-384. Winer, B. J. (1962). Statistical Principles in ExperiPoyser,

mental

York,

Design.

NY.

McGraw-hill

Book

Co.,

New