BIOLOGY

OF

46, 630-635

REPRODUCTION

(1992)

Multifactorial

Regulation

in Preovulatory RONALD

Department

G.

of Zoology,

of Prostaglandin

Goldfish

KELLNER

and

University

Ovarian GLEN

Guelph,

of

Synthesis Follicles1

VAN

DER

KRAAK2

Ontario,

Guelph,

Canada

NIG

2W1

ABSTRACT Goldfish

preovulatory

of activators phorbol

1 2-myristate

(10

50 g/ml)

and

ovarian

of different

13-acetate ionophore

of PKC.

produced

Although

A2, melittin

putative

inhibitor

elevate

skolin

.iM),

A2.

In

summary,

of cAMP

levels

dibutyryl

and

production of P611

A,,

production,

in part,

production

by goldfish

studies

blocked

the

phosphodiesterase attenuate that

of phospholipase

the

adenylate

A2. The

diacylglycerol the

(1P3/DAG)

regulation

mals.

FSH,

and

signalling

of ovarian

LH and

are

trisphosphate/

pathways

participate

known

cyclase, as well as drugs that els (cholera toxin, dibutyryl

Although

the

actions

pathways

production activators

the

ling ovarian PG tion of exogenous PGF production gonadotropins

mediate

the

species inhibit

follicles report

November

Received

July 8, 1991.

‘This

Research

work

cyclase/cAMP

poorly stimulate supports

guppy [20]. is evidence

Council

‘Correspondence.

of Canada FAX:

Goldfish,

the Natural

in the

regulation

ovarian

PG

common

14-16#{176}Cunder fed

stimulate

is inhibitory

PG

to PG

of PGE

follicles

and

PGF

the

goldfish.

from

production

AND

METHODS

variety,

were

Martinsville, tanks with photoperiod

trout

diet

once

purchased

IN. Fish flow-through (14L:

a day

from

were held water at

1OD).

Fish

were

to satiation.

Incubations

The dures

increase

deelby

protocol

for

previously

ovarian

follicle

incubations

described

steroidogenesis

by spinal Cortland’s

transection saline

for [10,22].

and [22].

studies Preovulatory

ovaries were Each experiment

followed on the

proce-

regulation

of

fish were

killed

placed in modified used full-grown

ovarian follicles (0.9-1.1 mm in diameter) obtained from a single fish; the oocytes had not undergone final maturation as the germinal vesicle occupied a central position. Fully grown follicles were separated from smaller vitellogenic

On the basis of a that the 1P3/DAG

and

a constant

a commercial

Follicle

defined control-

drugs that production

Sciences

or comet

and steroido-

follicles under a dissecting in groups of 20 to each plates ada).

for Engineering

(Falcon 3047; Fisher Immediately before

767-1656.

630

microscope. Follicles were added well of polystyrene tissue culture Scientific addition

replaced with were incubated

4 h at 18#{176}C; in preliminary

cubated for various ume was 1 ml. The

to G.V.D.I( (519)

pathways

medium was tinely, follicles from

for-

of phospholipase levels

pathway

Grassyforks Fisheries Co., in 1.2-m diameter circular

understood. Injecovarian PGE and the concept that

periovulatory

by a grant

calcium

signalling

mM),

Animals

15, 1991.

was supported

cyclase/cAMP

a to

Meittin-stianulated

activation

of intracellular

PM), known

(0.1-1.0

to the

distal

was

of phos-

(100 drugs

PGE production.

MATERIALS

pathway exerts a stimulatory effect on PGE and PGF production by brook trout ovarian follicles. The present study investigates the putative role of different signal transduc-

Accepted

activator

Several

had

in

cAMP levstimulate

of ovarian

[15-19]. However, PGE2 and PGF2

from the [21], there

of cAMP

elevation

in mam-

[13] have been well goldfish, the factors

production are gonadotropins [14, 15], which

scribed in several evate cAMP levels postpartum preliminary

adenylate

regulation

genesis [10-12] and ovulation in teleost species such as the

direct

chloroquine

production.

PKC,

activators

with

of adenylate

elevate intracellular cAMP [dbcAMP])

of the

on

The

whereas

PGE

by preovulatory

ovarian prostaglandin (PG) production [1-5]. Calcium ionophore A23 187 and either phorbol esters or synthetic diacylglycerols, which mimic the actions of 1P3 and DAG, respectively, also stimulate the production of ovarian PGs and prostacyclins in several mammalian species [5-9]. 1P3/DAG

not activate manner

3-isobutyl-1-methylxanthine

and

tion inositol

eicosanoid

which

A23 187.

and

+ A23 187-stimulated

adenylate

does

in a synergistic

production,

activators

1,2-dioctanoylglycerol

and

which

the actions

C (P1(C)

fofficles.

ovarian

cyclase/cAMP

by PMA PGE

action

of PKC

activation

acted

investigating kinase

g/ml),

didecanoate,

inhibitor PMA

protein

25

and

and

in

for studies

The

+ A23 187-stimulated

INTRODUCTION The

4s-phorbol

increase

PMA

suggesting that

(5

stimulated

was

utilized

production.

P611 production

PGF

and

5 mM)

dbcAMP,

demonstrate

activation

through

PGE,

basal

(dbcAMP;

inhibited by

was

these

phorbol

a dose-related

including

cAMP

inactive

than

stimulated

were

(P6)

1-oleoyl-2-acetylglycerol

saM) stimulated

amounts

saM),

phospholipase

intracellular

(10

the

187 (0.25-4.0

breakdown)

on prostaglandin

nM),

100-400

in lower

vesicle

to germinal

pathways

production;

PGE A23

(0.1-1.0 of

(prior

(PMA;

stimulated

no effect. Calcium

pholipase

follicles

transduction

signal

times medium

up

Co., Toronto, ON, of test compounds,

Canthe

fresh Cortland’s saline. with test compounds studies,

follicles

Rouin air

were

to 21 h. The incubation was then removed and

in-

volstored

OVARIAN at -30#{176}Cprior licate incubations Human thine

to analysis. There per treatment.

CG,

dbcAMP,

(IBMX),

were

forskolin,

phorbol-12,13-myristate

and

calcium

other

saline. follicle

The amounts incubations

bation basal

volume and or stimulated

A23187

in dimethylsulfoxide dissolved directly

of ethanol or did not exceed

DMSO 0.5%

at this concentration production of PGE

o V

1600

(PMA),

were

O 0

+

A23187

(4

U)

lU/mi)

1200

500

pur-

w 0 400 0

included in the of the final incunot not

nU)

hCG (10

4ct-

(DMSO). in Cortland’s

did (data

631

CONTROL #{149} PMA (400

1 -methylxan-

acetate

IN GOLDFISH

2000

rep-

Co. (St. Louis, MO). Forskolin whereas phorbols OAG, DOG,

were dissolved compounds were

A23187

four

PRODUCTION

1 -octanoyl-2-acetylgly(DOG), melittin,

ionophore

chased from Sigma Chemical was first dissolved in ethanol, and The

generally

3-isobutyl-

phorbol-1 2,13-didecanoate (PDD), cerol (OAG), 1,2-dioctanoylglycerol chloroquine,

PROSTAGIANDIN

influence shown).

I

I

I

I

I

4

5

12

16

20

5

12

16

20

750

E 500 0)

RIM

0..

PGE

and

PGF

levels

termined by highly vided by Drs. T.G.

in the

specific Kennedy

incubation

media

were

RIAs using antisera of the University

de-

kindly proof Western

250 0 0

Ontario (London, ON, Canada) and F.W. Goetz of the University of Notre Dame (South Bend, IN), respectively. Characteristics of these antisera were described previously [23, 15]. Both nedy

PGs were [23]. The

at 2 000 traction.

rpm and Samples

cessed

in

were says. were

using media

subsequently from the

duplicate.

the protocol given in Kenwere centrifuged for 2 mm

assayed directly same experiment

Intraassay

less than 6.6% Serial dilutions parallel to the

Statistical

and

without were

interassay

expro-

differences and Duncan’s

considered

at least

were determined using analysis of Multiple Range test. Ap value of