Cyclic AMP, ProstaglandinE2and Steroids: PossibleMediators in the ...

BIOLOGY OF REPRODUCTION

22, 289—296 (1980)

Cyclic AMP, ProstaglandinE2and Steroids: PossibleMediators in the Rat Cumulus Oophorus Mucification NAVA DEKEL1'2

and DAVID M. PHILLIPS3

The Rockefeller University,' New York,New York 10021 and The PopulationCouncil,3 The Rockefeller University, New York, New York 10021 ABSTRACT

The possible role of cyclic AMP (cAMP), prostaglandin E2 , progesterone and estradiol-17@ in in vitro induction of the rat cumulus oophorus mucification was studied with the scanning electron microscope. Follicular cumulus-oocyte complexes were isolated an early proestrus and cultured for 24 h. Cyclic AMP levels in the incubated complexes were elevated by addition of cAMP derivatives (dibutyryl cAMP or 8 bromo cAMP), the phosphodiesnerase inhibitor, 3-isobutyl-1-methylxanthine (MIX), or the adenylate cyclase stimulator, cholera enteronoxin. Under all these culture conditions the cumulus cells were stimulated to secrete a hyaluronidase-sensitive mucoid material which coated the cumulus-oocyne complexes. These complexes were similar in appearance to those incubated in the presence of gonadotropins. In the absence of the above agents extracellular material was not observed. In vitro cumulus mucificanion was non induced by prostaglandin E2; indomenhacin, an inhibitor of prosnaglandin synthenase, failed no block muciflcanion in complexes that have been incubated in the presence of LU. Cumulus complexes were not stimulated no mucify in vitro by progesterone or esnradiol-1 7(3. Therefore, the response of the cumulus cells to the gonadotropic stimulus appears no be mediated by cAMP. Prosnaglandin E2 and steroids seem not to be involved in this process. INTRODUCTION

Lindner,

Mucification in the rat cumulus oophorus in vivo has been shown to occur subsequent to the preovubatory

LH

surge

(Dekel

et

a!.,

1979).

By exposure of the intact follicles to LU in vitro, Uilbensjö et al. (1976) demonstrated that the maturationa! transformation of the cumlus

oophorus isolated

is cumuli

hormonally oophori

dependent. we have

Using

demonstrated

that these cells can be directly stimulated by gonadotropins (Dekeb et a!., 1976; Dekeb and Kraicer, 1978; Dekel and Phillips, 1979). Cyclic AMP and prostaglandins are bebived to be involved in the action of LU on ovarian

follicles in mammals

(Channing,

1970; Lindner

et al., 1974). Moreover, follicular levels of both of these substances, as well as of the ovarian sex steroids, increase about the time of ovulation (Barracbough et al., 1971 ; Bauminger and

Accepted October 17, 1979. Received June 2, 1979. 2Present address: Dept. of Biology, University, New York, NY 10003.

New York

1975; Nilsson et al., 1975; LeMaire et

a!., 1975; Bauminger et a!., 1978). ments described in this study were

The experi undertaken

to study the possible mediatory role of cAMP, prostaglandin E2 and steroids in the maturation of rat cumulus. Mucification of the cumulus has been

chosen

as an indicator

have found

the scanning

(SEM) cation Phillips,

for this process.

electron

We

microscope

to be a very sensitive tool for verifi of cumulus mucification (Dekel and 1979).

MATERIALS AND METHODS Young adult female Wisnar rats (Microbiological Co., NJ) were housed an 23°C with lights on from 0500 no 1900 h for at least 3 weeks before use. Only

rats with at least 2 consecutive 4 day cycles, as deter mined by vaginal smears, were employed in this study. Rats were killed by cervical dislocation an 1200 h on the day of proestrus. The ovaries were removed, rinsed and transferred to a depression slide containing Leibovitz's L-1 5 tissue culture medium (GIBCO, Grand Island, NY) (pH 7.2) supplemented with 15% fetal bovine serum (Reheis Chemical Co., Phoenix, AZ) and antibiotics (penicillin, 100 units/mI; snrepno mycin, 100 @&g/ml;GIBCO). The large preovulanory

follicles (usually 4—6/ovary)were punctured and their

289

DEKEL AND PHILLIPS

290

(Dekel and Phillips, 1979). The fixed complexes which

contents expelled into the medium. Following 3 transfers to fresh medium, the cumulus-oocyte com plexes ( 10—20) were incubated on a precleaned cover

did not remain attached

glass (22 X 22 mm, No. 2, Clay Adams, Parsippany,

10 mm in 0.1% aqueous solution of poly-L-lysine HBr (Sigma type 1-B) and washed in several changes of

NJ) in a 35 mm Petri dish (Falcon, Oxnard, CA) in 1.5 ml of medium, at 37°C, in air at relative humidity of 100%. Incubations ,were conducted in the presence or

water. Coverslips with attached complexes were dehydrated through a graded series of alcohols to acetone. Specimens were critical point dried with

absence of N6 ,O2 -dibutyryl 3': 5'-cyclic monophos

CO2 in a Sorvall critical-point drying apparatus, coated with gold in an Edwards 150 sputter coater and examined with an ETEC autoscan.

phate (dbcAMP), 8 bromoadenosine 3': 5'-cyclic monophosphate (8-Br-cAMP) (Sigma Chemical Co., St. Louis, MO), 3-isobutyl-1-methylxanthine (MIX) (Al drich Chemical Co. , Milwaukee, WI), cholera entero

toxin (Schwarz/Mann, Orangeburg, NY), prostaglandin E2 (gift

of Dr. John E. Pike, The Upjohn

RESULTS

Co., Kala

mazoo, MI), ovine LH (oLH), NIH LH 5-19, indo methacin

[1-(p-chlorobenzoyl)-5-methoxy-2

Follicular

methyl

San Diego, CA). At least 3 were conducted for each of the

above culture conditions. For each of the indicated culture conditions, complexes from 3 rats were isolated. After 24 h of incubation, the complexes were fixed overnight in 0.1 M collidine-buffered 2.5% glutaraldehyde

(pH

7.2).

Some

complexes

cellular

were

treated with hyaluronidase as previously described

@ @ @ @

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material

was

observed

on

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or between

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the culture and flattened

].,

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.@:,.

at the periphery.

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,@.,,

iso

these cells (Fig. 1). The effect of cAMP on the

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complexes

. -. .t@,;:':: , ... ..‘¿ .. .‘ r- .-, .@-.@ ; . @.-. . -.s,.',@ C@ .@ .: ‘¿-.@. •¿. .,! -.‘ ...,.

@

@

cumulus-oocyte

lated on the morning of proestrus formed a sphere of tightly packed cells (Dekel et at., 1978). During 24 h of incubation in control@ medium these cumuli lost their spherical structure; the cells attached to the dish, flat tened and formed a monolayer. No extra

indole-3-acetic acid] (Sigma), progesterone or estra diol-17@ (Calbiochem, individual experiments

to the dish were then placed

on a coverslip which had been presoaked for at least

.,

.,@ .

X 2000; insert same culture.

,@

X 150.

.

—¿

@

@, .@ —¿@

CUMULUS OOPHORUS MUCIFICATION cumulus

cells

was studied

the nucleotide

either

by addition

291

before ovulation, or incubated in vitro in the presence of LU or FSH (Dekel and Phillips,

of

derivatives to the incubation

medium or by elevation of their intracellular endogenous levels of cAMP. Incubated in either

1979). The extracellular material could not be

dbcAMP (0.5 or 5.0 mM) or 8-Br-cAMP (5.0

observed in complexes dase after culture.

mM) the complexes retained their spherical structure and did not attach to the dish. After 24 h in culture these cumuli were embedded in hyaline-bike matrix (Fig. 2). This extracelbular

riab was observed in cumuli incubated in prosta glandin E2 (10_6 M) (Fig. 5). Moreover, addi

material surface,

formed almost

a coating on the obscuring individual

No

tion

cumulus cumulus

effect

when the endogenous incubated cumulus cells

MIX (0.2 mM), stimulator, cholera

as mucoid-coated

cal

3,

structures

resembled

(Figs.

the

follicubar

4).

Their

both

spheri

terone

isolated

inhibitor,

of

the

gonadotropin

on

the

cumulus

culture

conditions

the

spherical

structure

(10_6

M) (Fig.

8) or estradiob-17f3

(10@

M) (Fig. 9). In the presence of these steroids

appearance

cumuli

synthetase

mate

was retained and the extracelbubar material was accumulated (Figs. 6, 7). Extraceblubar material accumulation was not observed in cumuli incubated in either proges

or the adenylate enterotoxin (100

ng/mb), also appeared

prostaglandin

extracebbular

mucification. The cumuli incubated with oLH in the presence or absence of indomethacin were indistinguishable in appearance. Under

were elevated. The cumuli incubated for 24 h in the presence of either the phosphodiesterase inhibitor, cyclase

of the

of the

with hya!uroni

indomethacin (5 @tg/mb), concomitantly with oLH (1 0 @zg/ml)failed to block the stimubatory

cells, although a few cells were exposed through occasional ruptures in the cumulus coating. In most cases, the surface of these cells were characterized by abundant blebs. This same response was observed levels of cAMP in the

accumulation

treated

just

the cumulus

cells attached

to the

dish

and

-.@

@

‘¿_.c.

. -‘.‘@.

@

@s-@-.

-@-@

S.

,2@T

.

@, - .

@

2

)@r

J@,

I

FIG. 2. Cumulus oophorus incubation with 5 mM dbcAMP for 24 h. Cells are covered with amorphous material. X 3000; insert same culture, X400.

DEKEL AND PHILLIPS

292

flattened forming a monolayer of the control cultures.

similar

to that

in stimulating adenybate cycbase activity (Cuat recasas et ab., 1975) has been reported to cause a significant

bated

DISCUSSION

In a previous SEM study of the mucification of the rat cumulus oophorus, we demonstrated that in vitro either LU or FSH can induce morphological transformations similar to those which occur' before ovulation in vivo (Dekel and Phillips, 1979). In the present study, we have shown that cAMP mimics the effect of the gonadotropins on the rat cumulus cells, while prostaglandin E2 , progesterone do not induce mucification.

suggest maturation

that

or estradiol-1 713 These findings

the hormonably-induced is mediated

by

the

cyclic

FIG. 3. Cumulus oophorus

incubated

choleragen

in

in cAMP

(Zor our

in rat

ovaries

et ab., 1972). present

study

incu

Using the as

another

effector of the cAMP have provided additional

intracellular pool, we evidence that cumulus

mucification levels of

correlated extracellular

is directly cAMP. The

with high material

accumulated in response to cAMP appears to have a nature similar to the mucus formed by LU stimulated cumuli, tivity to hyaluronidase 1979).

cumulus

The

nucleo

induction

of cumulus

with

view

tide, while neither prostaglandins nor steroids seem to be involved in this process. The stimulatory effect of cAMP on the cumulus cells found in this study is consistent with the observations made using the sensitivity of the cumulus to hyaluronidase as an indicator for its mucification (Dekeb and Kraicer, 1978). Cholera enterotoxin, a protein which is potent

x3000;insertsame culture, X400.

rise

in vitro

mediation the

as judged (Dekel

of cAMP that

in the gonadotropic

maturation this

by its sensi and Phillips,

nucleotide

is in accord plays

an

intermediate robe in the mechanism by which LU stimulates the mammalian ovary. However, the findings that prostaglandin E2 failed to stimulate the cumulus cells and that the inhi bition of prostaglandin synthesis by indo methacin did not block the LU-induced effect on this foblicular component, is somewhat

with 0.2 mM MIX for 24 h. Amorphous

material covers the cells.

@ @ @ @

-@ @:@,

.

I

-@

CUMULUS OOPHORUS MUCIFICATION @‘¿

293

1'@

@

., ‘¿V

I@

@

•¿

I:

,‘@

,@

@

. :. ‘¿ t,@ @5'..'Ip

@

¶@,

-@:‘:.

‘¿,@

@

@

F41@ “¿4@-@:

Naa- @—¿_____---_FIG. 4. Cumulus incubated

with 100 ng/ml cholera enterotoxin

for 24 h. Amorphous

material covers the

cumulus cells. X3000; insert same culture, X400.

:‘‘¿

(I

_@@;

.,‘ 4@_@

.

.,.@ @.

‘¿--.5 @‘¿.

*.

‘¿ ..@-

FIG. 5. Culture of cumulus cells after incubation in medium containing

ology is similar to control cultures. X3000; insert same culture, X125.

4. 10'

M prostaglandin E2 . The morph

DEKEL AND PHILLIPS

294

FIG. 6. Cumulus incubated in 10 pg/ml oLH with indomethacin (5 j@g/ml). X 2000. FIG. 7. Cumulus incubated in 10 @tg/mloLH without indomethacin. Spherical structures composed of blebby

cells and mucus, as in Fig. 6. X2000.

. @

d@t@

@

.@.(.

..5y, ,

@•¿@@“-..:‘-‘‘ . 4.. .. .@‘% ,@,: 1@@.

..

@.. ,,.. ‘¿ .@-. .-.‘..

,.

Sc. .-@.. %‘

I

@

..

;@i'i

.

-@

I

..‘,,

..

1@

-

‘¿5-

-4..,s

y.-i @-

-5

Th

.

@.

CUMULUS OOPHORUS MUCIFICATION

p

295

.

@

\5-

,@@__@___

a*@@4@.:T

4

FIG. 9. Cumulus cells exposed no 106 M estradiol-1 7P for 24 h. Cultures look similar to controls and estra

daiol appears no have no effect on mucificanion. X1500; insert same culture, X150.

unexpected since it has been shown that prostaglandins of the E type are able to repro duce many biological actions of LU on the ovary (Kuehl et a!., 1972; Lindner et ab., 1974; Pharriss and Shaw, 1974). In addition, it has been reported that fobbicubar levels of prosta glandins

increase

about

the

time

of ovulation

(LeMa,re et a!., 1973; Bauminger and Lindner, 1975; LeMaire et a!., 1975; Bauminger et a!., 1978). Moreover, a stimulatory effect of prosta glandin granubosa

E2 upon cells has

in vitro luteinization of been demonstrated (Chan

ning, 1972; Clark et al., 1978). The

fact

that

the granubosa

cells

do respond

to the prostagbandin while the cumulus cells are not affected by this substance is intriguing. Both cell populations originate from the membrana granubosa and are anatomically separated

only

development. accompany

at the oocyte

types secrete progesterone

to mucify

by gonadotropins

in vitro,

cells

not

Phillips,

1979). Moreover, bus complexes

activator land and Therefore,

are

(Dekel

unlike granubosa fail to secrete

in vitro in response

and

cells, cumu plasminogen

to FSH (Strick

Beers, 1976; Gibula et a!., 1978). our results suggest that the differen

tiation of cumulus and granubosa cells during late foblicular development results in the formation of exhibit different

two cell responses

same hormonal

environment.

populations when exposed

which to the

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while

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stimulated

granubosa

in response to LU in

stage

cells

of fobbicubar

the cumulus

cells

the granubosa

cells

stay in the ovary and luteinize. are similar

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