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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at the periphery.
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these cells (Fig. 1). The effect of cAMP on the
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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
REFERENCES
Barraclough, C. A., Collu, R., Massa, R. and Martini,
cell
changes in ovarian cyclic AMP and prostaglandins
while
to granubosa
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
1970; Nicosia and Mikhail, whereas cumulus cells are
L. (1971). Temporal interrelationships between plasma LH, ovarian secretion rats and peripheral plasma progestin concentration in the rat: Effect of Nembutal and exogenous gonado tropins. Endocrinology 88, 1437—1447. Bauminger, S., Koch, Y., Khan, I., Hillensjö, T., Nilsson, L. and Ahr@n, K. (1978). Preovulanory
tertiary
At ovulation the
vitro (Channing, 1975). However,
Cumulus
in that
both
cells
DEKEL AND PHILLIPS
296 in immature
rats injected
with PMSG. J. Reprod.
Fert. 52, 21—23. Bauminger, S. and Lindner, H. R. (1975). Periovu latory changes in ovarian prostaglandin
formation
and their hormonal control in the rat. Prosta glandins 91, 737—75 1. Channing, C. P. (1970). Influences
Hillensjö, T., Dekel, N. and Ahr@n, K. (1976).
of gonadotropins
Effect
on the cumulus oophorus of
isolated rat graafian follicle. Acta Physiol. Scand. 96, 558—568. Kuehl, F. A., Humes, J. L., Cirillo, V. J. and Ham, E. A. (1972). Cyclic AMP and prostaglandins in
of the in vivo and
hormone action. In: Advances in Cyclic Nucleo
the in vitro hormonal environment upon luteini
tide Research. (P. Greengard, P. Paoletti and G. A. Robinson, eds.). Raven Press, New York. p. 493. LeMaire, W. J., Yang, W.S.T., Behrman, H. H. and
zation of the granulosa cells in culture. Prog. Horm. Res. 26, 589—621.
Rec.
Channing, C. P. (1972). Stimulatory effects of prosta glandins upon luteinization of rhesus monkey granulosa cells cultures. Prostaglandins 2, 331—
349. Clark, M. R., Marsh, J. M. and LeMaire, W. J. (1978).
Mechanism of luteinizing hormone regulation of prostaglandin in rat granulosa Chem. 21, 7757—7761.
cells.
J.
Biol.
Cuatrecasas, P., Hollenberg, M. D., Chang, K. and Bennet, V. (1975). Hormone receptor complex and their modulation of membrane function. Rec. Prog. Horn). Res. 31, 37—94. Dekel, N., Hultborn, R. Hillensjö, T., Hamberger, L. and Kraicer, P. (1976). Effect of luteinizing
hormone
on respiration
of the preovulatory
cumulus oophorus of the rat. Endocrinology 98, 498—504. Dekel, N. and Kraicer, P. F. (1978). Induction in vitro
of mucification of rat cumulus oophorus by gonadotropins and c-AMP. Endocrinology 102, 1797—1802. Dekel, N., Kraicer, P. F., Phillips, D. M., Sanchez, R. and Segal, S. J. (1978). Cellular association in the rat oocyte-cumulus
cell complex:
Morphology
and ovulatory changes. Gamete Res. 1, 47—57. Dekel, N. and Phillips, D. M. (1979). Maturation of the rat cumulus oophorus: A scanning electron microscopic study. Biol. Reprod. 21, 9—18. Dekel, N., Hillensjö, T. and Kraicer, P. F. (1979). Maturational effects of gonadotropins on the cumulus-oocyte complex of the rat. Biol. Re prod. 20, 191—197. Gilula, N. B., Epstein, M. C. and Beers, W. H. (1978).
Cell-to-cell communication study of the cumulus-oocyte Biol. 78, 58—75.
and ovulation. complex.
A
J. Cell
Marsh, J. M. (1973). Preovulatory changes in the concentration of prostaglandins in rabbit graafian follicles. Prostaglandins 3, 367—376. LeMaire, W. J., Leidner, R. and Marsh, J. M. (1975). Pre- and post-ovulatory changes in the concen
tration of prostaglandins in the rat graafian folli des. Prostaglandins 9, 221—229. Lindner, H. R., Tsafriri, A., Lieberman, M. E., Zor, U., Koch, Y., Bauminger, S. and Barnea, A. (1974). Gonadotrophin action on cultured Graafian follicles: Induction of maturation divi sion of the mammalian oocyte and differen
tiation of the luteal cell. Rec. Prog. Horm. Res. 30, 79—138.
Nicosia, S. V. and Mikhail, G. (1975). Cumuli oophori in tissue culture: Hormone production, ultra structure and morphometry of early luteini zation. Fertil. Steril. 26, 427—448. Nilsson, L., Rosberg, S., Hillensjö, T. and Ahr@n, K. (1975). Preovulatory changes of ovarian cyclic AMP in the rat. Life Sci. 16, 5 17—524. Pharriss, B. B. and Shaw, J. E. (1974). Prostaglandins in reproduction. Am. Rev. Physiol. 36, 391—412.
Strickland, S. and Beers, W. H. (1976). Studies on the role of plasminogen activator in ovulation: In vitro tropins,
response of granulosa cells to gonado cyclic nucleotides and prostaglandins.
J. Biol. Chem. 251, 5694—5702. Zor, U., Lamprecht, S. A., Tsafriri, A., Pomerantz, S., Koch, Y. and Lindner, H. R. (1972). Stimu latory action of cholera enterotoxin on cAMP formation ovum maturation and ornithine decarboxylase activity in rat ovaries. Israel. J. Med. Sci. 8, 1774.