BIOLOGY OF REPRODUCTION 68, 516–523 (2003)
Published online before print 17 October 2002.
DOI 10.1095/biolreprod.102.009662
Role of Arachidonic Acid and Protein Kinase C During Maturation-Inducing
Hormone-Dependent Meiotic Resumption and Ovulation in Ovarian Follicles
of Atlantic Croaker1
Reynaldo Patiño,2,3,4 Goro Yoshizaki,5 Digbo Bolamba,4 and Peter Thomas6
U.S. Geological Survey,3 Texas Cooperative Fish & Wildlife Research Unit,4 Texas Tech University,
Lubbock, Texas 79409-2120
Department of Aquatic Biosciences,5 Tokyo University of Fisheries, Minato-ku, Tokyo 108-8477, Japan
University of Texas Marine Science Institute,6 Port Aransas, Texas 78373-1267
ABSTRACT
The roles of arachidonic acid (AA) and protein kinase C (PKC)
during in vitro maturation-inducing hormone (MIH)-dependent
meiotic resumption (maturation) and ovulation were studied in
ovarian follicles of Atlantic croaker (Micropogonias undulatus).
The requirement for cyclooxygenase (COX) metabolites of AA
was examined using a nonspecific COX inhibitor, indomethacin
(IM), as well as two COX products, prostaglandin (PG) F2a and
PGE 2, whereas the role of lipoxygenase (LOX) was investigated
using a specific LOX inhibitor, nordihydroguaiaretic acid
(NDGA). The involvement of PKC was examined using phorbol
12-myristate 13-acetate (PMA), a PKC activator, as well as
GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin-
olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of
protein kinases. Genomic mechanisms were examined with the
transcription-inhibitor actinomycin D (ActD) and the function-
ality of heterologous (oocyte-granulosa) gap junctions (GJ) with
a dye transfer assay. The AA (100 mM) and PGF2a (5 mM) did
not induce maturation, and NDGA (10 mM) did not affect MIH-
dependent maturation. However, IM (100 mM) partially inhib-
ited MIH-dependent maturation. Conversely, AA and both PGs
induced, and IM and NDGA inhibited, MIH-dependent ovula-
tion in matured follicles. The PMA (1 mg/ml) did not induce
maturation but caused ovulation in matured follicles, whereas
PKC inhibitors (GF, 5 mM; H7, 50 mM) did not affect MIH-de-
pendent maturation but inhibited MIH- and PMA-dependent
ovulation. The PMA-dependent ovulation was inhibited by IM
but not by NDGA. In addition, ActD (5 mM) blocked MIH-de-
pendent, but not PMA-dependent, ovulation, and PGF2a restored
MIH-dependent ovulation in ActD-blocked follicles. The AA and
PGs did not induce, and GF did not inhibit, MIH-dependent
heterologous GJ uncoupling. In conclusion, AA and PKC medi-
ate MIH-dependent ovulation but not meiotic resumption or
heterologous GJ uncoupling in croaker follicles, but a permissive
role of COX products of AA during maturation is possible. A
novel model of MIH-dependent ovulation is proposed in which
1) LOX and COX metabolites of AA are both required for ovu-
lation, but at upstream and downstream sites of the pathway,
1
Funding for this work was provided by the U.S. Department of Agricul-
ture (NRICGP Animal Reproduction, 00-35203-9135) and Japan Society
for the Promotion of Science’s Research for the Future program
(97L00902). The U.S. Geological Survey, Texas Tech University, Texas
Parks and Wildlife Department, and the Wildlife Management Institute
jointly sponsor the Texas Cooperative Fish and Wildlife Research Unit.
2
Correspondence. FAX: 806 742 2946; e-mail: r.patino@ttu.edu
Received: 22 July 2002.
First decision: 14 August 2002.
Accepted: 28 August 2002.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
516
respectively, relative to PKC, and 2) PKC is downstream of ge-
nomic activation.
follicle, meiosis, ovary, ovulation
INTRODUCTION
The role of arachidonic acid (AA) during the meiotic
resumption (maturation) of oocytes is not fully understood.
Stimulation of maturation by AA or its metabolites has
been reported in some starfishes [1], European sea bass [2],
mouse [3–5], and sheep [6]. Conversely, inhibition of mei-
otic resumption by cyclooxygenase (COX) products (pros-
taglandins [PGs]) of AA metabolism was observed in
mouse [7] and Xenopus laevis [8]. Furthermore, in other
species of starfishes, AA and its metabolites appeared to
have no involvement in oocyte maturation [1]. Concerning
ovulation, it is generally believed that AA is required for
this process in most vertebrates, including teleost fishes, but
the relative importance of the COX and lipoxygenase
(LOX) metabolic pathways is the subject of disagreement
among species or studies [9–15].
The involvement of protein kinase C (PKC) in oocyte
maturation is also unclear. For example, negative [16, 17]
as well as positive [18–21] regulation of meiotic resump-
tion by PKC has been reported in several different species.
Nevertheless, general agreement is found in the literature
that ovulation in most vertebrates, including teleost fishes,
requires PKC-dependent pathways [10, 22–27].
The process of LH-dependent ovarian follicle maturation
can be described in two distinct stages for some teleost
fishes [28, 29]. The basic mechanisms of each stage have
been relatively well studied in Atlantic croaker and other
sciaenid species [28]. Namely, the competence of ovarian
follicles to produce steroidal maturation-inducing hormone
(MIH) and of the oocyte to resume meiosis in response to
MIH is acquired during the first stage [30]. These events
are accompanied by increases in heterologous (granulosa
cell-oocyte) gap junctions (GJ) [31] and MIH-receptor ac-
tivity [32–34]. Activation of protein kinase A (PKA) is suf-
ficient and necessary for LH induction of the first stage of
maturation [35]. Production of MIH marks the beginning
of the second stage [30]. The MIH binds to a high-affinity
receptor on the surface of the oocyte [32, 34], and this
hormone-receptor complex associates with a pertussis tox-
in-sensitive inhibitory G protein [36]. Oocyte cAMP and
PKA activity levels presumably decline and, eventually,
lead to meiotic resumption in sciaenid species, as has been
reported for other teleosts [37]. To our knowledge, the role
of AA in follicular maturation has not been investigated for
 MECHANISMS OF MATURATION AND OVULATION IN CROAKER OVARIAN FOLLICLES
Atlantic croaker. Furthermore, although pharmacological
activation of PKC does not induce meiotic maturation in
Atlantic croaker [35, 38], the role of MIH-regulated PKC
is also unknown for this species. It has been reported that
heterologous GJ decline during the periovulatory period in
croaker follicles [31, 38], but the mechanisms of this down-
regulation are uncertain.
We are unaware of the mechanisms of ovulation having
been examined in Atlantic croaker. However, in spotted sea-
trout [39] and in yellow perch [40], MIH-dependent ovu-
lation involves genomic mechanisms regulated by a nuclear
receptor [39]. In addition, as noted already, AA and its
metabolites as well as PKC are generally believed to be
part of the transduction pathway of hormonally induced
ovulation across vertebrate species. Although AA and its
metabolites also are effective uncouplers of GJ channels in
a variety of tissues [41–43], to our knowledge the role of
AA in the periovulatory uncoupling of heterologous GJ has
never been examined.
The present study examined the role of AA and PKC
during MIH-dependent meiotic resumption and ovulation
in Atlantic croaker. The results obtained suggest that AA
and PKC are primary transducers of MIH-dependent ovu-
lation but not of maturation. These results also suggest a
novel transduction pathway for MIH-induced ovulation.
MATERIALS AND METHODS
Fish and Tissue Collection
Atlantic croaker (Micropogonias undulatus) were collected in the fall
near Port Aransas, Texas, and were maintained in indoor circular tanks
under standard conditions as previously described [30]. During sampling
for tissue collection, fish were deeply anesthetized with quinaldine sulfate
and spinally transected. Ovaries were removed into Dulbecco modified
Eagle medium (DME) supplemented with streptomycin sulfate (100 mg/
L) and penicillin (60 mg/L) at pH 7.6. All procedures were reviewed and
approved by the animal care and use committees of Texas Tech University
and the University of Texas at Austin.
Chemicals
General chemicals, DME, antibiotics, hCG, AA, PGF2a, PGE 2, indo-
methacin (IM), nordihydroguaiaretic acid (NDGA), phorbol 12-myristate
13-acetate (PMA), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7),
bisindolylmaleimide GF109203X (GF), and actinomycin D were obtained
from Sigma Chemical (St. Louis, MO). 17a,20b,21-Trihydroxy-4-preg-
nen-3-one (MIH of Atlantic croaker) was purchased from Steraloids, Inc.
(Newport, RI), and Lucifer Yellow from Molecular Probes (Eugene, OR).
The carrier solution was ethanol for AA, PGs, and MIH and dimethyl
sulfoxide for IM, NDGA, PMA, H7, and GF. The final concentrations of
carrier solutions in incubation medium never exceeded 0.1% (v/v). All
other chemicals were dissolved in incubation medium.
Experimental Design
The roles of AA and PKC in MIH-dependent maturation and ovulation
were investigated with in vitro bioassays using a variety of compounds
that act at different sites along the AA metabolic pathway as well as drugs
that alter PKC activity. Namely, the involvement of COX products of AA
metabolism was examined using a nonspecific inhibitor of COX, IM, as
well as two COX products, PGF2a and PGE 2, whereas the role of the LOX
pathway of AA metabolism was investigated using a specific inhibitor of
LOX, NDGA. The role of PKC was studied using PMA, a PKC activator,
as well as GF, a specific inhibitor of PKC, and H7, a nonspecific inhibitor
of protein kinases. The requirement for genomic mechanisms was exam-
ined with actinomycin D, a transcription inhibitor.
Human chorionic gonadotropin was used as a homologue of LH. All
incubations were carried out at 258C under gentle agitation. Two series of
experiments were conducted. The first series focused primarily on meiotic
resumption, although ovulation was also recorded. For these experiments,
fragments of ovarian lamella containing approximately 50 full-grown ovar-
ian follicles were incubated in 1 ml of DME with hCG (5 IU/ml) for 12–
517
14 h to induce maturational competence (first stage of maturation). Folli-
cles were then incubated with MIH (290 nM) or the appropriate experi-
mental reagent for an additional 22–24 h (total incubation time, 36 h).
Although MIH-dependent germinal vesicle breakdown (GVBD) under
these conditions is first observed in some follicles within 4–5 h [38], a
22- to 24-h incubation with MIH was used, because pilot observations
indicated that most ovulation occurs within 36 h of total incubation time.
When inhibitors were used, they were added 45–60 min before the addi-
tion of MIH (or other stimuli) to allow adequate time for their action. At
the end of the incubations, ovulation was determined by counting the
number of oocytes that were expelled from their follicles. Meiotic re-
sumption was then determined by measuring the incidence of GVBD in
follicles cleared with Serra solution (ethanol:formalin:glacial acetic acid,
60:30:10 [v/v]). These observations were made using a binocular stereo-
scope.
The ability of croaker ovarian follicles to ovulate in response to MIH
is LH-dependent and follows the acquisition of maturational competence
(unpublished observations). Thus, the second series of experiments, fo-
cusing on ovulation, used ovarian follicles at a more advanced stage of
follicular maturation to ensure adequate ovulatory responses. For this pur-
pose, the length of the priming incubation with hCG was increased to 14–
18 h, until follicles showed signs of endogenous MIH-dependent ooplas-
mic clarification and hydration (stages D and E according to Yoshizaki et
al. [38]), which correspond to the second (and irreversible) stage of mat-
uration [28]. Also, for experiments concerning PMA-induced ovulation,
follicles were first incubated with exogenous MIH (290 nM) for 30 min
immediately after the hCG incubation and before adding the appropriate
inhibitors (45–60 min) or PMA. This strategy was used to ensure that the
process of maturation was well underway and to prevent the atypical mor-
phology of croaker follicles that results when PMA is added early during
the maturational process (unpublished data). A similar protocol was used
in a study of MIH-dependent ovulation with spotted seatrout [39]. Total
incubation times in this second series of experiments were also 36 h.
All treatments within an experiment were conducted with quadruplicate
incubates using ovarian fragments obtained from a single fish. Except
when noted, all experiments were repeated with tissues from three or more
fish.
Dye Transfer Assay for Heterologous Coupling
The methods of Yoshizaki et al. [38] were used for this procedure.
Briefly, following their appropriate treatment, ovarian follicles were iso-
lated using watchmaker forceps under a binocular stereoscope. The iso-
lated follicles were held in grooves made on an agarose plate, and ap-
proximately 10 nl of a Lucifer Yellow (457.2 daltons) solution (0.1% in
DME) were microinjected into the cytoplasm of the oocytes using a glass
micropipette (outer diameter, ;10 mm). The position of the micropipette
was controlled using a micromanipulator (IM-5B; Narishige, East Mead-
ow, NY). After an equilibration period of 15 min, the follicular wall con-
taining the thecal cell layer, basement membrane, and granulosa cell layer
was manually peeled off the oocyte surface using watchmaker forceps
under the binocular stereoscope and carefully rinsed. The isolated follic-
ular cell layer was observed under a fluorescence microscope (Nikon
E600; Nikon Instruments, Inc., Lewisville, TX) with B-filter set (excitation
filter, 465–495 nm; emission filter, 515–555 nm). The appropriate treat-
ment was continued during the entire procedure to eliminate the possibility
of reversal of effects on coupling. The presence or absence of fluorescence
in the follicle wall was interpreted as indicating the presence or absence
of functional heterologous coupling, respectively.
Data Analysis
Percentage GVBD (100 3 number of GVBD 4 number of full-grown
follicles) and percentage ovulation (100 3 number ovulated eggs 4 num-
ber of GVBD) were scored in all replicates. The results of all four repli-
cates per treatment per fish were averaged to obtain an individual fish
value, and sample size for statistical analyses was considered to be the
number of fish. The variability in percentage ovulation among individual
fish was sometimes high, but this variability was greatly reduced with data
transformed relative to stimulated values. The among-fish variability in
GVBD was also generally reduced when using relative values. Thus, ex-
cept when noted, relative values were used for one-way ANOVA followed
by, when appropriate, Duncan multiple-range tests. This data transforma-
tion has been previously used in studies of ovarian follicle maturation with
Atlantic croaker [35, 44]. All analyses were conducted using the Statistica
for Windows 1998 software package (StatSoft, Inc., Tulsa, OK).
518 PATIÑO ET AL.
FIG. 1. Effects of IM and NDGA on MIH-dependent GVBD and ovula-
tion in ovarian follicles of Atlantic croaker. Ovarian fragments containing
approximately 50 follicles were each incubated in 1 ml of medium with
hCG (5 IU) for 12–14 h (upper panel) or 14–18 h (lower panel). Following
a brief rinse period, follicles were incubated with or without IM (100 mM)
or NDGA (10 mM) for 45–60 min and then with or without MIH (290
nM) for the appropriate length of time for a total of 36 h of incubation
(exposure to IM or NDGA continued until the end of the incubation). The
incidence of ovulation and GVBD were determined. Results are expressed
relative to the values obtained in the presence of MIH. Bars indicate the
mean (6 SEM) of three (upper panel) or eight (lower panel) separate ex-
periments, and those associated with common letters (large, GVBD;
small, ovulation) are not significantly different (one-way ANOVA and
Duncan multiple-range test; P , 0.05). Follicles incubated for 14–18 h
showed relatively high levels of ‘‘spontaneous’’ GVBD and ovulation
when subsequently placed in untreated medium, indicating that consid-
erable production of endogenous MIH occurred as result of the longer
hCG incubation time. Average MIH-dependent GVBD and ovulation were
38% and 5% (upper panel), respectively, and 86% and 40% (lower
panel), respectively.
RESULTS
Preliminary experiments indicated that IM (1–100 mM),
NDGA (1–50 mM), GF (0.05–5 mM), and H7 (5–50 mM)
inhibited MIH-dependent ovulation in a dose-dependent
manner. However, NDGA at 50 mM occasionally caused
atypical follicular appearance, presumably resulting from
toxic effects. Thus, in subsequent experiments, we used IM
at 100 mM, NDGA at 10 mM, GF at 5 mM, and H7 at 50
mM. The effective dose-response ranges for GF and H7
observed in the present study are similar to those reported
in an earlier study of maturational competence in croaker
follicles [35].
FIG. 2. Effects of GF and H7 on MIH-dependent GVBD and ovulation
in ovarian follicles of Atlantic croaker. Ovarian fragments containing ap-
proximately 50 follicles were each incubated in 1 ml of medium with
hCG (5 IU) for 12–14 h. Following a brief rinse period, follicles were
incubated with or without GF (5 mM) or H7 (50 mM) for 45–60 min and
then with or without MIH (290 nM) for the appropriate length of time for
a total of 36 h (exposure to GF or H7 continued until the end of the
incubation). The incidence of ovulation and GVBD were determined. Re-
sults are expressed relative to the values obtained in the presence of MIH.
Bars indicate the mean (6 SEM) of three separate experiments, and those
associated with common letters (large, GVBD; small, ovulation) are not
significantly different (one-way ANOVA and Duncan multiple-range test;
P , 0.05). Average MIH-dependent GVBD and ovulation were 82% and
17%, respectively.
Ovarian Follicles During Early Maturation
When added before the irreversible commitment to mei-
otic resumption in most follicles, IM (100 mM), a nonse-
lective inhibitor of COX, partially inhibited MIH-dependent
GVBD, but NDGA (10 mM), a selective inhibitor of LOX,
was without effect (Fig. 1, upper panel). However, neither
AA (0, 1, 10, and 100 mM; one-way ANOVA, P . 0.05,
n 5 4) nor PGF2a (0, 0.5, and 5 mM; one-way ANOVA, P
. 0.05, n 5 2) was able to induce GVBD (in both instanc-
es, negative controls [untreated medium] yielded negligible
or no GVBD, whereas positive controls [MIH-induced]
showed near 50% GVBD). The selective inhibitor of PKC,
GF (5 mM), as well as the nonselective inhibitor of protein
kinases, H7 (50 mM), did not affect MIH-dependent GVBD
(Fig. 2). In contrast, inhibitors of AA metabolism (IM and
NDGA) and of PKC activity (GF and H7) completely
blocked the small incidence of MIH-dependent ovulation
observed in these follicles (Figs. 1, upper panel, and 2).
The PKC-stimulator PMA, but not AA, PGF2a, or PGE 2,
induced the uncoupling of heterologous GJ after a 6-h in-
cubation, which is the time period necessary for MIH- and
PMA-dependent uncoupling of heterologous GJ in croaker
follicles (Table 1) [38]. However, MIH-dependent meiotic
resumption and uncoupling of heterologous GJ were not
affected by coincubation with GF (PKC inhibitor). Namely,
all follicles that underwent GVBD after a 10-h incubation
with MIH had completely lost their heterologous GJ cou-
pling regardless of the presence or absence of GF (a longer
incubation time was used in this second experiment to en-
sure complete maturation of the ovarian follicles).
Ovarian Follicles During Late Maturation
High incidences of ‘‘spontaneous’’ maturation were ob-
served when follicles preincubated for 14–18 h in hCG
 MECHANISMS OF MATURATION AND OVULATION IN CROAKER OVARIAN FOLLICLES 519

TABLE 1. Effects of PMA, AA, PGF2a, and PGE2 on heterologous gap-

junction coupling in maturationally competent ovarian follicles of Atlantic
croaker. Each treatment was applied to 12 hCG-pretreated (12–14 h) ovar-
ian follicles per individual fish (experiment), and each experiment was
repeated with ovarian follicles from three fish.

Length of treatment (h)

Treatment 0 3 6
Untreated medium 95 6 3* 95 6 3 (A)** 97 6 3 (A)
PMA (1 mg/ml) — 36 6 14 (B) 28 6 5 (B)
AA (100 mM) — 95 6 3 (A) 97 6 3 (A)
PGF2a (5 mM) — 100 6 0 (A) 97 6 3 (A)
PGE2 (5 mM) — 89 6 3 (A) 100 6 0 (A)
* Percent follicles coupled (mean 6 SEM, n 5 3 fish).
** Values within individual columns associated with common letters are
not significantly different (one-way ANOVA and Duncan multiple range
test; P , 0.05).

FIG. 4. Effects of GF, IM, and NDGA on AA-dependent ovulation in

ovarian follicles of Atlantic croaker. Ovarian fragments containing ap-
proximately 50 follicles were each incubated in 1 ml of medium with
hCG (5 IU) for 14–18 h. Following a brief rinse period, follicles were
incubated with or without GF (5 mM), IM (100 mM), or NDGA (10 mM)
for 45–60 min and then with or without AA (100 mM) for the appropriate
length of time for a total of 36 h (exposure to inhibitors continued until
the end of the incubation). The incidence of ovulation was determined.
Results are expressed relative to the values obtained in the presence of
AA (average ovulation, 31%). Bars indicate the mean (6 SEM) of three
separate experiments, and those associated with common letters are not
significantly different (one-way ANOVA and Duncan multiple-range test;
P , 0.05). This experiment was conducted in conjunction with that for
AA shown in Figure 3.

were subsequently placed in untreated medium. This spon-

FIG. 3. Effects of AA (upper panel) and PGF2a (lower panel) on ovulation
in ovarian follicles of Atlantic croaker. Ovarian fragments containing ap-
proximately 50 follicles were each incubated in 1 ml of medium with
hCG (5 IU) for 14–18 h. Following a brief rinse period, follicles were
incubated with or without AA (1–100 mM) or PGF2a (0.5–5 mM) for the
appropriate length of time for a total of 36 h. The incidence of ovulation
was determined. Results are expressed relative to the values obtained in
the presence of AA at 100 mM (average ovulation, 31%) or PGF2a at 5
mM (average ovulation, 34%). Bars indicate the mean (6 SEM) of three
(upper panel) or two (lower panel) separate experiments, and those as-
sociated with common letters are not significantly different (one-way AN-
OVA and Duncan multiple-range test; P , 0.05).
taneous maturation is caused by the onset of endogenous
MIH production after prolonged hCG incubations [30, 45].
As noted already, the longer preincubation times were to
ensure adequate development of gonadotropin-dependent
ovulatory competence. In these follicles, IM (COX inhibi-
tor) inhibited the small increase in (exogenous) MIH-de-
pendent GVBD, but NDGA (LOX inhibitor) was without
effect (Fig. 1, bottom panel). Both IM and NDGA effec-
tively blocked MIH-dependent ovulation (Fig. 1, bottom
panel).
Ovulation was induced in a dose-dependent manner with
AA and PGF2a (Fig. 3). In addition, PGE 2 (5 mM) induced
ovulation, but its potency seemed to be lower than that of
PGF2a (data not shown). The AA-induced ovulation was
inhibited by IM and the PKC inhibitor, GF, but not by
NDGA (Fig. 4). Ovulation was also induced by activation
of PKC with PMA (1 mg/ml), and like AA-induced ovu-
lation, PMA-induced ovulation was suppressed by IM and
GF but not by NDGA (Fig. 5).
The MIH-induced ovulation was inhibited by the tran-
scription inhibitor, actinomycin D (5 mM), and restored by
cotreatment with PGF2a (Fig. 6). The AA-induced ovula-
tion, but not the PMA- or PGF2a-induced ovulation, was
also inhibited by actinomycin D (Fig. 7). Finally, PMA-
and AA-induced ovulation were completely inhibited by
GF, but PGF2a-induced ovulation was only partially inhib-
ited by GF (Fig. 7).
DISCUSSION
Maturation
At concentrations that induce ovulation (see later dis-
cussion), AA was insufficient to induce meiotic resumption
520 PATIÑO ET AL.
FIG. 5. Effects of GF, IM, and NDGA on PMA-dependent ovulation in
ovarian follicles of Atlantic croaker. Ovarian fragments containing ap-
proximately 50 follicles were each incubated in 1 ml of medium with
hCG (5 IU) for 14–18 h. Following a brief rinse period and a 30-min
incubation with MIH (290 nM), follicles were incubated with or without
GF (5 mM), IM (100 mM), or NDGA (10 mM) for 45–60 min and then
with or without PMA (1 mg/ml) for the appropriate length of time for a
total of 36 h (exposure to inhibitors continued until the end of the incu-
bation). The incidence of ovulation was determined. Results are expressed
relative to the values obtained in the presence of PMA (average ovulation,
65%). Bars indicate the mean (6 SEM) of three separate experiments, and
those associated with common letters are not significantly different (one-
way ANOVA and Duncan multiple-range test; P , 0.05).
FIG. 6. Blockage of MIH-dependent ovulation by actinomycin D (ActD)
and rescue by PGF2a in ovarian follicles of Atlantic croaker. Ovarian frag-
ments containing approximately 50 follicles were each incubated in 1 ml
of medium with hCG (5 IU) for 14–18 h. Following a brief rinse period,
follicles were incubated with or without ActD (5 mM) for 45–60 min and
then with or without MIH (290 nM) or PGF2a (5 mM) for the appropriate
length of time for a total of 36 h (exposure to ActD continued until the
end of the incubation). The incidence of ovulation was determined. Re-
sults are expressed relative to the values obtained in the presence of MIH
(average ovulation, 46%). Bars indicate the mean (6 SEM) of three sep-
arate experiments, and those associated with common letters are not sig-
nificantly different (one-way ANOVA and Duncan multiple-range test; P
, 0.05).
FIG. 7. Effects of actinomycin D (ActD) and GF on PMA-, AA-, and
PGF2a-dependent ovulation in ovarian follicles of Atlantic croaker. Ovar-
ian fragments containing approximately 50 follicles were each incubated
in 1 ml of medium with hCG (5 IU) for 14–18 h. Following a brief rinse
period and a 30-min incubation with MIH (290 nM), follicles were in-
cubated with or without ActD (5 mM) and GF (5 mM) for 45–60 min and
then with or without PMA (1 mg/ml), AA (100 mM), or PGF2a (5 mM) for
the appropriate length of time for a total of 36 h (exposure to inhibitors
continued until the end of the incubation). The incidence of ovulation
was determined. Results are expressed relative to the values obtained in
the presence of PMA (average ovulation, 86%). Bars indicate the mean
(6 SEM) of four separate experiments, and those within experimental
groups (indicated by a line over the bars) associated with common letters
in reference to the negative control (medium) are not significantly differ-
ent (one-way ANOVA and Duncan multiple-range test on all treatments;
P , 0.05).
in maturationally competent ovarian follicles of Atlantic
croaker. Ovulation-inducing concentrations of PGF2a (COX
product) were also insufficient to induce maturation. Fur-
thermore, ovulation-inhibiting concentrations of NDGA
(LOX inhibitor) did not affect MIH-dependent maturation.
These results differ from those obtained with another teleost
fish, the European sea bass, in which exogenous AA as well
as the COX products, PGF2a and PGE 2, were sufficient to
induce meiotic resumption in maturationally competent
ovarian follicles [2]. The mechanisms of AA-induced oo-
cyte maturation were not examined in European sea bass,
however, and whether the action of AA was indirect, via
regulation of follicular MIH production, or direct, as trans-
ducer of MIH action, is unclear [2]. The possibility of in-
direct effects via induction of MIH production is suggested
by the results of studies with goldfish showing that exog-
enous AA can enhance the steroidogenic activity of ovarian
follicles [46]. Nevertheless, in the present study, IM seemed
to partially inhibit MIH-induced meiotic resumption. Al-
though the latter observation allows the possibility that an
intact COX pathway is necessary to facilitate MIH-induced
maturation in Atlantic croaker, caution must be used when
interpreting results obtained with IM, because effects of this
reagent on cell functions unrelated to AA metabolism have
been described [47]. Overall, the results of the present study
strongly suggest that AA and its metabolites are insufficient
to directly or indirectly induce meiotic resumption in ovar-
ian follicles of Atlantic croaker, but the possibility of a
permissive role of COX metabolic pathways in MIH-de-
pendent meiotic resumption cannot be ruled out.
Studies with vertebrates other than teleosts also have
yielded inconsistent observations regarding the involve-
ment of AA and its metabolites in oocyte maturation. For
example, the LOX pathway of AA metabolism was linked
 MECHANISMS OF MATURATION AND OVULATION IN CROAKER OVARIAN FOLLICLES 521

FIG. 8. Major steps of the proposed transduction pathway for MIH-induced ovulation in ovarian follicles of Atlantic croaker. The MIH binds to a
nuclear receptor (nMIHR), possibly in granulosa or thecal cells, and activates genomic mechanisms. Enzymes associated with the LOX pathway of AA
metabolism are consequently synthesized or activated, and an undefined product or products (X) of LOX directly or indirectly induce the activity of
preexisting PKC. Increased PKC activity in turn induces COX and increases the synthesis of prostaglandins (PGF2a) from AA. The PGF2a causes rupture
of the follicle and consequent expulsion of the oocyte (ovulation). Lines ending with solid circles point toward steps along the pathway that are blocked
by the inhibitors used in the present study (see text). The PMA directly activates PKC and bypasses the actinomycin D (ActD)- and NDGA-sensitive
steps of ovulation.

to hormonally induced oocyte maturation in some, but not
all, species of starfishes [1]. Also, PGE1 and PGE 2 inhibited
progesterone-dependent meiotic resumption in Xenopus oo-
cytes [8], whereas positive [3–6], negative [7], or no [15]
involvement of AA or its metabolites was suggested in oo-
cytes of a number of mammalian species. Although it is
possible that conditions associated with specific experimen-
tal systems can lead to artifactual results, the information
available also suggests that differences exist in the roles
and mechanisms of AA during oocyte maturation among
species.
In croaker follicles, activators of PKC (PMA) do not
seem to induce meiotic resumption ([35, 38]; present
study), and inhibitors of PKC (GF and H7) do not affect
MIH-induced meiotic resumption at concentrations that
suppress ovulation (see later discussion). Also, treatment of
croaker follicles with PMA before MIH stimulation appears
to be toxic to the oocytes (data not shown). These obser-
vations indicate that MIH-dependent meiotic resumption in
croaker oocytes does not involve PKC and that premature
PKC activation may be inhibitory or toxic to maturation.
A maturation-suppressing effect of PKC was also suggested
for mouse oocytes [17] and Xenopus oocytes [16]. Con-
versely, pharmacological activation of PKC was reported
to promote oocyte maturation in species such as the rat
[18], the amphibian Rana dybowskii [19], and the teleost
killifish [20, 21]. However, like in Atlantic croaker, PKC
activation was found to be unnecessary for MIH-dependent
oocyte maturation in killifish [21]. Thus, PKC does not
seem to have a physiological role in the process of MIH-
dependent maturation in those teleost species examined to
date. Preliminary information (unpublished data) suggests
that specific inhibitors of PKA (H89) also do not affect
MIH-induced meiotic resumption in croaker follicles at
concentrations that inhibit hCG-dependent maturational
competence (first stage of maturation) [35]. Furthermore,
PKA inhibitors are sufficient to induce maturation in killi-
fish follicles [21]. These observations are consistent with
the concept that a decline in PKA activity is involved in
MIH-dependent meiotic resumption of teleost oocytes [37].
Correlations between meiotic resumption and uncou-
pling of granulosa cell-oocyte GJ have been observed in
all vertebrate species examined, including Atlantic croaker
[31, 38]. Given the recently proposed involvement of AA
in oocyte maturation of some teleosts [32] and the inhibi-
tory effect of AA on GJ coupling in a number of tissues
[41–43], the present study examined the hypothesis that AA
is part of the signaling pathway for MIH-dependent uncou-
pling of heterologous GJ. However, the present results
showed that neither AA nor its COX metabolites, PGE 2
and PGF2a, are able to uncouple heterologous GJ in mat-
urationally competent croaker follicles within the time
frame necessary for MIH-dependent uncoupling and mat-
uration [38]. Furthermore, although pharmacological acti-
vation of PKC (by PMA) caused heterologous GJ to un-
couple in croaker follicles ([38]; present study), specific
inhibitors of PKC (GF) had no effect on MIH-dependent
GJ uncoupling. Thus, AA and PKC do not seem to partic-
ipate in the MIH-dependent periovulatory uncoupling of
heterologous GJ.
Ovulation
In contrast to maturation, MIH-dependent ovulation in
croaker follicles seems to require functional PKC and intact
COX and LOX pathways of AA metabolism. Namely, in-
hibitors of PKC (GF and H7), COX (IM), and LOX
(NDGA) all blocked MIH-dependent ovulation, whereas
PMA, AA, PGF2a, and PGE 2 were all sufficient to induce
ovulation. Furthermore, actinomycin D blocked MIH-de-
pendent ovulation, and coincubation with PGF2a restored
ovulation. (Transcription is not required for MIH-dependent
meiotic resumption in croaker follicles [30].) These various
findings are generally consistent with those obtained in oth-
er vertebrates. For example, a requirement for de novo tran-
scription during MIH-dependent ovulation has been previ-
ously reported in spotted seatrout [39] and yellow perch
[40]. This transcription requirement suggests that MIH-de-
pendent ovulation in teleost fishes occurs via interaction
with a nuclear (classical) steroid receptor recently charac-
terized in teleost ovaries [39]. Similar conclusions were
made regarding progesterone-dependent ovulation in rats
[48]. Furthermore, PKC has been previously linked to ovu-
lation in other teleosts [10, 22–24], amphibians [27], and
mammals [10, 25, 26]. Finally, as observed in the present
study with Atlantic croaker, AA seems to be involved in
the ovulation process of most other vertebrates that have
been studied to date [9–15].
The PMA-dependent ovulation was completely blocked
by a specific PKC inhibitor (GF), thus confirming that PMA
acts via activation of PKC. Furthermore, PMA-dependent
ovulation was inhibited by IM but not by actinomycin D
or NDGA. These latter observations suggest that the re-
quirements for transcription and LOX during MIH-depen-
dent ovulation are both upstream of PKC activation, where-
as the requirement for COX is downstream of PKC (Fig.
8). This proposed sequence of events implies that AA-de-
pendent ovulation should not be markedly affected by the
522 PATIÑO ET AL.
LOX inhibitor, NDGA (because AA-derived PGs produced
by COX would bypass the upstream requirement for LOX),
or by actinomycin D. The present results are consistent with
the first condition, but the inhibitory effect of actinomycin
D on AA-dependent ovulation is seemingly inconsistent
with the proposed model. However, because PKC inhibitor
(GF) completely blocked AA-induced ovulation, it can be
postulated that MIH-induced PKC activity not only serves
to increase the activity of existing COX enzymes but also
to slow down their turnover rate or that of their mRNAs.
In such a scenario, the combination of a transcription block-
age (as when in the presence of actinomycin D) and con-
ditions of basal PKC activity would lead to a gradual de-
cline in COX protein and activity and, thus, to a reduced
follicular capacity to metabolize AA into the ovulation-ac-
tive products, PGs. The overall outcome would be a re-
duced ability of AA to induce ovulation, thus explaining
the observation of the present study. Alternatively, tran-
scription-mediated induction of COX by AA could also ex-
plain the inhibition of AA-dependent ovulation by actino-
mycin D, but this scenario would be difficult to reconcile
with the findings that the IM-sensitive site of ovulation
(COX) is downstream of PKC and that PKC-dependent
ovulation is insensitive to actinomycin D.
The proposed transduction pathway (Fig. 8) also indi-
cates that PG-dependent ovulation should not be affected
by transcription inhibitors. This condition was met by the
present results. Interestingly, PGF2a-induced ovulation was
partially diminished in the presence of a concentration of
GF that completely blocked PMA-induced ovulation. Thus,
it appears that PKC activity facilitates, but is not strictly
required for, PG-induced ovulation. Although endogenous
PGs were not measured in the present study, ovarian PGEs
and PGFs have been observed in all vertebrate species for
which measurements have been taken [10]. In teleost fishes,
PGF2a has consistently induced ovulation, whereas the ef-
fects of PGEs have been variable [10]. In Atlantic croaker,
exogenous PGF2a and PGE 2 both induced ovulation, but
PGF2a seemed to be slightly more potent.
The transduction pathway for MIH-induced ovulation
proposed here for Atlantic croaker (Fig. 8) is remarkably
consistent with the following observations in the rat: 1) LH-
induced PG production and ovulation seem to be mediated
by progesterone production and interaction with a classical
progesterone receptor [48], 2) LOX metabolites (leukotri-
enes) are produced earlier than COX products (PGs) during
hCG-induced ovulation [49], 3) treatment of perfused ova-
ries with LOX inhibitor (NDGA) inhibits LH-dependent PG
production and ovulation [12], and 4) certain LOX metab-
olites can induce PG production by ovarian granulosa cells
[50]. These observations suggest that LH (progesterone)-
dependent ovulation in rat follicles is mediated by LOX
product-dependent synthesis of PGs. Furthermore, although
its relative position in the transduction pathway is un-
known, PKC is also thought to be required for LH-depen-
dent ovulation in rat follicles [26, 51]. In yellow perch, the
COX and LOX pathways of AA metabolism have been
implicated in ovulation as well [52], but unlike in Atlantic
croaker, PMA-induced ovulation is inhibited only by
NDGA and not by IM [22]. Thus, the requirements for
LOX and COX during perch ovulation seem to be down-
stream and upstream of PKC, respectively, which is the
reverse situation relative to the proposed croaker ovulation
pathway (Fig. 8).
Conclusion
The present observations suggest that AA and PKC are
key elements of the transduction pathway for MIH-depen-
dent ovulation but not meiotic resumption or heterologous
GJ uncoupling in ovarian follicles of Atlantic croaker.
However, these results are inconclusive regarding the pos-
sibility of a permissive role of COX products during MIH-
dependent maturation. A novel model of MIH-dependent
ovulation is proposed for Atlantic croaker in which LOX
and COX metabolites of AA are both required for ovulation
but at upstream and downstream sites of the pathway, re-
spectively, relative to PKC and in which PKC is down-
stream of genomic activation. This heuristic model is ame-
nable to experimental testing and, if verified, would allow
further studies focusing on specific steps of the pathway
and their respective mechanisms.
ACKNOWLEDGMENTS
Dr. Frederick W. Goetz and Mr. Naresh Pandey provided critical com-
ments on a draft version of this manuscript.
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