General and Comparative Endocrinology 159 (2008) 46–57

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General and Comparative Endocrinology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y g c e n

An investigation into the role of prostaglandins in zebrafish oocyte maturation

and ovulation
A.L. Lister, G. Van Der Kraak *
Depart­ment of Inte­gra­tive Biol­ogy, Uni­ver­sity of Guelph, Guelph, Ont., Can­ada N1G 2W1

a r t i c l e i n f o a b s t r a c t

Article history: This study explored the potential for ovar­ian-derived pros­ta­glan­dins (PGs) to be involved in the reg­u­
Received 24 March 2008 la­tion of oocyte mat­u­ra­tion and ovu­la­tion in zebrafish. It was dem­on­strated that cul­tured vitel­lo­genic
Revised 25 July 2008 fol­li­cles have the capac­ity to pro­duce pros­ta­glan­din E2 (PGE2) and PGF2a in response to ara­chi­donic acid
Accepted 28 July 2008
(AA) in a con­cen­tra­tion-depen­dent man­ner, and that AA stim­u­lates the in vitro pro­duc­tion of 17b-estra­
Available online 3 August 2008

diol (E2). The pro­duc­tion of AA-stim­ul­ ated PGF2a was sig­nif­i­cantly reduced by treat­ment with the non-
selec­tive cyclo­ox­y­gen­ase (COX) inhib­i­tor, indo­meth­a­cin (INDO). Treat­ment of full-grown fol­li­cles with
AA did not induce oocyte mat­u­ra­tion as assessed by ger­mi­nal ves­i­cle break­down, but INDO sig­nif­i­cantly
Key­words:
Zebrafish
decreased the rate of spon­ta­ne­ous mat­ur­ a­tion. Using Real-Time PCR, it was shown that fol­li­cles of dif­fer­
Repro­duc­tion ent devel­op­men­tal size clas­ses (primary growth and pre-vitel­lo­genic, early-vitel­lo­genic, and mid- to full-
Pros­ta­glan­dins grown vitel­lo­genic) express enzymes that release (cyto­solic phos­pho­li­pase A2 (cPLA2); phos­pho­li­pase
Cyclo­ox­y­gen­ase Cc1) or metab­o­lize (COX-1, COX-2, and pros­ta­glan­din syn­thase-2) AA to PG metab­o­lites. The expres­sion
Ovu­la­tion of cPLA2 was found to be sig­nif­i­cantly greater in full-grown fol­li­cles com­pared to fol­li­cles of the pre-
and early-vitel­lo­genic stages. In vivo stud­ies dem­on­strated that breed­ing groups of zebrafish exposed to
100 lg/L INDO exhib­ited reduced spawn­ing rates and clutch sizes com­pared with con­trol and 1 lg/L INDO
exposed fish. In other stud­ies, it was shown that nat­ur­ ally spawn­ing groups of females exhibit increased
ovar­ian lev­els of PGF2a, E2, and 17a,20b-dihy­droxy-4-preg­nen-3-one (a mat­u­ra­tion-induc­ing hor­mone
in zebrafish) near the time of ovu­la­tion com­pared with non-breed­ing females. Col­lec­tively, these exper­
i­ments indi­cate that the AA path­way in zebrafish ova­ries is involved in the reg­ul­ a­tion of oocyte mat­u­ra­
tion and ovu­la­tion and a non-selec­tive inhib­i­tor of COX dis­rupts these pro­cesses.
© 2008 Else­vier Inc. All rights reserved.

1. Intro­duc­tion ‘oocyte mat­u­ra­tional com­pe­tence’. The oocyte resumes mei­o­sis as

The mat­u­ra­tion and ovu­la­tion of tel­e­ost oocytes are com­plex,
gon­a­do­tro­pin (lutein­iz­ing hor­mone (LH))-ini­ti­ated events that are
known to involve numer­ous reg­u­la­tory fac­tors, includ­ing ovar­ian-
derived growth fac­tors, ste­roids, and metab­o­lites of ara­chi­donic
acid (AA) (Pa­ti­no et al., 2001; Pa­ti­no and Sul­li­van, 2002; Ya­ron
and Sivan, 2006). Pa­ti­no et al. (2003) sug­gest that mat­u­ra­tion and
ovu­la­tion of oocytes are two events best con­cep­tu­ally viewed as a
con­tin­uum. For oocyte mat­u­ra­tion to occur, a surge in LH secre­tion
must stim­u­late fol­lic­u­lar pro­duc­tion of a pro­ges­tin that acts as the
mat­ur­ a­tion-induc­ing hor­mone (MIH). In zebrafish (Danio re­rio), the
ste­roid 17a,20b-dihy­droxy-4-preg­nen-3-one (17a,20b-P) has been
iden­ti­fied as an effec­tive MIH through its abil­ity to induce oocyte
mat­ur­ a­tion in in vitro stud­ies (Sel­man et al., 1994). Also, oocytes
must acquire the abil­ity to respond to MIH, which has been termed
* Cor­re­spond­ing author. Fax: +1 519 767 1656.
E-mail address: gvan­derk@uo­guelph.ca (G. Van Der Kraak).
a con­se­quence of the MIH that is pro­duced by the fol­lic­u­lar cell
lay­ers (Pa­ti­no and Thomas, 1990; Pa­ti­no et al., 2001) and the onset
of mat­u­ra­tion is marked by the migra­tion of the ger­mi­nal ves­i­cle
(GV) from its cen­tral loca­tion toward the ani­mal pole of the oocyte,
fol­lowed by its break­down (GVBD) (Sel­man et al., 1994). Ovu­la­
tion is the last step of oogen­es­ is and involves the expul­sion of the
mature ovum. Pre­vi­ous stud­ies have con­cluded that gon­a­do­tro­
pin-induced ovu­la­tion is also depen­dent on the pro­duc­tion of MIH
­(Pin­ter and Thomas, 1999; Pa­ti­no and Sul­li­van, 2002).
The role of AA and its cyclo­ox­y­gen­ase (COX) metab­o­lites, pros­
ta­glan­dins (PGs) in oocyte mat­u­ra­tion or ovu­la­tion is not well
under­stood in fish and only lim­ited stud­ies have been con­ducted.
In regards to the mat­u­ra­tional pro­cess, these stud­ies have yielded
incon­sis­tent results, which sug­gest that spe­cies dif­fer­ences may
exist (Pa­ti­no et al., 2003). For exam­ple, AA, PGF2a and PGE2 induce
GVBD in Euro­pean sea bass (Dicen­trar­chus lab­rax) (Sor­ber­a et al.,
2001), but were inef­fec­tive at induc­ing mat­u­ra­tion of Atlan­tic
croaker (Micr­o­po­go­nias un­dul­a­tus) fol­li­cles (Pa­ti­no et al., 2003).
The reg­u­la­tion of ovu­la­tion in tel­eo
­ sts is known to be more spe­cific

0016-6480/$ - see front matter © 2008 Else­vier Inc. All rights reserved.
doi:10.1016/j.ygcen.2008.07.017
 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57 47
than mat­u­ra­tion; gen­er­ally, it is lim­ited to stim­uli that increase the
activ­ity of pro­tein kinase C and AA metab­o­lism (reviewed in Goe­tz
et al., 1991; Pa­ti­no and Sul­li­van, 2002). It has been dem­on­strated
through in vitro stud­ies of yel­low perch (Perca flaves­cens) (Brad­ley
and Goe­tz, 1994) and Atlan­tic croaker (Pa­ti­no et al., 2003), that
MIH-stim­u­lated ovu­la­tion is inhib­ited by the non-selec­tive COX
inhib­i­tor, indo­meth­a­cin (INDO), and exog­e­nous treat­ment with
AA or PGF2a often induces ovu­la­tion. Fur­ther sup­port of PG involve­
ment in ovu­la­tion is sug­gested by the obser­va­tion that brook trout
(Salv­eli­nus fon­ti­nal­is) ova­ries pro­duce PGs at the time of ovu­la­tion
(Goe­tz and Cet­ta, 1983; Goe­tz and Gar­czyn­ski, 1997) and the abil­
ity of PGF2a to induce ovu­la­tion in gold­fish (Sta­cey, 1976).
Recent stud­ies have con­firmed this gon­a­do­tro­pin-depen­dent
model of oocyte mat­u­ra­tion in zebrafish (Pang and Ge, 2002), and
sev­eral stud­ies have dem­on­strated that locally derived growth fac­
tors influ­ence gon­a­do­tro­pin-stim­u­lated or spon­ta­ne­ous mat­u­ra­
tion of zebrafish oocytes in vitro (Pang and Ge, 1999, 2002; Wang
and Ge, 2004a; Clel­land et al., 2006). To our knowl­edge, there are
no pub­lished stud­ies that explore the role of AA and its metab­o­
lites in the reg­ul­ a­tion of ovar­ian fol­li­cle mat­u­ra­tion or ovu­la­tion in
the zebrafish. It is unknown whether a fully func­tional AA cas­cade
exists within zebrafish ova­ries, but recent stud­ies have described
the expres­sion of sev­eral puta­tive AA path­way genes in var­i­ous tis­
sues of the zebrafish, includ­ing mem­bers of the phos­pho­li­pase A2
(PLA2, Far­ber et al., 1999) and phos­pho­li­pase C (PLC, Law­son et al.,
2003) fam­i­lies, which directly or indi­rectly act to release ara­chid­o­
nate, respec­tively. PGs are formed through the sequen­tial actions
of COX-1 or COX-2 and spe­cific PG syn­thases; genes encod­ing
for some of these enzymes have been char­ac­ter­ized in zebrafish
(Grosser et al., 2002; Pini et al., 2005; Is­hik­a­wa et al., 2007), as well
as PG recep­tor homo­logues (Vil­la­blan­ca et al., 2007).
The bur­geon­ing molec­u­lar research on the AA cas­cade in
zebrafish com­bined with the ease at which zebrafish oocytes can
be col­lected and induced to mature in vitro, make this pop­u­lar
model tel­eo ­ st suit­able for stud­ies that exam­ine the role of PG in
oocyte mat­u­ra­tion. As reviewed in Spence et al. (2008), zebrafish
ovu­la­tion is depen­dent on female expo­sure to male gonadal pher­
o­mones, but males must be pres­ent for spawn­ing. This allows for
exper­i­ments to be designed that exam­ine puta­tive mech­a­nisms
con­trol­ling ovu­la­tion, despite the inabil­ity of mature, healthy and
intact zebrafish oocytes to ovu­late in vitro (Li et al., 1993; Sel­man
et al., 1994).
The pur­pose of this study was to inves­ti­gate the ovar­ian pro­duc­
tion of PG in zebrafish and to study the potential roles of PG in
the mat­ur­ a­tion and ovu­la­tion of fol­li­cles by using in vitro and in
vivo approaches. Most nota­bly, we exam­ined: (1) the capac­ity of
fol­li­cles of dif­fer­ent size clas­ses to pro­duce PG in response to exog­e­
nous AA treat­ment, (2) the effec­tive­ness of dif­fer­ent COX inhib­i­tors
on AA-stim­ul­ ated PG lev­els and on the rate of GVBD, (3) mRNA
tran­script lev­els of genes within the AA cas­cade in fol­li­cles of dif­fer­
ent size clas­ses, (4) the in vivo effects of INDO on egg pro­duc­tion,
spawn­ing rate, and tis­sue PG and COX activ­ity lev­els of breed­ing
female fish, and (5) ovar­ian lev­els of PGF2a, E2, and 17a,20b-P in
breed­ing and non-breed­ing female zebrafish.
2. Mate­ri­als and meth­ods
2.1. Ani­mals
Adult zebrafish were pur­chased from DAP Inter­na­tional
(Eto­bi­coke, ON, Can­ada) and held for at least 1 month in an A-HAB
fish con­tain­ment unit (Aquatic Hab­i­tats, Apo­pka, FL) at 28 °C on
a 12 h light:12 h dark cycle. Fish were main­tained in re­cir­cu­lat­ed
well water and fed com­mer­cial salmon fry pel­lets (Mar­tin Mills,
Elmira, ON, Can­ada) twice daily with occa­sional sup­ple­ments of
blood­worms (Ore­gon Des­ert Brine Shrimp Co., Lake­view, OR). All
exper­i­ments were per­formed in accor­dance with ani­mal care pro­
to­cols approved by the Uni­ver­sity of Guelph Ani­mal Care Com­mit­
tee on behalf of the Cana­dian Coun­cil on Ani­mal Care.
2.2. Fol­li­cle Incu­ba­tions
Gravid female zebrafish were iden­ti­fied by their enlarged abdo­
mens and eutha­nized by an over­dose of MS-222 fol­lowed by cer­
vi­cal tran­sec­tion prior to the removal of the ova­ries. The ovar­ian
fol­li­cles were iso­lated and incu­bated sim­i­larly to the pro­ce­dure
described by Pang and Ge (2002) with the stag­ing of fol­li­cles
based on Wang and Ge (2004a). Ova­ries were placed in a 35-mm
cul­ture dish con­tain­ing room tem­per­a­ture 60% L-15 (Gib­co Invit­
ro­gen, Carls­bad, CA) sup­ple­mented with 100 U/ml pen­i­cil­lin G
and 100 lg/ml strep­to­my­cin sul­phate (Invit­ro­gen). Fol­li­cles were
sep­a­rated with fine-tipped for­ceps and a sur­gi­cal blade. Each incu­
ba­tion was con­ducted at 28 °C for 18 h, at which time media was
col­lected and stored at ¡80 °C until anal­y­ses. The test com­pounds
were all dis­solved in eth­a­nol, and the con­trol treat­ments con­tained
an equal amount of eth­a­nol.
In the ini­tial exper­i­ment, mid-vitel­lo­genic to imma­ture full-
grown fol­li­cles (termed Stage III in this study) were iso­lated from
the ova­ries of 10 fish and ran­domly placed in groups of 40 in 1 ml of
60% L-15 in 24-well tis­sue cul­ture plates (Corn­ing) in rep­li­cates of
4. Prior to treat­ment with ara­chi­donic acid (AA; Cay­man Chem­i­cal,
Ann Arbor, MI), the media in each well was replaced with 990 ll of
fresh 60% L-15 and then 10 ll of AA dis­solved in eth­a­nol was added
to each well to final con­cen­tra­tions of 7.6, 30.5, and 121.8 lg/ml. A
com­pa­ra­ble AA dose response exper­i­ment was repeated and sim­i­
lar results were obtained.
To exam­ine the capac­ity of fol­li­cles at dif­fer­ent stages of devel­
op­ment to pro­duce PGF2a in response to AA, three dif­fer­ent devel­
op­men­tal size clas­ses of fol­li­cles were iso­lated from the ova­ries of
16 fish. The dif­fer­ent clas­ses of fol­li­cles were then incu­bated in a
total vol­ume of 0.25 ml of 60% L-15 in 96-well tis­sue cul­ture plates
(Corn­ing) in trip­li­cate. Each well of the Stage I fol­li­cles con­tained
approx­i­mately 3 mg of ovar­ian frag­ments dom­i­nated by primary
growth and small pre-vitel­lo­genic fol­li­cles, but min­i­mal amounts
of con­nec­tive tis­sues were also pres­ent as these were dif ­fi­cult
to sep­a­rate. The Stage II ‘early-vitel­lo­genic’ size class had 25 fol­
li­cles per well and the Stage III ‘vitel­lo­genic’ size class con­tained
the same stage of fol­li­cles used in the first exper­i­ment, except that
15 fol­li­cles were placed in each well. AA was added as a 2.5 ll ali­
quot to a final con­cen­tra­tion of 0.3, 3.0, and 30 lg/ml. To assess the
effects of COX inhib­i­tors on AA-stim­u­lated PGF2a, the incu­ba­tion
pro­ce­dure and den­sity of vitel­lo­genic fol­li­cles described above was
used, except that the fol­li­cles were obtained from a pool con­tain­
ing fol­li­cles from 6 fish. The final con­cen­tra­tions of the com­pounds
tested were: 0.5 and 5.0 lg/ml indo­meth­a­cin (INDO) (Sigma) and
0.25, 2.5, and 12.5 lg/ml of NS-398 or SC-560 (Cay­man Chem­i­
cal). Sim­i­lar inhib­i­tory effects of these com­pounds were observed
in a repeated exper­i­ment. For all exper­i­ments, the con­trol wells
received an equal amount of eth­a­nol.
To assess the impact of the same COX inhib­i­tors (INDO, NS-
398, and SC-560) plus a com­pound known to inhibit COX and
lipoxy­ge­nase (LOX) path­ways of AA metab­o­lism, ei­co­sate­tray­
noic acid (ETYA) (Cay­man Chem­i­cal) on spon­ta­ne­ous oocyte mat­
u­ra­tion, 10 lg/ml of each test com­pound was added to wells con­
tain­ing approx­i­mately 40 full-grown fol­li­cles in 1 ml of 60% L-15
in 24-well tis­sue cul­ture plates. Each exper­i­ment uti­lized a pool
of fol­li­cles sep­a­rated from 3 to 4 fish. The method of iden­ti­fy­ing
mature fol­li­cles was based on Pang and Ge (2002). The fol­li­cles
that under­went mat­u­ra­tion after 18 h of incu­ba­tion were iden­
ti­fied by their trans­par­ency due to GVBD. It is widely believed
that oocyte mat­u­ra­tion in tel­e­osts is marked by the onset of GV
migra­tion from its cen­tral loca­tion toward the ani­mal pole of the
48 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57

oocyte, fol­lowed by GVBD. As dis­cussed in Sel­man et al. (1994), Table 2

zebrafish fol­li­cles can be cred­i­bly scored for GVBD with only the
aid of a dis­sect­ing micro­scope, as was used in this study. This
exper­i­ment was repeated three times.
Rates of spawn­ing (%) of groups of breed­ing zebrafish in the con­trol or indo­meth­a­
cin (INDO; 1.0 or 100 lg/L) treat­ments dur­ing the 6 days pre-expo­sure and 16 days
expo­sure peri­ods
% Spawn­ing rate

2.3. Gene expres­sion of fol­li­cles at dif­fer­ent stages of devel­op­ment Pre-expo­sure Expo­sure

Con­trol 45.0 ± 7.6 35.6 ± 5.1

Gene expres­sion of enzymes respon­si­ble for the mobi­li­za­tion INDO 1 lg/L 61.7 ± 6.0 60.0 ± 6.3
of ara­chi­donic acid or its metab­o­lism to pros­ta­glan­dins were quan­ INDO 100 lg/L 35.0 ± 8.1 20.0 ± 3.3*
ti­fied in the three stages of fol­li­cles as described above with the Spawn­ing rates were cal­cu­lated by con­firm­ing the num­ber of groups of fish per
excep­tion that the Stage I size class did not con­tain con­nec­tive tis­ treat­ment that spawned and then was expressed as a per­cent­age of the total sam­ple
sues. size (N = 10). An * denotes a sig­nif­i­cant dif­fer­ence between the treat­ment and con­
On six dif­fer­ent occa­sions, pools of ovar­ian tis­sue from 3 to trol group within the expo­sure period (ANOVA, Dun­nett’s).

4 fish were used to iso­late the dif­fer­ent stages of fol­li­cles. Sim­

i­lar pro­ce­dures for tis­sue sam­pling, RNA extrac­tion, reverse
tran­scrip­tion reac­tions and Real-Time PCR meth­od­ol­o­gies can
be found in Ings and Van Der Kra­ak (2006). Briefly, fol­li­cle sam­ 2.4. Indo­meth­a­cin in vivo expo­sure and tis­sue prep­a­ra­tions
ples were extracted for total RNA using a gua­ni­dine thio­cy­a­nate
phe­nol chlo­ro­form extrac­tion method (Chom­czyn­ski and Sac­chi, To inves­ti­gate the role of PG in spawn­ing, breed­ing groups
1987) and the RNA was resus­pended in 10–20 ll of DEPC-treated of zebrafish were exposed to INDO and the output of eggs were
water fol­lowed by quan­ti­fi­ca­tion and then reverse tran­scrip­tion assessed, along with whole body PGE2 and COX activ­ity lev­els and
(RT). The RT reac­tion used 2 lg of total RNA for each sam­ple and ovar­ian lev­els of COX activ­ity. Mature adult zebrafish were sexed
was mixed with 2 U of DNase I (AMP-D1, Sigma, St. Louis, MO) and 2 females and 1 male were placed into 1.2 L dou­ble-walled plas­
accord­ing to the man­u­fac­turer’s instruc­tions. Then 0.1 ng of tic con­tain­ers (spawn­ing bas­kets) that had a mesh screen across
random prim­ers (Promega, Mad­i­son, WI) were incu­bated with the bot­tom. This set up allowed for daily egg counts as spawned
each sam­ple for 5 min at 70 °C and then iced. As found in Ings eggs dropped to the bot­toms of the spawn­ing bas­kets through the
and Van Der Kra­ak (2006), the total reac­tion vol­ume was 25 ll mesh screen­ing, and out of the water col­umn that con­tained the
and con­tained the fol­low­ing final con­cen­tra­tions: 5£ RT Buffer fish. Each treat­ment had 10 spawn­ing bas­kets, which con­tained
(50 mM Tris–HCl, 75 mM KCl, 3 mM MgCl2; Invit­ro­gen), DTT 1 L of Hagen Aqua­lab well water and an air stone. The spawn­ing
(10 mM, Invit­ro­gen), dNTPs (0.5 mM; Roche Molec­ul­ ar Bio­chem­ bas­kets were set inwto water baths and the tem­per­a­ture was main­
i­cals, La­val, PQ, Can­ada), M-MLV reverse trans­crip­tase (200 U, tained at 28 °C with a 12 h light:12 h dark pho­to­pe­riod. Fish were
Invit­ro­gen) and DEPC-treated ster­ile water. Geno­mic DNA con­ held for 4 days in these spawn­ing bas­kets prior to the begin­ning
trols were prepared by omit­ting M-MLV from par­al­lel sam­ples of the exper­i­ment. Dur­ing the first 6 days of the exper­i­ment (pre-
to con­firm the absence of DNA. The RT reac­tion was per­formed expo­sure period), all groups of fish had 100% of the water replaced
at 37 °C for 1 h fol­lowed by 5 min at 90 °C. Prim­ers were designed with clean water daily. For next 16 days (expo­sure period), the
as per Ings and Van Der Kra­ak (2006). Table 1 shows the primer spawn­ing bas­kets received either 50 ll of eth­a­nol (con­trol groups),
sequences used in this study and each ampli­con was between 72 or 50 ll of INDO such that the final con­cen­tra­tion was 1 or 100 lg/
and 86 base pairs. In this study, expres­sion of the endog­e­nous L. Dur­ing the pre-expo­sure and the expo­sure peri­ods, treat­ments
con­trol genes b-actin did not remain com­pletely con­stant among were renewed daily with 100% water changes after the eggs were
the three size clas­ses; yet, b-actin input val­ues across size clas­ses removed from the outer bas­ket with a plas­tic Pas­teur pipette. Fish
were not sig­nif­i­cantly dif­fer­ent from each other. Sta­tis­ti­cal anal­y­ were fed twice daily through­out the exper­i­ment and received both
ses con­ducted on both non-nor­mal­ized arbi­trary input val­ues of com­mer­cial trout pel­lets and blood­worms. At the end of the exper­
the genes of inter­est and on data nor­mal­ized to b-actin expres­ i­ment, fish were anaes­the­tized with MS-222 and killed by cer­vi­cal
sion lev­els yielded sim­il­ ar results and so the data are pre­sented trans­ac­tion. Their ova­ries (for COX activ­ity) and bodies (COX activ­
as nor­mal­ized val­ues . ity and PGE2 lev­els) were weighed and fro­zen in liquid nitro­gen
and then stored at ¡80 °C until homog­en ­ ates were prepared for
fur­ther anal­y­ses.
Table 1 A ran­domly cho­sen sub-sam­ple of females (N = 5) from each
Primer sequences used in the Real-Time PCR assays
treat­ment was used to pro­vide match­ing whole body PGE2 and
Gene Primer sequence Acces­sion COX activ­ity lev­els. Ova­ries were only mea­sured for COX activ­ity
No. lev­els. First, ovar­i­oec­tom­ized bodies were placed into liquid nitro­
b-Actin For­ward 59-ACA­GGGAAAAGATGACACAGATCA-39 AF025305 gen and then pul­ver­ized with a chilled ceramic mortar and pes­
Reverse 59-CAG­CCTGGATGGCAACGTA-39 tle. The tis­sue was scraped into a 10 ml glass homog­e­niz­ing tube
PLC-c1 For­ward 59-AC­CAGTTTTCCAGCGAGTCGT-39 NM_
and 2 ml of ice-cold 0.1 M Tris–HCl buffer con­tain­ing 1 mM EDTA
194407.1
Reverse 59-AG­CAGTCCA­ACTC­GATG­CATC-39 (pH 7.8) was added and mixed with the tis­sue frag­ments. The con­
cPLA2 For­ward 59-TGCTCTTGGAAGTTTGCGC-39 NM_ tents of the homog­e­niz­ing tube were poured through a coarse cell
131295.1 strainer placed over absor­bent KIM™ wipes in order to remove
Reverse 59-TCTGCGTGTCTGCATGAA­CAG-39 blood-tainted buffer. The tis­sue frag­ments were replaced into the
COX-1 For­ward 59-AAGGTCTGGGTCACGGAGTG-39 NM_ homog­e­niz­ing tube and 3 ml of buffer was added. The sam­ple was
153656.1
homog­e­nized on ice with a Tef­lon P-E pes­tle attached to a drill and
Reverse 59CA­AGAGAATCTCCATAAATGTGTCCA-39
divided into two ali­quots. For PGE2 anal­y­ses, one 600 ll ali­quot had
COX-2 For­ward 59-GTTAAAAGATGGAAAGCTTAAA­TAC­CAGG-39 NM_
153657.1 20 ll of 10 lM indo­meth­a­cin added to pre­vent the ex vivo metab­
Reverse 59-GGGTACACCTCACCATCC­ACA-39 o­lism of PG and was placed at ¡80 °C until PGE2 anal­y­ses. The
PGES-2 For­ward 59-GCAGAGAAAGAATCT­GACG­CATC-39 NM_ remain­ing homog­e­nate was cen­tri­fuged at 10,000g for 15 min at
200280.1 4 °C and the super­na­tant was col­lected and stored at ¡80 °C until
Reverse 59-GCAAAACGGACAGGTCTTATACTGA-39
mea­sure­ment of COX activ­ity. For mea­sure­ment of COX activ­ity,
 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57 49
each ovary was homog­e­nized with a hand-held homog­e­nizer in a
1.5 ml micro­cen­tri­fuge tube con­tain­ing homog­e­ni­za­tion buffer at a
ratio of 50 mg tis­sue: 100 ll of buffer and then cen­tri­fuged and the
super­na­tant col­lected as described. The anal­y­ses were com­pleted
within 7 days (see below).
2.5. Prep­ar­ a­tion of ova­ries sam­pled from spawn­ing and
non-spawn­ing fish
In this exper­i­ment, we pro­filed the lev­els of the sex ste­roids,
17b-estra­diol (E2) and 17a,20b-P, and the pros­ta­glan­din PGF2a of
both spawn­ing and non-spawn­ing females. Two tanks of female-
only fish (N = 10 per tank) and two tanks of mixed sexes were sam­
pled at 12:00 or 8:00 AM. The 12:00 and 8:00 AM mixed sex tanks
held 11 females/13 males and 16 females/8 males, respec­tively. The
males and females were of sim­i­lar body size. The tanks were set up
for 4 days prior to sam­pling and each tank con­tained a mesh screen
along the bot­tom to con­firm the absence or pres­ence of spawned
eggs. Under our exper­i­men­tal con­di­tions, sex­u­ally mature females
typ­ic­ ally spawn around the time the lights come on, which was at
7 AM. At sam­pling, female bodies and ova­ries were weighed and
fro­zen in liquid nitro­gen, then stored at ¡80 °C. Ovar­ian homog­e­
nates were prepared by son­i­cat­ing approx­i­mately 50 mg of ovar­
ian tis­sue in 100 ll of phos­phate buf­fered saline (80 mM Na2HPO4,
20 mM NaH2PO4, 100 mM NaCl; pH 7.4) con­tain­ing 1 mM EDTA and
10 lM INDO. The pieces of ovary were son­i­cated briefly in 1.5 ml
micro­cen­tri­fuge tubes and kept on ice until the start of the meth­a­
nol extrac­tion pro­ce­dure described below.
2.6. Meth­a­nol extrac­tions of tis­sue homog­e­nates for PG and ste­roid
mea­sure­ments
Whole body and ovar­ian homog­e­nates were treated with 4£
meth­a­nol (v/v) and vor­texed, placed at 4 °C for 1 h, and then cen­
tri­fuged at 3000g for 5 min at 4 °C. The pellet was fro­zen with
dry ice and then the upper meth­a­nol phase was decanted to 7 ml
glass vials. This pro­ce­dure was repeated two more times with
the thawed pellet, except the sam­ples were placed at 4 °C for
30 min. All three meth­an ­ ol phases were com­bined into a sin­gle
vial and were treated with a stream of nitro­gen gas until dry.
Dried whole body and ovary extrac­tion vials had 600 ll and
300 ll of 50 mM ace­tate buffer (2.35 ml gla­cial ace­tic acid, 1.23 g
sodium ace­tate tri­hy­drate in 1 L; pH 4.0) added, respec­tively.
These sam­ples were passed through Am­prep C-18 octa­decyl
mini col­umns (Amersham Bio­sci­ences, Little Chal­font, England)
accord­ing to the man­uf­ ac­turer’s instruc­tions for non-polar ana­
lytes. The final elu­ate was col­lected with 2 ml of ethyl ace­tate
con­tain­ing 1% meth­an ­ ol. The ethyl ace­tate frac­tion was dried
with nitro­gen gas. Whole body sam­ples from the INDO expo­sure
and ovar­ian sam­ples from fish sam­pled around ovu­la­tion were
recon­sti­tuted in 500 and 800 ll of EIA buffer (Cay­man Chem­i­cal),
respec­tively, and were stored at ¡80 °C. The extrac­tion effi­ciency
of this method was tested by spik­ing five ovar­ian homog­e­nates
with 5000–6000 cpm of tri­ti­ated PGF2a or E2 and count­ing the
recov­ered radio­ac­tiv­ity. There was greater than 90% recov­ery of
PGF2a and 86% recov­ery of E2.
2.7. Radio­im­mu­no­as­say and enzyme immuno assay pro­ce­dures
Media sam­ples were ana­lyzed for PG or E2 with­out extrac­tion. In
the first in vitro exper­im
­ ent, the media lev­els of PGE2, PGF2a, and E2
were mea­sured by radio­im­mu­no­as­say (RIA) as per Van Der Kra­ak
and Chang (1990). The PGE2 and PGF2a anti­bod­ies were obtained
from Sigma (St. Louis, MO) and the E2 anti­body was obtained from
MP Bio­med­i­cals (Irvine, CA), and the tri­ti­ated com­pounds were
obtained from Amersham (Baie d’Urfe, PQ). The E2 lev­els ana­lyzed
from ovary sam­ples of fish sam­pled around ovu­la­tion were also via
this RIA pro­ce­dure.
All other PGF2a lev­els that appear in this study, as well as the
lev­els of 17a,20b-P were ana­lyzed by enzyme immuno assays (EIA;
Cay­man Chem­i­cal) as per the man­u­fac­turer’s instruc­tions. Greater
sen­si­tiv­ity, while using less sam­ple vol­ume was made pos­si­ble
by the use of the EIA. Regard­less of the assay used, par­al­lel­ism of
pooled and diluted sam­ples was dem­on­strated for both PG and
ste­roids of media and extracted tis­sue sam­ples, which indi­cated
that the sam­ples did not con­tain com­pounds that inter­fered in the
assays.
2.8. Cyclo­ox­y­gen­ase (COX) activ­ity of tis­sue homog­e­nates
Total COX activ­ity of whole body and ovar­ian sam­ples were mea­
sured with the COX Activ­ity Assay kit (Cay­man Chem­i­cal) accord­
ing to the man­u­fac­turer’s instruc­tions. The sam­ple vol­umes added
to the assay were 10 ll of whole body sam­ple and 40 ll of ovar­ian
sam­ple; the prep­a­ra­tion of these sam­ples is described above. Par­al­
lel­ism of diluted sam­ples was also dem­on­strated in this assay.
2.9. Sta­tis­ti­cal anal­y­ses
Data were ini­tially screened for homo­ge­ne­ity of var­i­ance with
a Le­vene’s test prior to a one-way ANOVA. When nec­es­sary, gene
expres­sion data were log trans­formed to meet the require­ments
of Le­vene’s homo­ge­ne­ity of var­i­ance test. Tu­key’s Hon­estly Sig­nif­
i­cant Dif­fer­ence test for multiple com­par­i­sons was used to deter­
mine sig­nif­i­cant dif­fer­ences among groups. Dif­fer­ences in ovar­ian
lev­els of ste­roids or PGF2a between 12:00 and 8:00 AM were com­
pared with a Stu­dent’s t-test. A fac­to­rial ANOVA of the three com­
bined exper­i­ments that exam­ined the pres­ence of GVBD indi­cated
a sig­nif­i­cant inter­ac­tion; there­fore, these exper­i­ments were ana­
lyzed indi­vid­u­ally with ANOVA fol­lowed by Dun­nett’s to indi­cate
sig­nif­i­cant dif­fer­ences between the con­trol and treat­ment groups.
The GVBD per­cent­age data under­went arc­sine trans­for­ma­tion
prior to anal­y­ses. In all sta­tis­ti­cal anal­y­ses, the soft­ware pack­age
SPSS (v. 15) was used and dif­fer­ences were con­sid­ered sig­nif­i­cant
if p < 0.05.
3. Results
3.1. In vitro incu­ba­tions of fol­li­cles treated with ara­chi­donic acid
There are no pub­lished stud­ies that dem­on­strate that zebrafish,
which are capa­ble of daily spawn­ing and have asyn­chro­nous ova­
ries, are a suit­able model for the study of the role(s) of pros­ta­glan­
dins in reg­u­lat­ing ovar­ian repro­duc­tive pro­cesses like fol­lic­u­lar
devel­op­ment, oocyte mat­u­ra­tion, or ovu­la­tion. Thus, our first
exper­i­ments were con­ducted to inves­ti­gate the effects of graded
doses of AA (7.6, 30.5, and 121.8 lg/ml) on the pro­duc­tion of PGE2,
PGF2a, and E2 by vitel­lo­genic (mid- to full-grown) fol­li­cles. Fig.
1a–c shows that PGE2, PGF2a, and E2 were sig­nif­i­cantly increased
by AA treat­ment in a dose-depen­dent man­ner, while basal lev­els
of these com­pounds were non-detect­able in incu­ba­tions of fol­li­
cles at a den­sity of 40/ml. The next exper­i­ment dem­on­strated that
the capac­ity of fol­li­cles to respond to AA treat­ment is not lim­ited
to the vitel­lo­genic stage of devel­op­ment. Lower doses of AA (0.3,
3.0, and 30 lg/ml) were shown to sig­nif­i­cantly increase PGF2a pro­
duc­tion by two other devel­op­men­tally dis­tinct stages, primary
growth/pre-vitel­lo­genic Stage I (Fig. 2a) and early-vitel­lo­genic
Stage II (Fig. 2b) fol­li­cles. Fig. 2c shows the AA-stim­u­lated pro­duc­
tion of PGF2a by mid- to full-grown vitel­lo­genic fol­li­cles (Stage III).
All stages responded in a dose-depen­dent man­ner; yet, it is the
ear­li­est stage of fol­li­cles that are capa­ble of pro­duc­ing the great­est
quan­tity of PGF2a in response to AA treat­ment. The approx­i­mate
50 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57
Fig. 1. Pros­ta­glan­din E2 (PGE2) (a), Pros­ta­glan­din F2a (PGF2a) (b) or estra­diol (E2) (c)
pro­duced by vitel­lo­genic zebrafish ovar­ian fol­li­cles cul­tured in vitro for 18 h at 25 °C
in the pres­ence of increas­ing con­cen­tra­tions of ara­chi­donic acid (AA). Fol­li­cles not
treated with AA had non-detect­able (ND) lev­els of PGE2, PGF2a, and E2 in the media.
Data rep­re­sent means ± SEM of 4 rep­li­cate wells. The exper­i­ment uti­lized fol­li­cles
pooled from 10 indi­vid­u­als. Sta­tis­ti­cal anal­y­ses (ANOVA, Tu­key’s) were per­formed
within AA treat­ments and dif­fer­ent let­ters denote sig­nif­i­cance.
weight of 3 mg was cho­sen for this incu­ba­tion as that is sim­i­lar
to what 20–25 early-vitel­lo­genic or 15 full-grown vitel­lo­genic fol­li­
cles weigh. As the 3 mg frag­ments of tis­sue were dense with a mix
of pre-vitel­lo­genic fol­li­cles, and few primary growth and con­nec­
tive tis­sue com­po­nents, it is rea­son­able to assume that this size
group­ing has the great­est capac­ity to con­vert exog­e­nous AA into
pros­ta­glan­dins due to hav­ing the high­est cell den­si­ties.
To ver­ify that com­pounds known to inhibit PG syn­the­sis were
effec­tive at reduc­ing AA-stim­u­lated PG pro­duc­tion by zebrafish
ovar­ian fol­li­cles, we tested var­i­ous con­cen­tra­tions of INDO (0.5 and
5.0 lg/ml) and NS-398 and SC-560 (.25, 2.5, and 12.5 lg/ml). Only
treat­ments of INDO and the high­est dose of SC-560 resulted in a
sig­nif­i­cant reduc­tion in AA-stim­u­lated PGF2a pro­duc­tion; although,
there was a slight ten­dency for a reduc­tion in all treat­ments (Fig. 3).
Fig. 2. In vitro pros­ta­glan­din F2a (PGF2a) pro­duc­tion by ovar­ian fol­li­cles at dif­fer­ent
stages of devel­op­ment after treat­ment with increas­ing con­cen­tra­tions of ara­chi­
donic acid (AA; 0.3, 3.0, and 30 lg/ml). The wells con­tained either 3 mg of primary
growth/pre-vitel­lo­genic fol­li­cles with sur­round­ing con­nec­tive tis­sues (a; Stage I),
20–25 early-vitel­lo­genic fol­li­cles (b; Stage II), or 15 vitel­lo­genic fol­li­cles (c; Stage
III) in 0.25 ml of L-15 media for 18 h at 25 °C. Ovar­ian tis­sues from 16 fish were
pooled allow­ing for each treat­ment to be con­ducted in trip­li­cate. Data rep­re­sent
means ± SEM. Dif­fer­ent let­ters denote sig­nif­i­cance (ANOVA, Tu­key’s).
3.2. Inhi­bi­tion of spon­ta­ne­ous oocyte mat­u­ra­tion
As shown in Pang and Ge (2002), zebrafish fol­li­cles mature
spon­ta­ne­ously under in vitro con­di­tions; yet, the mat­u­ra­tional com­
pe­tence is great­est in ovar­ian fol­li­cles that are full-grown. Three
pan­els (A–C) are pre­sented in Fig. 4, which rep­re­sents three exper­
i­ments whereby the chem­i­cals INDO, NS-398, SC-560, and ETYA
were tested at 10 lg/ml for their effects on the rate of spon­ta­ne­ous
mat­u­ra­tion of full-grown fol­li­cles. Despite efforts to be con­sis­tent
in our selec­tion of only imma­ture, full-grown fol­li­cles in each of
these exper­i­ments, a fac­to­rial ANOVA indi­cated a sig­nif­i­cant inter­
ac­tion as dif­fer­ing effects of treat­ment on GVBD occurred among
 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57 51
Fig. 3. Ara­chi­donic acid (AA)-stim­u­lated pros­ta­glan­din F2a (PGF2a) pro­duc­tion by vitel­lo­genic zebrafish fol­li­cles cul­tured in vitro for 18 h at 25 °C in the pres­ence of the known
cyclo­ox­y­gen­ase (COX) inhib­i­tors: indo­meth­ac­ in (0.5 and 5.0 lg/ml; non-selec­tive inhib­i­tor), NS-398 (0.25, 2.5, and 25 lg/ml; COX-2 inhib­i­tor), and SC-560 (0.25, 2.5, and
25 lg/ml; COX-1 inhib­i­tor). Data rep­re­sent means ± SEM of 4 rep­li­cate wells of fol­li­cles pooled ran­domly from 6 indi­vid­u­als. Dif­fer­ent let­ters denote sta­tis­ti­cal sig­nif­i­cance
(ANOVA, Tu­key’s).
the exper­i­ments, which pre­cludes com­bin­ing the data. How­ever,
INDO in all three exper­i­ments sig­nif­i­cantly inhib­ited the rate of
oocyte mat­ur­ a­tion (Uni­var­i­ate Anal­y­sis of Var­i­ance, Dun­nett’s). A
subsequent exper­i­ment showed that treat­ment with AA (0.3, 3.0,
and 30 lg/ml) did not affect the rate of spon­ta­ne­ous mat­u­ra­tion
(data not shown).
3.3. Gene expres­sion of fol­li­cles at dif­fer­ent stages of devel­op­ment
Fig. 5 shows the expres­sion of cyto­solic phos­pho­li­pase A2
(cPLA2), phos­pho­li­pase C c1 (PLCc1), cyclo­ox­y­gen­ase-1 (COX-1),
cyclo­ox­y­gen­ase-2 (COX-2), and pros­ta­glan­din E syn­thase-2 (PGES-
2) in sam­ples of fol­li­cles at three stages of devel­op­ment: primary
growth/pre-vitel­lo­genic (Stage I), early-vitel­lo­genic (Stage II) and
mid- to full-grown vitel­lo­genic (Stage III) fol­li­cles. Fur­ther descrip­
tion and images of these stages can be found in Wang and Ge
(2004a). Expres­sion of the selected genes was eas­ily detected in
the dif­fer­ent stages of fol­li­cles, but COX-2 expres­sion tended to be
much lower in sev­eral sam­ples. Our other stud­ies have shown that
ovar­ian COX-2 expres­sion changes dra­mat­ic­ ally in mature fol­li­cles
or in whole ova­ries sam­pled around ovu­la­tion, and are not read­
ily detect­able in other ovar­ian sam­ples (Lister and Van Der Kra­ak,
unpub­lished). It is pos­si­ble that man­ual manip­u­la­tion dur­ing sort­
ing of fol­li­cles in this study has influ­enced the expres­sion of COX-2
due to the role of ara­chi­donic acid metab­ol­ ites in inflam­ma­tion.
The expres­sion of cPLA2 in vitel­lo­genic fol­li­cles was sig­nif­i­cantly
greater than the expres­sion lev­els detected in primary growth and
pre-vitel­lo­genic fol­li­cles (Fig. 4, top panel). As in the study by Ings
and Van Der Kra­ak (2006), the expres­sion lev­els of the endog­en ­ ous
con­trol gene, b-actin tended to decrease as the fol­li­cles grew from
Stages I to III, but this decrease was not sta­tis­ti­cally sig­nif­i­cant in
this study. The nor­mal­ized and non-nor­mal­ized data were com­
pared, and the sta­tis­ti­cal out­comes were sim­i­lar, which is why
we have pre­sented nor­mal­ized data in this case. In both data sets,
the only sig­nif­i­cant out­come was found in the expres­sion lev­els of
cPLA2.
3.4. In vivo expo­sure of spawn­ing groups to indo­meth­a­cin
To eval­ua
­ te the involve­ment of PG in ovu­la­tion and spawn­ing,
groups of male and female zebrafish were treated with 0, 1, or
100 lg/L INDO and the num­bers of eggs spawned were quan­ti­fied
daily. The exper­i­ment began with a 6 days pre-expo­sure period to
deter­mine whether the treat­ment groups exhib­ited inher­ent dif­fer­
ences in their aver­age spawn­ing rates (cal­cu­lated as the num­ber
of groups of fish that spawned out of the total num­ber of groups
within a treat­ment) or clutch size (num­ber of eggs pro­duced per
female within a treat­ment). The aver­age spawn­ing rates for both
the pre-expo­sure and the expo­sure period are pre­sented in Table 2.
There were no sig­nif­i­cant dif­fer­ences in spawn­ing rates among the
treat­ments dur­ing the pre-expo­sure period. How­ever, the spawn­
ing rate of the 100 lg/L INDO treat­ment group was sig­nif­i­cantly
reduced com­pared with the con­trol group dur­ing the expo­sure
period. Although the aver­age spawn­ing rates were reduced dur­ing
the 16 days expo­sure period com­pared with the 6 days pre-expo­
sure period of the con­trol and 100 lg/L INDO treat­ment groups,
these reduc­tions were not sta­tis­ti­cally sig­nif­i­cant.
Fig. 6 shows the accu­mu­lated num­ber of eggs pro­duced per
female for each treat­ment and Fig. 7 shows the aver­age clutch size
in each treat­ment. The aver­age clutch size of the females in the
100 lg/L INDO treat­ment group was sig­nif­i­cantly reduced com­
pared with the other treat­ments. These data indi­cate that spawn­
ing rate and clutch size are adversely affected by INDO, which fur­
ther indi­cates a reg­u­la­tory role of pros­ta­glan­dins in the pro­cess of
mat­u­ra­tion and ovu­la­tion of oocytes in zebrafish.
To eval­u­ate whether INDO was effec­tive at block­ing the syn­the­
sis of pros­ta­glan­dins in exposed fish, lev­els of PGE2 were quan­ti­
fied in whole body tis­sues. PGE2 was cho­sen as it has been pre­vi­
ously shown to be the pre­dom­i­nant PG in whole body sam­ples of
zebrafish (Grosser et al., 2002). INDO at 100 lg/L led to a sig­nif­i­cant
decrease in tis­sue lev­els of PGE2 (Fig. 8a), but cor­re­spond­ing COX
activ­ity lev­els in the whole body tis­sue prep­a­ra­tions (Fig. 8b) were
unaf­fected by treat­ment. Like­wise, there was no effect of treat­ment
on COX activ­ity within the ova­ries (data not shown).
3.5. PG and ste­roid lev­els in ova­ries of spawn­ing and non-spawn­ing
zebrafish
By sam­pling gravid females dur­ing the dark period (12:00 AM)
and soon after dawn (8:00 AM) that were held alone or in the pres­
ence of males, we were able to dem­on­strate that intra-ovar­ian lev­
els of PGF2a, E2, and 17a,20b-P were sig­nif­i­cantly ele­vated in spawn­
ing females (Fig. 9a) com­pared with non-spawn­ing females (Fig.
9b). Also, the ovar­ian lev­els of PGF2a and E2 were higher in the non-
52 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57

Fig. 4. Effect of 10 lg/ml of the cyclo­ox­y­gen­ase inhib­i­tors indo­meth­a­cin (INDO; non-selec­tive), NS-398 (COX-2 spe­cific), and SC-560 (COX-1 spe­cific) and of the non-selec­tive
inhib­i­tor of cy­clo­ox­y­gen­as­es and lip­ox­y­gen­as­es, ei­co­sate­tray­noic acid (ETYA) on spon­ta­ne­ous oocyte mat­u­ra­tion as deter­mined by the pres­ence of ger­mi­nal ves­i­cle break­
down (GVBD) in three sep­ar­ ate exper­i­ments (a–c). GVBD was scored after 18 h at 25 °C from wells con­tain­ing approx­i­mately 40 fol­li­cles. Data rep­re­sent means ± SEM of 3–4
rep­li­cate wells and an aster­isk denotes sig­nif­i­cant dif­fer­ence between the con­trol and treated group (ANOVA, Dun­nett’s).

breed­ing females com­pared with the females housed with males.
The use of mod­i­fied A-HAB tanks that con­tained a mesh screen
across the bot­tom allowed for the con­fir­ma­tion of spawned eggs
or the absence of eggs, as in the case of the female-only tanks. It is
our expe­ri­ence that female zebrafish when well fed, but iso­lated
from males for sev­eral days will con­tinue to grow their ova­ries and
even­tu­ally may ovu­late matured oocytes into the abdo­men; yet,
these fish will not spawn with­out the pres­ence of males (reviewed
in Spence et al., 2008). This sim­ple exper­im­ en­tal design allows for
a direct com­par­i­son of ovar­ian hor­mone lev­els in spawn­ing vs non-
spawn­ing fish.
4. Dis­cus­sion
The pur­pose of this study was to inves­ti­gate the ovar­ian pro­duc­
tion of PG and to study the potential roles of PG and MIH in the mat­
u­ra­tion and ovu­la­tion of zebrafish fol­li­cles by using in vitro and in
vivo approaches. In tel­e­osts, PG are impli­cated in the reg­u­la­tion of
ovar­ian func­tion, namely gonadal ste­roi­do­gen­e­sis (Van Der Kra­ak
and Chang, 1990; Wade and Van Der Kra­ak, 1993), oocyte mat­u­
ra­tion (Sor­ber­a et al., 2001) and ovu­la­tion (Pa­ti­no and Sul­li­van,
2002; Pa­ti­no et al., 2003), and sex­ual behav­iours by act­ing as pher­
o­mones (Ko­bay­ash­i et al., 2002). It is well estab­lished in mam­mals
that PGs act as local medi­a­tors of ovu­la­tion, fer­til­iza­tion, implan­ta­
tion, and par­tu­ri­tion (Lim et al., 1997; Sales and Jab­bour, 2003). To
our knowl­edge, this is the first study to exam­ine the pro­duc­tion of
PG by zebrafish ovar­ian tis­sues and to exam­ine their potential role
in reg­u­lat­ing oocyte mat­u­ra­tion and ovu­la­tion.
By treat­ing cul­tured ovar­ian fol­li­cles at dif­fer­ent stages of devel­
op­ment with an exog­en ­ ous source of AA, it was dem­on­strated that
zebrafish fol­li­cles increased their pro­duc­tion of PGE2 and PGF2a in a
dose-depen­dent man­ner. Vitel­lo­genic fol­li­cles treated with AA also
increased their pro­duc­tion of E2. Sev­eral stud­ies have doc­u­mented
the capac­ity of tel­eo ­ st ovar­ian fol­li­cles to pro­duce PG under dif­fer­
ent in vitro cul­ture regimes (Goe­tz et al., 1989; Brad­ley and Goe­tz,
1994; Mer­cure and Van Der Kra­ak, 1996). There are ­sev­eral sites
within the ovary with the capac­ity to pro­duce PG, includ­ing the
extra-fol­lic­u­lar (EF) tis­sues, which were shown in Goe­tz et al.
(1989) to con­trib­ute sub­stan­tially to the over­all pro­duc­tion of PGE2,
but pro­duced very little PGF2a. In this study, it is also likely that EF
 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57 53

Fig. 5. Quan­ti­ta­tive Real-Time PCR anal­y­sis of stage depen­dent expres­sion of genes within the pros­ta­glan­din bio­syn­thetic path­way: cyto­solic phos­pho­li­pase A2 (cPLA2),
phos­pho­li­pase C c1(PLCc1), cyclo­ox­y­gen­ase-1 (COX-1), cyclo­ox­y­gen­ase-2 (COX-2), and pros­ta­glan­din E syn­thase-2 (PGES-2). The expres­sion lev­els were nor­mal­ized to that
of b-actin and expressed as the fold change of the lev­els of the Stage I primary growth/pre-vitel­lo­genic fol­li­cles. Stage II and Stage III fol­li­cles are early-vitel­lo­genic and vitel­
lo­genic (mid- to full-grown) fol­li­cles, respec­tively. Val­ues rep­re­sent means ± SEM of 6 deter­mi­na­tions. Each deter­mi­na­tion was gen­er­ated from a pool of fol­li­cles sep­a­rated
from 3 to 4 fish. Dif­fer­ent let­ters denote sta­tis­ti­cal sig­nif­i­cance (ANOVA, Tu­key’s).

tis­sues con­trib­uted in a small way to the pro­duc­tion of PGF2a mea­
sured from the ‘primary growth/pre-vitel­lo­genic’ stage of fol­lic­u­lar
growth, as this stage con­tained small amounts of con­tam­i­nat­ing
EF. We included the EF tis­sues in this stage in order to min­i­mize
the man­ual dis­rup­tion of the tis­sue as it is dif ­fi­cult to sep­a­rate the
small­est stage of fol­li­cle from the sur­round­ing tis­sues.
The reg­u­la­tion of ste­roid and PG pro­duc­tion in tel­e­ost ova­ries
is not fully under­stood; yet, sev­eral stud­ies pro­vide evi­dence that
their pro­duc­tion should not be viewed as entirely sep­a­rate events.
For exam­ple, AA and PGE2 stim­u­late sex ste­roid (i.e., tes­tos­ter­one)
pro­duc­tion by gold­fish ovar­ian fol­li­cles likely through an increase
in cAMP pro­duc­tion (Van Der Kra­ak and Chang, 1990; Mer­cure
and Van Der Kra­ak, 1996) and prog­es­tins, E2, and tes­tos­ter­one neg­
a­tively affect the in vitro pro­duc­tion of basal and AA-stim­u­lated
PGE2 by gold­fish fol­li­cles (Kell­ner, 1993). The mech­a­nism by which
exog­e­nous AA affects gonadal ste­roid pro­duc­tion in tel­e­osts may
involve the actions of the COX prod­uct PGE2 (Mer­cure and Van Der
Kra­ak, 1996), which is not sur­pris­ing as many of the effects of AA
have been attrib­uted to its con­ver­sion by oxy­gen­ases to PG or leu­
ko­tri­enes. But free AA also exhib­its bio­ac­tiv­ity includ­ing effects on
ion chan­nels (e.g., Ca2+) (reviewed in Brash, 2001) that may con­trib­
ute to its ste­roi­do­genic effects when admin­is­tered in vitro. It has
already been estab­lished that the induc­tion of ste­roi­do­gen­e­sis by
PGE2 depends on cal­cium (Mer­cure and Van Der Kra­ak, 1996) and
cal­cium is needed for eicos­a­noid syn­the­sis by tel­eo ­ st ovar­ian fol­li­
cles (Kell­ner and Van Der Kra­ak, 1992).
Two PG syn­the­sis inhib­i­tors were found in this study to sig­nif­
i­cantly inhibit the pro­duc­tion of AA-stim­u­lated PGF2a by zebrafish
vitel­lo­genic fol­li­cles. These two com­pounds were the non-selec­
tive inhib­i­tor of COX enzymes, INDO (0.5 and 5.0 lg/ml) and the
COX-1 selec­tive inhib­i­tor SC-560, which was effec­tive at 12.5 lg/
ml. INDO treat­ments resulted in a 57% reduc­tion in PGF2a pro­duc­
tion. The COX-2 selec­tive inhib­i­tor, NS-398 did not sig­nif­i­cantly
reduce the AA-stim­u­lated PGF2a lev­els. We obtained the same
sta­tis­ti­cal results in a repeat of this exper­i­ment. Pre­vi­ous in vitro
stud­ies have shown that INDO was effec­tive at reduc­ing stim­u­
54 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57
Fig. 6. Accu­mu­la­tive egg pro­duc­tion by groups of sex­ua
­ lly mature zebrafish (N = 10
groups per treat­ment) over a 22-day period and adjusted for the num­ber of females
per group. Eggs were col­lected from fish held in clean water for 6 days to estab­lish
a base­line level of egg pro­duc­tion, fol­lowed by 16 days of egg col­lec­tion from fish
exposed to eth­a­nol (Con­trol) or indo­meth­a­cin (INDO) at 1.0 or 100 lg/L. Breed­ing
groups typ­i­cally con­sisted of 3 fish: 1 female and 2 males.
Fig. 7. Aver­age num­ber of eggs per treat­ment pro­duced by each female dur­ing the 6
days pre-expo­sure period (clean water only) or dur­ing the 16 days of treat­ment to
either eth­an­ ol (Con­trol) or to indo­meth­ac­ in (Indo) at 1.0 or 100 lg/L. Neg­at­ ive num­
bers rep­re­sent pre-expo­sure days. Data rep­re­sent means ± SEM of 10 rep­li­cate breed­
ing groups per treat­ment. Dif­fer­ent let­ters denote sta­tis­ti­cal sig­nif­i­cance (ANOVA,
Tu­key’s).
lated PGE2 lev­els of cul­tured gold­fish fol­li­cles (Mer­cure and Van
Der Kra­ak, 1996) and reduc­ing PGE2 and PGF2a lev­els of cul­tured
yel­low perch fol­li­cles, which cor­re­sponded to decreased ovu­la­tion
(Brad­ley and Goe­tz, 1994). These results sug­gest that COX-1 activ­
ity may be the pre­dom­i­nant COX enzyme pres­ent in early to full-
grown vitel­lo­genic fol­li­cles and acts to con­vert AA to PGs. Fur­ther
evi­dence for this is pro­vided by the tran­script lev­els of COX-1 and
COX-2 gen­er­ated from the Real-Time PCR results; COX-2 expres­
sion was nearly non-detect­able com­pared with COX-1 and the
other genes exam­ined (raw data not shown). The min­i­mal level of
COX-2 expres­sion may have been due to man­ual manip­u­la­tion of
the tis­sues dur­ing the iso­la­tion of fol­li­cles, as we have found in
our ongo­ing stud­ies that adult female zebrafish rarely have detect­
able lev­els of COX-2 in ovar­ian sam­ples, while COX-1 is rou­tinely
detected. The excep­tion is that COX-2 tran­script lev­els are strongly
induced in mature fol­li­cles and are read­ily detect­able in ova­ries of
fish sam­pled at ovu­la­tion (Lister and Van Der Kra­ak, unpub­lished).
Con­versely, a study by Grosser et al. (2002) con­cluded that the
Fig. 8. Pros­ta­glan­din E2 (a) (PGE2; pg/mg tis­sue) and total cyclo­ox­y­gen­ase enzyme
activ­ity (U/mg tis­sue) (b) in ovar­i­oec­tom­ized bodies of con­trol and indo­meth­a­cin (1
or 100 lg/L) treated female fish. Data rep­re­sent means ± SEM of 5 indi­vid­u­als from
each treat­ment. Dif­fer­ent let­ters denote sta­tis­ti­cal sig­nif­i­cance (ANOVA, Tu­key’s).
PGE2 lev­els detected in whole body zebrafish homog­e­nates were
the result of COX-2 metab­ol­ ism, as both INDO and NS-398 expo­
sure sig­nif­i­cantly reduced PGE2 lev­els. The pre­dom­i­nant COX prod­
uct of adult zebrafish is PGE2 (Grosser et al., 2002), and although a
par­tic­u­lar tis­sue source is unknown, there is con­sti­tu­tive expres­
sion of COX-2 in blood ves­sels and intes­tines (Grosser et al., 2002).
It is pos­si­ble that in the ovary of zebrafish, the induc­ible COX-2
gene plays a highly spe­cific role at the time of ovu­la­tion, while the
con­sti­tu­tive expres­sion of COX-1 is respon­si­ble for AA metab­o­lism
dur­ing fol­lic­u­lar devel­op­ment.
In addi­tion to COX-1 and COX-2, three other genes were quan­
ti­fied that either directly (cPLA2) or indi­rectly (PLCc1) lead to
the release or pro­duc­tion of free ara­chid­o­nate, or its subsequent
metab­o­lism to PGE2 (PGES-2). All of the genes, except COX-2 as
pre­vi­ously dis­cussed, were read­ily detect­able in the three stages
of fol­lic­u­lar devel­op­ment that were exam­ined in this study, which
sug­gests that a fully func­tional AA cas­cade is pres­ent in zebrafish
ovar­ian fol­li­cles. Only tran­scripts of cPLA2 exhib­ited a sig­nif­i­cant
dif­fer­ence in expres­sion lev­els across fol­lic­u­lar devel­op­ment. In
other spe­cies stud­ied, PLA2 plays a piv­otal role in the ovu­la­tory
pro­cess. In rodents, the expres­sion and activ­ity of cPLA2 of gran­
u­losa cells were shown to be induced by gon­a­do­tro­pin (Kur­usu
et al., 1998), and an increased expres­sion of cPLA2 has been doc­
u­mented in cows fol­low­ing the gon­a­do­tro­pin surge, where it is
thought that phos­pho­li­pase (termed PLA2G4a in cows) and COX-2
are respon­si­ble for the pro­duc­tion of PG that are required for ovu­la­
tion and oocyte mat­u­ra­tion (Dio­uf et al., 2006). Duf­fy et al. (2005)
showed that cPLA2 is pre­dom­i­nantly respon­si­ble for the gon­a­do­
tro­pin-stim­u­lated mobi­li­za­tion of AA nec­es­sary for PG pro­duc­
 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57 55
Fig. 9. Ovar­ian lev­els (pg/mg tis­sue) of pros­ta­glan­din F2a (PGF2a), 17b-estra­diol (E2),
and 17a, 20b-dihy­droxy-4-preg­nen-3-one (17a,20b-P) in spawn­ing (a) and non-
spawn­ing (b) females sam­pled dur­ing the night at 12:00 AM and soon after dawn
at 8:00 am. In (a), data rep­re­sent means ± SEM of 11 and 16 indi­vid­u­als at 12:00 and
8:00 AM, respec­tively. In (b), data rep­re­sent means ± SEM of 10 indi­vid­u­als at each
time point. Stu­dent t-tests were per­formed and an aster­isk denotes sig­nif­i­cance for
each mea­sure­ment between time peri­ods.
tion by pe­riov­ul­ a­to­ry fol­li­cles in cyno­mol­gus macaques (Macaca
­fas­cic­u­lar­is). There­fore, we can hypoth­e­size that in zebrafish, cPLA2
expres­sion lev­els increase in the grow­ing ovar­ian fol­li­cle due to its
role in the mobi­li­za­tion of AA, which sub­se­quently allows for the
pro­duc­tion of PG that are required for nor­mal oocyte mat­u­ra­tion
and ovu­la­tion. This hypoth­e­sis is sup­ported in another tel­e­ost by
the study of Sor­ber­a et al. (2001) that dem­on­strated a sup­pres­sion
of gon­a­do­tro­pin-induced mat­u­ra­tion by a PLA2 blocker, quin­a­crine
in oocytes of the Euro­pean sea bass. Stud­ies are now under­way in
our lab­o­ra­tory to estab­lish the role of cPLA2 and COX-2 in oocyte
mat­u­ra­tion and ovu­la­tion.
A role for PG in medi­at­ing oocyte mat­u­ra­tion and ovu­la­tion in
zebrafish is sup­ported in the pres­ent study through in vitro and
in vivo results. First, cul­tured full-grown fol­li­cles treated with PG
inhib­it­ing com­pounds, nota­bly indo­meth­a­cin, exhib­ited a reduced
abil­ity to undergo spon­ta­ne­ous mat­u­ra­tion. Full-grown fol­li­cles of
zebrafish undergo spon­ta­ne­ous mat­u­ra­tion after iso­la­tion from
the ovary (Sel­man et al., 1994); this devel­op­men­tal stage exhib­
its the great­est respon­sive­ness to 17a,20b-P as they have already
become mat­u­ra­tion­al­ly com­pe­tent within the ovary prior to dis­sec­
tion (Pang and Ge, 1999; Pang and Ge, 2002). Yet, exog­e­nous treat­
ment of full-grown fol­li­cles with AA (0.3, 3.0, and 30 lg/ml) had
no effect on the rate of spon­ta­ne­ous mat­u­ra­tion in this study (data
not shown). The rate of spon­ta­ne­ous mat­u­ra­tion of the con­trol
groups used in this study aver­aged 45% after 18 h of incu­ba­tion.
Going by the descrip­tion of fol­li­cle sizes that undergo GVBD after
48 h in Sel­man et al. (1994), we strongly believe that the zebrafish
fol­li­cles used in these exper­i­ments would have been in a late stage
of mat­u­ra­tion and should have been mat­u­ra­tion­al­ly com­pe­tent.
This means that the response of zebrafish fol­li­cles appears to be
sim­i­lar to that of Atlan­tic croaker, where Pa­ti­no et al. (2003) estab­
lished that AA and its metab­o­lites were insuf ­fi­cient to induce mei­
otic resump­tion in cul­tured fol­li­cles but indo­meth­a­cin still inhib­
ited mat­u­ra­tion-induc­ing hor­mone-stim­u­lated oocyte mat­u­ra­tion.
Pa­ti­no et al. (2003) con­cluded that AA and its metab­o­lites are
involved in medi­at­ing ovu­la­tion and not mei­otic resump­tion, but
that COX prod­ucts may play a per­mis­sive role dur­ing mat­u­ra­tion.
AA metab­o­lites have been impli­cated in the reg­u­la­tion of oocyte
mat­u­ra­tion in the Euro­pean sea bass, where exog­en ­ ous AA, PGF2a,
and PGE2 induced mei­otic resump­tion in mat­u­ra­tion­al­ly com­pe­
tent ovar­ian fol­li­cles (Sor­ber­a et al., 2001), but the mech­a­nism by
which AA induces GVBD is unclear as it may act directly via the
reg­u­la­tion or the trans­duc­tion of fol­lic­u­lar mat­u­ra­tion-induc­ing
ste­roid. In this study, con­sis­tent results were not obtained by the
other COX inhib­i­tors (NS-398, SC-560) or by ETYA, an inhib­i­tor of
COX and lipoxy­ge­nase metab­o­lism of AA. It should be noted that a
trend of decreased% of GVBD was obtained by all the treat­ments in
each exper­i­ment. Perhaps in zebrafish, PG medi­ate oocyte mat­ur­ a­
tion at a point prior to the fol­li­cles becom­ing fully mat­u­ra­tion­al­ly
com­pe­tent, which would require an inves­ti­ga­tion of the effects of
COX inhib­i­tors or treat­ments with AA metab­o­lites on gon­a­do­tro­
pin- or 17a,20b-P-stim­u­lated mat­u­ra­tion.
Sec­ondly, breed­ing groups of fish exposed to 100 lg/L INDO
spawned sig­nif­i­cantly fewer oocytes (‘clutch size’), and spawned
less fre­quently (‘spawn­ing rate’) com­pared with the con­trol and
1 lg/L INDO groups dur­ing the expo­sure period of the exper­i­ment.
These results pro­vide fur­ther evi­dence for the involve­ment of PG
in medi­at­ing oocyte mat­u­ra­tion and/or ovu­la­tion in zebrafish.
How­ever, as both males and females were exposed to INDO in our
study, it is not pos­si­ble to dis­cern whether the non-ste­roi­dal anti-
inflam­ma­tory drug (NSAID) exerted its neg­a­tive effects solely on
the abil­ity of the eggs to mature or ovu­late within the females, or
whether the inhi­bi­tion of PG syn­the­sis also affected sex­ual behav­
iours of the males, which may have con­trib­uted to the reduc­tions
in egg pro­duc­tion. Expect­edly, PGE2 lev­els mea­sured in whole
body homog­e­nates were reduced in the 100 lg/L INDO group; yet,
we did not detect any change in the activ­ity lev­els of COX in the
same sam­ples or in ovar­ian sam­ples com­pared with con­trols. Even
though expo­sure to INDO was shown to reduce whole body lev­els
of PGE2 in the study by Grosser et al. (2002), the author’s did not
con­firm a con­com­i­tant reduc­tion in COX enzyme activ­ity, which is
the known mech­a­nism of action of this NSAID. Like­wise, a recent
study by Is­hik­a­wa et al. (2007) tested the COX activ­ity of expressed
zebrafish COX iso­forms using the same COX Activ­ity Assay as this
study, but the abil­ity to inhibit the activ­ity of these iso­forms by a
phar­ma­co­log­i­cal agent was not tested. The reason for the lack of
inhi­bi­tion of COX activ­ity in sam­ples from fish exposed to 100 lg/
L INDO in vivo remains unknown and pub­lished stud­ies have not
yet dem­on­strated the abil­ity of INDO to inhibit fish COX activ­ity,
despite its long-stand­ing use in the mod­u­la­tion of repro­duc­tive
func­tion and inhi­bi­tion of PG syn­the­sis of fish. INDO has been pre­vi­
ously shown to sup­press the in vitro ovu­la­tion of fol­li­cles in the yel­
low perch (Brad­ley and Goe­tz, 1994), and to neg­a­tively affect ovu­
la­tion of fol­li­cles of the amphib­ian Rana dy­bow­skii depend­ing on
sea­sonal vari­a­tions (Chang et al., 1995). In all mam­mals stud­ied to
date, INDO and other NSA­IDs cause ovu­la­tory dys­func­tion, which
has been attrib­uted pri­mar­ily to the inhi­bi­tion of COX-2 activ­i­ties
(Gay­tan et al., 2006a; reviewed in Gay­tan et al., 2006b).
56 A. Lister, G. Van Der Kra­ak / General and Comparative Endocrinology 159 (2008) 46–57
Lastly, intra-ovar­ian lev­els of PGF2a, as well as 17a,20b-P (MIH)
and E2 are sig­nif­i­cantly increased in the whole ova­ries of breed­ing
females at or near the time of ovu­la­tion (8:00 AM) com­pared with
breed­ing females sam­pled ear­lier in the dark cycle (12:00 AM) and
non-breed­ing females at either time point. In lower ver­te­brates, it is
widely accepted that the pre-ovu­la­tory surge in LH pro­motes oocyte
mat­u­ra­tion by stim­u­lat­ing the fol­li­cle to pro­duce and secrete MIH.
In turn, MIH is thought to cause an increase in pros­ta­glan­din syn­
the­sis, which is nec­es­sary for MIH-induced ovu­la­tion of the fol­li­cle
(Goe­tz, 1997; Pin­ter and Thomas, 1999). It has been shown that MIH
and PGF2a act to increase cer­tain pro­te­o­lytic enzyme activ­it­ ies in
gold­fish fol­li­cles or col­lag­e­no­lytic pro­te­ase in yel­low perch, which
might aid in the deg­ra­da­tion of the fol­lic­u­lar wall and form a site
through which the mature ovum emerges (reviewed in Ya­ron and
Sivan, 2006). Cur­rently, we are con­duct­ing exper­i­ments that inves­ti­
gate the potential reg­u­la­tion of ovar­ian PG syn­the­sis by gon­a­do­tro­
pin (human cho­ri­onic) and ste­roids, includ­ing 17a,20b-P.
Although stud­ies have char­ac­ter­ized 17a,20b-P as an MIH in
zebrafish based on its effec­tive­ness to induce mat­u­ra­tion in vitro
(e.g., Sel­man et al., 1994), there is a pau­city of infor­ma­tion on its
endog­e­nous reg­u­la­tion by LH. In sal­mo­nids, the pre­ovu­la­tory gon­
a­do­tro­pin surge induces the expres­sion of 20b-hydroxy­ster­oid
dehy­dro­ge­nase (20b-HSD), which is the enzyme that con­verts
17a-hydrox­y­pro­ges­ter­one to 17a,20b-P in gran­u­losa cells prior to
oocyte mat­u­ra­tion (Young et al., 1986; Na­ga­ha­ma, 1997). But in
zebrafish, 20b-HSD expres­sion remained unchanged among fol­li­
cles through­out devel­op­ment and tran­script lev­els do not change
in response to gon­a­do­tro­pin (Wang and Ge, 2002). Sim­i­lar neg­a­
tive results in zebrafish (a cyp­ri­nid) were found with the upstream
enzyme, Cyto­chrome P450c17 that con­trols the pro­duc­tion of the
MIH pre­cur­sor, fur­ther high­light­ing the dif­fer­ences in reg­u­la­tion
of ovu­la­tion between sal­mo­nids and the cyp­ri­nids (as dis­cussed in
Wang and Ge, 2002, 2004b). How­ever, there is evi­dence that gon­a­
do­tro­pin tran­script lev­els undergo a pre­dict­able surge dur­ing the
time at which ovar­ian fol­li­cles undergo GVBD and mat­u­ra­tion (So
et al., 2005), which would have occurred in the breed­ing females
used in this study at or soon after the 12:00 AM time period. By
dem­on­strat­ing that 17a,20b-P lev­els are increased in the ova­ries of
nat­ur­ ally spawn­ing zebrafish soon after ovu­la­tion, this study has
fur­ther sub­stan­ti­ated a role for this ste­roid in induc­ing mat­u­ra­tion
and ovu­la­tion in zebrafish.
In this study, the phys­io ­ ­log­i­cal sig­nif­i­cance of the increased lev­
els of PGF2a and E2 observed in the ova­ries of spawn­ing zebrafish
are spec­u­la­tive based on pre­vi­ous stud­ies of the roles of PG and
sex ste­roids in repro­duc­tive func­tion­ing. It is pos­si­ble that the
increased pro­duc­tion of ovar­ian PG at the time of ovu­la­tion facil­
i­tates the par­a­crine reg­u­la­tion of fol­lic­u­lar rup­ture (reviewed in
Soren­sen and Goe­tz, 1993). Although plasma lev­els of PG were not
mea­sured in the spawn­ing or non-spawn­ing fish used in this study,
it is pos­si­ble that cir­cu­lat­ing lev­els of F pros­ta­glan­dins in the female
fish would have been increased in cir­cu­la­tion, as stud­ies in other
tel­e­osts have shown that these PG rise at the time of ovu­la­tion and
act within the brain to elicit female sex­ual behav­iours (reviewed in
Soren­sen and Goe­tz, 1993 and Ko­bay­ash­i et al., 2002). PGF2a may
also be metab­o­lized in the females to an active pher­om ­ one (15-
keto PGF2a) and released to the water where it func­tions as a sex
pher­om ­ one that stim­u­lates male sex­ual behav­iour (Ko­bay­ash­i et
al., 2002; Sta­cey et al., 2003). Perhaps the increased level of E2 that
was pres­ent in the ova­ries of the spawned fish that were sam­pled
soon after ovu­la­tion is indic­at­ ive of vitel­lo­gen­e­sis that was under­
taken to grow the next cohort of eggs. A typ­i­cal char­ac­ter­is­tic of
asyn­chro­nous ovar­ian devel­op­ment in tel­e­osts is the con­tin­u­ous
recruit­ment of oocytes that are under­go­ing vitel­lo­genic growth,
with some oocytes being driven to mat­u­ra­tion and ovu­la­tion (Wal­
lace and Sel­man, 1981). In the cur­rent study, it is expected that a
large pro­por­tion of oocytes were already matured and were sub­
se­quently spawned because the females had pre­vi­ously been iso­
lated to ensure substantial ovar­ian devel­op­ment and to aid in the
positive iden­ti­fi­ca­tion of sex. Com­pared with the pre-ovu­la­tory
state of the ovary, the spawn­ing event would have resulted in an
ovary that was pre­dom­i­nantly under­go­ing recru­des­cence with a
high rate of vitel­lo­gen­es­ is that is depen­dent upon the pro­duc­tion
of E2. This is in agree­ment with a pre­vi­ous study of another cyp­
ri­nid with asyn­chro­nous ovar­ian devel­op­ment, gold­fish (Car­as­sius
aura­tus), which found that the gon­a­do­tro­pin surge was not asso­
ci­ated with a decline in E2 pro­duc­tion and E2 lev­els were found
to be higher post-ovu­la­tion com­pared with pre-ovu­la­tion lev­els
(Ko­bay­ash­i et al., 1988).
5. Sum­mary
The results of our study imply a func­tional AA bio­syn­thetic path­
way in zebrafish ova­ries through the detec­tion of selected gene
tran­scripts in the intact fol­li­cles, as well as the quan­ti­fi­ca­tion of
PGs from ovar­ian fol­li­cles cul­tured in vitro. The inhi­bi­tion of PG
lev­els con­com­i­tant with a decrease in the num­ber of spawned eggs
by fish exposed to 100 lg/L INDO strongly sug­gests a role for PG in
the nor­mal mat­u­ra­tion or ovu­la­tion of zebrafish oocyte. As well,
the increased ovar­ian pro­duc­tion of PGF2a and 17a,20b-P detected
in nor­mally spawn­ing zebrafish com­pared to non-spawn­ing fish
strongly sug­gests their involve­ment in the ovu­la­tory pro­cess. In
con­clu­sion, this study has estab­lished the zebrafish as an appro­pri­
ate and con­ve­nient model of an asyn­chro­nous tel­e­ost in which to
fur­ther study the reg­u­la­tion of the ovar­ian AA path­way and its role
in oocyte mat­u­ra­tion and ovu­la­tion.
Acknowl­edg­ments
This study was sup­ported by an NSERC Dis­cov­ery Grant to
G.V.D.K. and by the Sal­a­man­der Foun­da­tion. A.L. was sup­ported by
NSERC and Ontario Grad­u­ate Schol­ar­ships.
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