Group Title: Reproductive Biology and Endocrinology 2004, 2:14
Title: Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays
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Title: Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays
Series Title: Reproductive Biology and Endocrinology 2004, 2:14
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Creator: Lin YWP
Petrino TR
Wallace RA
Publication Date: 38070
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Fundulus heteroclitus gonadotropins.5: Small scale chromatographic
fractionation of pituitary extracts into components with different
steroidogenic activities using homologous bioassays
Yu-Wai Peter Lin* Teresa R Petrinol and Robin A Wallace2

Address: 'Barry University, School of Natural & Health Sciences, Miami Shores, Florida 33161, USA and 2Whitney Laboratory, University of
Florida, St. Augustine, Florida 32086, USA
Email: Yu-Wai Peter Lin*; Teresa R Petrino; Robin A Wallace
* Corresponding author

Published: 24 March 2004
Reproductive Biology and Endocrinology 2004, 2:14

Received: 09 January 2004
Accepted: 24 March 2004

This article is available from:
2004 Lin et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media
for any purpose, provided this notice is preserved along with the article's original URL.

Fractionation and characterization of gonadotropins (GtH) from Fundulus heterocitus pituitary
extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia
FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction
of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-
exclusion chromatography eluted gonadotropic activity in one major protein peak (Mr- 30,000).
Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of
17beta-estradiol (E2)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity
with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed
by HIC) resolved pituitary extracts into two active fractions; both induced E2 synthesis, but one
was relatively poor in eliciting DHP and testosterone production. Thus, using homologous
bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E2-
promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has
a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F.
heteroditus pituitaries by a variety of chromatographic procedures. This study complements
previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH
function in a multiple-spawning teleost.

During a breeding season, many fish known as fractional
spawners have been found to undergo periodic reproduc-
tive activity correlated with the lunar cycle [1], including
Fundulus heteroclitus killifishh) [2,3]. Such activity involves
a complicated interplay of gonadotropic hormones
(GtHs) and ovarian steroidogenic events as clutches of
oocytes are periodically recruited into vitellogenic, matu-
rational, and ovulatory processes [3,4].

The presence of two distinct GtHs in teleosts has been
demonstrated in several species including chum salmon
[5,6], coho salmon [7], killifish [8-10], common carp
[11], Atlantic croaker [12], tuna [13], bonito [14], red sea-
bream [15], striped bass [16], yellowfin porgy [17], gold-
fish [18]and Japanese eel [19]. These GtHs, distinguished
by their 3-subunits and termed GtH I and GtH II, are sim-
ilar (38-48% sequence identity) to tetrapod follicle stim-
ulating hormone (FSH) and luteinizing hormone (LH),
respectively [20,21]. The biological significance of the two
GtHs has been intensively studied in salmonids and other

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Reproductive Biology and Endocrinology 2004, 2

annual spawners. However, much less information is
available from other commercially important species that
are fractional spawners such as tuna, flounder, red drum,
croaker, and halibut. Due to habitat destruction and/or
overfishing, the populations of some of these species are
declining. Fundulus is also a fractional spawner and can act
as an inexpensive, easily manipulated model system.

In order to understand the operant mechanisms involved
in cyclic teleost reproduction and to provide a broader
basis for comparative study of the teleost GtHs, we have
focused our studies on the F. heteroclitus GtHs and their
role in controlling the cyclic reproductive activity charac-
teristic of this species and common to a large class of com-
mercially important species other than salmonids. These
efforts have included the development of a homologous
bioassay for gonadotropic hormones using oocyte matu-
ration and steroid production by isolated ovarian follicles
[22], the characterization of steroidogenic responses to an
F. heteroclitus pituitary extract (FPE) by the ovarian follicle
[23-26]; specifically, the identification of 17a-
hydroxy,20p-dihydroprogesterone (DHP) as the matura-
tion-inducing substance (MIS) for F. heteroclitus upon
stimulation by gonadotropin [27], and the cloning and
sequencing of the two B-subunits of the GtHs from a
cDNA library [8]. The a-subunit, shared by the two GtHs
and thyrotropin hormone (TSH), has also been
sequenced from the same cDNA library [28]. More
recently, we reported the preparation and use of specific
antibodies to the two B-subunits ofF. heteroclitus GtHs [9].
Each antibody recognizes a different subset of pituitary
cells in the central (GtH I) and peripheral (GtH II) proxi-
mal pars distalis, respectively, regions that display the typ-
ical tinctorial properties of gonadotrops. Similarly,
Shimizu and Yamashita [10] also reported the prepara-
tion of antibodies against the a-subunit and the two P-
subunits, and the purification ofF. heteroclitus GtHs using
an immunochemical assay. Thus, the molecular and
immunochemical data available for F. heteroclitus show
the presence of two distinct GtHs; however, a detailed
analysis on the duality of GtH function is still lacking in
this and other fractional spawners.

To further investigate the possible differential biological
activities of the two distinct GtHs in F. heteroclitus may
require the use of specific homologous bioassays. The
intention of the present study was to discern any discrete
differences in terms of maturational and steroidogenic
activities of the two gonadotropins (GtH I and GtH II)
shown to be present in F. heteroclitus. To accomplish this
objective, we carried out the purification and characteriza-
tion of GtH activities from Fundulus pituitary by fast pro-
tein liquid chromatography (FPLC) as determined by
homologous bioassays.

Materials and methods
Animals and chemicals
Animals were collected from salt marshes in the Matanzas
River, St. Augustine, Florida. Routine husbandry proce-
dures were used to maintain the fish in the laboratory:
captured fish (average weight = 7 g) were kept in a temper-
ature-regulated (25 C) running sea water aquarium with
a controlled photoperiod (14L: 10D) and were fed with an
enriched diet (mixture of boiled chicken eggs and dry
flake food). Under this regimen, the laboratory-main-
tained fish had responsive ovarian follicles and active
pituitaries throughout the year [29]. In effect, the ovarian
follicles retrieved from these fish were responsive to pitu-
itary extract stimulation, and underwent oocyte matura-
tion normally in vitro. In addition, the pituitary glands
from these laboratory fish also retained high gonado-
tropic potencies even outside of the normal breeding sea-
son. The care and use of, as well as all procedures
involving, animals have been approved by Barry Univer-
sity's Institutional Animal Care and Use Committee
(IACUC), in accordance with the guidelines of the IACUC
of the National Institutes of Health (NIH).

Absolute grade (NH4)2SO4 was obtained from Research
Plus, Inc. All other chemicals were obtained from Sigma
unless specified otherwise.

Preparation of Fundulus pituitary extract (FPE)
Pituitaries from sexually mature fish {gonadosomatic
index (GSI) = gonad weight eviscerated body weight x
100; GSI = ~10% for female and ~5% for male} were iso-
lated immediately after decapitating the animals and
stored frozen at -80 C (for up to one year) before the
extraction procedure. The pituitary glands were collected
from animals used in this and other studies in the labora-
tory over a period of three years. The pituitaries (0.2 mg
wet wt per gland; 40 to 500 glands) were homogenized
with a Teflon-coated pestle at 4 C in the starting buffer
used for each chromatographic run. Homogenates were
then centrifuged (13,000 g) for 1 h at 40C and the super-
natants were collected. The pellets were extracted once
more with the starting buffer and recentrifuged. Supema-
tants from both extracts were combined to provide FPE
and filtered through a 0.2-gm Nylon centrifugal microfil-
ter (Centrex, Schleicher & Schuell) before injection onto
chromatographic columns.

Pituitaries from both male and female fish were used. Due
to a report of sexual differences in GtH [30], pituitaries
from both sexes were not mixed. However, numerous
experiments in our lab indicated that similar chromato-
grams are obtained from pituitary extracts of either sex
(data not shown), so an indication of sex for the sample
source is not provided.

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Reproductive Biology and Endocrinology 2004, 2

Size-exclusion chromatography (SEC) was carried out
using two columns (Pharmacia Superose 12 HR 10/30
and Superose 6 HR 10/30) connected in series on a Phar-
macia FPLC system equipped with absorbance monitors
at 214 and 280 nm (HR 10 flow cells). The columns were
equilibrated with 50 mM NH4HCO3, pH 7.5, before the
application of FPE. The same buffer was used to elute the
sample isocratically with a flow rate of 0.5 ml/min. One-
ml fractions were collected and evaporated to dryness by
vacuum centrifugation (Savant SpeedVac Concentrator).

For anion-exchange chromatography (AEC), the column
(Pharmacia Mono Q HR 5/5) was initially equilibrated
with 50 mM Tris-HC1 buffer (pH 7.5). FPE was applied to
the column and unadsorbed proteins were allowed to per-
colate through the column using the same starting buffer.
Elution of the adsorbed sample was then carried out with
a linear, increasing gradient of NaCl (0 0.33 M) in 50
mM Tris-HC1, pH 7.5. The flow rate was 1.0 ml/min, the
fraction size was 2.0 ml/tube, and eluted fractions were
dialyzed overnight at 40C against distilled water.

The starting buffer for hydrophobic-interaction chroma-
tography (HIC) was 1.2 M (NH4)2SO4 in 0.1 M sodium
phosphate, pH 6.8. The hydrophobic-interaction column
(Bio-Rad Bio-Gel TSK Phenyl-5-PW, 7.5 x 75 mm) was
equilibrated with starting buffer before the application of
FPE and a linear gradient of decreasing (NH4)2SO4 con-
centration was begun after the unadsorbed protein eluted
from the column. Eluted fractions of 1.0 ml were collected
at a flow rate of 0.5 ml/min and dialyzed against distilled

For chromatofocusing, the column (Pharmacia Mono P
HR 5/20) was initially equilibrated with 25 mM Bis-Tris-
HC1, pH 6.3. After application of FPE, elution was carried
out with 40 ml 10% Polybuffer 74 (Pharmacia), pH 4.0,
and this was followed by a 1.0-M NaCl wash to elute the
remaining adsorbed protein. Fractions of 2.0 ml were col-
lected at a flow rate of 1.0 ml/min. Eluted fractions were
pooled into five samples (I-V) and each sample was
adjusted to 1.2 M (NH4)2SO4 and rechromatographed on
the hydrophobic-interaction column. Eluted fractions
were dialyzed against distilled water. All dialyzed samples
were evaporated to dryness by vacuum centrifugation,
tightly sealed, and stored at 4 C for up to a month prior
to reconstitution.

Dried fractions were reconstituted with 75% L-15 and
measured for gonadotropic activities by RIA determina-
tions of the amounts of 17a-hydroxy,20p-dihydroproges-
terone (DHP), testosterone (T), and 17B-estradiol (E2)
generated after follicle culture at 25 C for 24 h and by

scoring cultured follicles for oocyte maturation [germinal
vesicle breakdown (GVBD)] after 48 hr as previously
described [22]. For each bioassay, prematurational ovar-
ian follicles (1.2 1.4 mm in diameter) were pooled from
10 to 20 animals.

Each of the following chromatographic procedures has
been performed at least ten times. Representative chroma-
tographs are illustrated in the following results.

Size-exclusion chromatography (SEC)
SEC yielded several adsorbance peaks that were not well
separated from each other (Fig. 1A). Oocyte maturation
bioassays of the eluted fractions indicated a single peak
with a maximum activity of 80% GVBD (similar to the
maximum response of positive controls to unfractionated
FPE) (Fig. 1B). Steroidogenic activities were also meas-
ured by RIA and were found to elute in a similar position
(corresponding to an Mr of approx. 30,000) (Fig. 1C);
maximum activities were also comparable to those stimu-
lated by unfractionated FPE. The peak ofT and DHP pro-
duction coincided with the peak of oocyte maturation
activity, although steroidogenic activity in earlier eluting
fractions were indicated for both DHP- and E2-produc-
tion. None of the activities indicated in Figures 1B and 1C
corresponded precisely with the peaks provided by the
adsorbance trace (Fig. 1A).

Anion-exchange chromatography (AEC)
AEC of FPE provided a complex elution profile (Fig. 2A).
Bioassays for oocyte maturation also indicated that activ-
ity was distributed throughout the eluted fractions (Fig.
2B), as was also the case for the various steroidogenic
activities (Fig. 2C). Nevertheless, well defined peaks of E2
and DHP-promoting activity were eluted at NaCl concen-
trations of about 0.15 and 0.23 M, respectively (Fig. 2C).
No steroidogenic activities were detected before the start
of the NaCl gradient. Similarly, a 1.0-M NaCl wash eluted
little additional activity from the anion-exchange column.

Hydrophobic-interaction chromatography (HIC)
HIC of FPE also yielded numerous UV-adsorbing compo-
nents (Fig. 3A). Bioassays for oocyte maturation indicated
that activity was restricted to fractions eluting near the end
of the gradient (Fig. 3B). No steroidogenic activities were
detected in unabsorbed material (before the start of the
gradient) or during the early part of the chromatographic
process, but were also confined to the second half of the
gradient [(NH4)2SO4 concentration < 0.6 M] (Fig. 3C).
Two distinct peaks of E2-producing activities were
detected at an (NH4)2SO4 concentration of about 0.31 M
and 0.17 M (peaks a and b, respectively) and heterogene-
ity was not particularly evident. T-promoting activity was
broadly associated with both peaks, while DHP-promot-

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Reproductive Biology and Endocrinology 2004, 2

Table I: Maturation-inducing and steroidogenic activities in active fractions derived from a combination of chromatofocusing followed
by HICa

pg per well



GVBD (%)

38 7
40 5
12 12
29 8
26 15

5,850 + 2,000
4,450 + 2,000
3,150 1,204
3,300 + 450
2,120 + 701
4,483 + 393


264 48
240 56
147 53
145 14
60 60
271 23

X 100


332 68
312 48
126 18
139 49
43 15
339 91



I Results presented as means SEM derived from three chromatographic series. In the absence of added gonadotropin, no GVBD took place and T-
and DHP-production were undetectable, while E2-production averaged 1,150 pg per well. The latter baseline value was subtracted from the induced
E2 data to provide the indicated values for E2. b Thawed, immediately added to L- 15 medium, and tested for activity at a concentration of 0.5 pit.
equiv./well (0.25 pit. equiv./ml). cThawed, stored for up to a week at 4C, dialyzed overnight against distilled water, dried by vacuum centrifugation,
reconstituted with L- 15 medium, and tested for activity at a concentration of 0.5 pit. equiv./well. d Roman numerals refer to portions of the
chromatofocusing runs (Fig. 4) while Arabic numerals refer to active fractions found after subsequent HIC (Fig. 5).

ing activity co-eluted primarily with peak b. It is impor-
tant to note that this DHP-promoting activity also
coincided with the peak of oocyte maturation (Fig. 3B).

Chromatofocusing of FPE with a decreasing pH gradient
gave a complex but reproducible elution profile of 280-
nm adsorbing material followed by a major protein peak
eluted with the high salt wash (Fig. 4). The presence of
Polybuffer in the eluant precluded adsorptivity measure-
ments at 214 nm; it also proved to be toxic to cultured fol-
licles so that bioassays could not be performed directly on
eluant fractions. Polybuffer was therefore removed by
rechromatographing five major portions (indicated as I to
V in Fig. 4) on a Phenyl-5-PW column (Fig. 5 I-V). The
Polybuffer, which was not adsorbed on the Phenyl-5-PW
column, eluted at the beginning of each chromatogram.
Various fractions from each hydrophobic-interaction run
(indicated as 1 to 19 in Fig. 5) were then tested for ster-
oidogenic activity and ability to induce maturation, and
positive results were obtained for four fractions (indicated
by shaded bars in Fig. 5). This exercise was performed
three times and the pooled results for the active fractions
together with appropriate controls are provided in Table
1. These latter data indicate that the GtHs in FPE survive
the manipulative procedures associated with chromato-
graphic fractionation, dialysis, vacuum centrifugation and
reconstitution for up to a week at 40C (perhaps a slight
loss in E2-promoting activity is suggested). Among the
four active fractions (1-1, 11-8, III-10, V-19) eluted chroma-
tographically (Fig. 5, Table 1), fraction III-10 had rela-
tively poor DHP-promoting and maturation-inducing
activities but had E2-promoting activity, while fraction V-
19 appeared enriched in DHP-promoting activity. In gen-

eral, T-promoting activity tended to elute with DHP-pro-
moting activity (Table 1).

GtH bioassay
Fundulus pituitary extract (FPE) was fractionated by vari-
ous biocompatible liquid chromatographic procedures in
an attempt to purify the GtHs and characterize their bio-
logical activities. Chromatographic fractions were tested
for their gonadotropic activities by using a homologous
bioassay system which utilized intact F. heteroclitus ovar-
ian follicles in vitro [22]. Two indicators of gonadotropic
activity were employed, one being the ability of the frac-
tions to stimulate prematurational oocytes (1.2-1.4 mm
in diameter), which are arrested at prophase I of meiosis,
to resume the meiotic process by undergoing GVBD. The
other indication of gonadotropic activity was the ability of
the eluted fractions to stimulate the ovarian follicles to
produce three reproductively important steroid hormones
(DHP, T, and E).

This homologous bioassay system has been extensively
verified and shown to be sensitive and specific for F. hete-
roclitus GtHs [22]. It thus avoided pitfalls that may have
arisen using a heterologous bioassay system [31].
Although a report has appeared that prolactin and growth
hormone stimulate ovarian steroidogenesis when injected
into F. heteroclitus [32], no biological activity was detected
for either hormone using our in vitro bioassay system
(data not shown). Another advantage of our homologous
bioassay system was that many fractions could be assayed
simultaneously with a large number of appropriate-sized
follicles that can be pooled from several fish and rand-
omized, thus eliminating most of the between-animal

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Reproductive Biology and Endocrinology 2004, 2

1.6 C
1 2


20 30
Effluent Volume (ml)

Figure I
Size-exclusion chromatography (SEC) of FPE. Approximately
78 pituitary equivalents in a volume of 200 [il were applied to
the Superose columns and the effluent was monitored for
UV-absorbance (A). One-ml fractions were collected and
tested for (B) maturational and (C) steroidogenic activity.
For this and subsequent figures, negative controls (no gona-
dotropic extracts or fractions added) had 0-5% GVBD, DHP
and T secretion were not detectable, and E2 secretion aver-
aged less than 0.5 ng/2-ml well. For this experiment only,
positive controls (FPE added at a concentration of 0.5 pit.
equiv./well) were 65% GVBD with DHP-, T-, and E2-secre-
tion of 1.25, 0.75, and 7.4 ng/well, respectively.

variation in the procedure. To further minimize the inher-
ent variation in the responsiveness of ovarian follicles to
gonadotropin and to assure the availability of large num-
bers of sensitive follicles and active pituitary glands
throughout the year, we also developed a routine hus-
bandry procedure to maintain a large population of repro-
ductively healthy F. heteroclitus in the laboratory [29].
With this husbandry procedure, we were able to proceed
with the FPE fractionation and to carry out homologous
bioassays throughout the year.

Chromatographic resolution of gonadotropic activity
Size-exclusion chromatography (SEC) produced several
UV-absorbing peaks, none of which correlated precisely
with biological activity found around 30 kDa (Fig. 1).
Active fractions induced oocyte maturation in high fre-
quency and stimulated the production of all three steroids
tested (DHP, T, and E2), with estradiol being predominant
(as was the case for unfractionated FPE). The size ofF. het-
eroclitus GtHs thus approximates previous lower estimates
(ranging in molecular weight from 25 to 62 kDa) made by
SEC for fully glycosylated, nondenatured teleost GtHs
[5,7,30,33-36]. Recently, a molecular weight of about 40
kDa (gel filtration) was reported for F. heteroclitus (Arasaki
strain, Japan) GtH I and II [10]. The large variation in
these molecular weights may be population-specific or
due to the presence of aggregates or to differences in the
methods used for their estimation.

After anion-exchange chromatography (AEC) (Fig. 2A),
maturational activity was found to be spread throughout
the chromatogram (Fig. 2B). Similarly, steroidogenic
activities for all three steroids tested were distributed
among the various eluted fractions, although very distinct
peaks of E2- and DHP-activity were discerned at NaCl con-
centrations of 0.15 and 0.24 M, respectively (Fig. 2C).
Hence at least two different GtHs, one with predomi-
nantly E2-producing activity and the other with relatively
high DHP-producing activity, could be distinguished by
the steroidogenic bioassay data. However, the appearance
of all types of gonadotropic activity throughout the AEC
profile implies the presence of charge-heterogeneity in the
F. heteroclitus GtHs. Such heterogeneity has previously
been well documented for a variety of teleost species [37-
39] as well as other vertebrates [40-45]. Charge-heteroge-
neity in GtHs has been previously explained by
differences in sialic acid content [46] or by post-transla-
tional modifications of amino acid residues [43], but the
precise differences between various isohormones are not
well understood. In most cases, however, electrostatically
distinct GtHs of the same type (i.e., FSH-like or LH-like)
have been considered to be qualitatively identical in bio-
logical action.

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.o0 A





oS (

L Sample applied

Reproductive Biology and Endocrinology 2004, 2

0 10

, B

10 20 30 40

0.6- C ;

0.4- 04

0.2 '

c--- - - - - 4

0 10 20 30 40
Effluent Volume (ml)


.3 ""

2 "

Figure 2
Anion-exchange chromatography (AEC) of FPE. Approximately 40 pituitaries were extracted twice with 0.5 ml 50 mM Tris-
HCI (pH 7.5) and the extracts were combined, centrifugally filtered, and applied to a Mono Q column via the 10-ml Superloop.
The effluent was monitored for UV-absorbance (A) and a gradient of NaCI was applied after non-adsorbing material percolated
through the column. Two-ml fractions were collected and tested for (B) maturational and (C) steroidogenic activity. For this
experiment only, positive controls (FPE added at a concentration of 0.5 pit. equiv./well) were 43% GVBD with DHP-, T-, and
E2-secretion of 0.68, 0.54, and 3.4 ng/well, respectively.

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I: 0



io 0.25


S I I I I F r I I

Reproductive Biology and Endocrinology 2004, 2


008 T
0.06 2

- 0.04 c

tSomple applied









0 0


0 10
Effluent Volume (ml

20 30 40

Figure 3
Hydrophobic-interaction chromatography (HIC) of FPE. Approximately 56 pituitaries were extracted twice with 0.5 ml 1.2 M
(NH4)2SO4 in 0. M sodium phosphate buffer (pH 6.8) and the extracts were combined, centrifugally filtered, and applied to a
Phenyl-5-PW column via the 10-ml Superloop. The effluent was monitored for UV-absorbance (A) and a linear gradient of
decreasing (NH4)2SO4 concentration was started after the unadsorbed protein eluted from the column. Fractions of 1.0 ml
were collected and tested for (B) maturational and (C) steroidogenic activity. Two peaks of E2-generating activity are indicated
(a, b). For this experiment only, positive controls (FPE added at a concentration of 0.5 pit. equiv./well) were 50% GVBD with
DHP-, T-, and E2-secretion of 1.3, 0.8, and 7.4 ng/well, respectively.

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Reproductive Biology and Endocrinology 2004, 2


0 10 20 30 40 50

L Gradient started

Effluent Volume (ml)

Figure 4
Chromatofocusing of FPE. Approximately 500 pituitaries were extracted twice with 0.5 ml 25 mM Bis-Tris-CI (pH 6.3) and the
extracts were combined, centrifugally filtered, and applied to a Mono P column via the I ml Superloop. After the absorbance
recording returned to the baseline (tracing not shown; no gonadotropic activity was present in collected fractions), 10% Poly-
buffer 74 was pumped onto the column via the 50ml Superloop and this was followed by a salt wash. The effluent was moni-
tored for absorbance at 280 nm. Two-ml fractions were collected and measured for pH. Several fractions (indicated as I-V)
were also pooled and subjected to further hydrophobic-interaction chromatography.

Gonadotropic fractions relatively rich in either E2- or
DHP-promoting activity were also discerned either by
hydrophobic-interaction (HIC) elutingg at -0.31 and-
0.17 M (NH4)2SO4, respectively; Fig. 3C) or chromatofo-
cusing followed by HIC (in order to remove Polybuffer).
Fractions relatively enriched in E2- and DHP-promoting
activity were found to elute from the chromatofocusing
column at pH 4.5 and after the salt wash, respectively, and
these activities subsequently eluted from the Phenyl-5-PW
column at (NH4)2SO4 concentrations of -0.34 M and -
0.12 M, respectively (Figs. 4, 5; Table 1). Thus a consistent
elution pattern by HIC is indicated. The need to elute
DHP-promoting activity from the chromatofocusing col-

umn with a salt wash is also consistent with its relatively
late elution during AEC (Fig. 2C).

Based on previous reports that employed similar proce-
dures, it would appear that the gonadotropic activity puri-
fled by Swanson et al. [33] from coho salmon pituitaries
primarily corresponds to those fractions described here
that lack preferential steroidogenic activity (i.e., Table 1,
fractions I-1 and II-8), while the gonadotropin described
by Copeland and Thomas [34] for Atlantic croaker is sim-
ilar to our DHP-promoting gonadotropin (Table 1, frac-
tion V-19). None of the fractions we have analyzed from
any single chromatographic run have yielded single,

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Reproductive Biology and Endocrinology 2004, 2

2 3 4





02- -



I" --o03

o C

( 02

o -
n |






13 14 -004



10 20 30 40 0

10 20 30 40




0 ,
| oh O|

Effluent Volume (ml)



S 02-


12 0.04


-0.02 w

yI 001

Effluent Volume (ml)

Figure 5
Mono-P fractions #1-V (containing Polybuffer) were readjusted with start buffer [1.2 M (NH4)2SO4 in 0. M sodium phosphate,
pH 6.8]. Each individual Mono-P fraction was rechromatographed on a Phenyl-5-PW column. A linear gradient of decreasing
salt concentration was started after the Polybuffer eluted. Shaded areas indicate fractions containing gonadotropic activities
(see Table I).

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Reproductive Biology and Endocrinology 2004, 2

silver-stained bands at the expected size on electro-
phoretic gels (data not shown). This may indicate that the
amount of GtH protein present in the chromatographic
fractions was below the detection level (<5 ng) of silver
staining [47]. On the other hand, the homologous oocyte
maturation and steroid production assays used in this
study are far more sensitive in discerning biological

A series of articles have attested to the duality of the GtHs
in teleosts [7-10,12,19,48]. These studies provided ample
biochemical and immunological evidence that there are
two chemically distinct GtHs that reside in separate pitui-
tary gonadotrops and have different ontogenies. Unfortu-
nately, attempts to distinguish different steroidogenic
activities between GtH I and GtH II have been less defini-
tive because biological activities of GtHs overlap consider-
ably. Although GtH II seems to be more potent than GtH
I in stimulating DHP-production by salmon ovarian folli-
cles, no significant difference in E2-steroidogenic activity
has been found between GtH I and GtH II [7,48-50]. As a
corollary, therefore, GtH I appear to preferentially pro-
mote E2-production in salmonids. Since E2 and DHP are
primarily involved in vitellogenic processes and the
resumption ofmeiotic maturation, respectively, GtH I and
GtH II would seem to correspond to the vitellogenesis-
promoting "carbohydrate-poor" and maturation-promot-
ing "carbohydrate-rich" GtHs described by Idler and his
colleagues for salmon and flounder [51-54]. Based on
this, our own results for F. heteroclitus appear to indicate
that GtH I-like and GtH II-like activities reside in fractions
III-10 and V-19, respectively, derived from chromatofo-
cusing of FPE (Table 1). Similar activities were found in
fractions obtained by AEC (Fig. 2C) and HIC (Fig. 3C).

The chromatographic separation of the GtHs evaluated by
bioassay cannot directly discern which fraction is GtH I
and GtH II, and even though there is an overlap in the
steroidogenic activity, the homologous bioassay data
clearly distinguish two well defined peaks of different bio-
logical activities. Additionally, our HIC profiles as deter-
mined by the specific biological activities (oocyte
maturation and steroidogenesis) are in agreement with
those reported by Shimizu and Yamashita [10] who iden-
tified the F. heteroclitus GtH I and GtH II using an immu-
nochemical analysis. We conclude, therefore, that at least
two gonadotropic activities are present in F. heteroclitus
pituitaries: an E2-promoting gonadotropin (GtH I-like)
and a DHP-promoting gonadotropin (GtH II-like) which
has a lower isoelectric point but greater hydrophobicity
than the former. Consistent with our previous findings
that DHP is the maturation-inducing steroid in F. hetero-
clitus [27] the DHP-promoting gonadotropin is also asso-
ciated with high maturation-inducing activity (Fig. 3).
Taken together, the homologous bioassay data presented

here, and our previous immunochemical study indicating
that each of the two GtHs could be released selectively [9],
help to substantiate the duality of GtH function in a tele-
ost which is a multiple spawner.

Using homologous bioassay, at least two quantitatively
different gonadotropic (steroidogenic) activities: an E-
promoting gonadotropin (GtH I-like) and a DHP-pro-
moting gonadotropin (GtH II-like), which has a lower
isoelectric point but greater hydrophobicity than the
former, can be distinguished from F. heteroclitus pituitaries
by a variety of chromatographic procedures. This study
complements previous biochemical and molecular data
in F. heteroclitus and substantiates the duality of GtH func-
tion in a multiple spawner.

This study was supported by NSF grant No. DCB-8819005 awarded to
RAW, by NSF grant No. DBI-01 1608 awarded to TRP, and by NIH-MBRS
SCORE Grant GM45455-08 awarded to Y-WPL. The authors wish to thank
Lynn Milstead (Whitney Lab) and Linda Cahill (Barry CELT) for their assist-
ance with the figures and Scott van Arnam for the collection of animals.

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