Fish Physiology and Biochemistry 20: 143–154, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

143

Effects of accelerated photoperiod regimes on the reproductive cycle of the

female rainbow trout: II Seasonal variations of plasma gonadotropins
(GTH I and GTH II) levels correlated with ovarian follicle growth and egg
size

E. Bon1,2 , B. Breton3 , M. S. Govoroun3,4 and F. Le Menn1

1 Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des Fac-
ultés, F-33405 Talence Cedex, France (Fax: 33 0 556 999 060; E-mail: elizabeth.bon@ibgc.4-bordeaux2.fr);
2 Salmonidés d’Aquitaine, Route de Taller, F-40260 Castets, France; 3 INRA, Laboratoire de Physiologie des

Poissons, Campus de Beaulieu, F-35042 Rennes Cedex, France; 4 Institute of Agricultural Biotechnology of Russia,
Moscow, Russia

Accepted: June 11, 1998

Key words: biological clock, oogenesis, vitellogenesis

Abstract
Female rainbow trout were exposed over their second reproductive cycle to a simulated natural photoperiod (control
group) and to two accelerated photoperiod regimes (S9 and S6 groups). Early spawning was achieved in both
accelerated groups, coupled, however, with a reduction of mean egg size. To investigate this reduction of egg size,
circulating levels of GTHs and two indices of ovarian growth (gonadosomatic index and follicle diameter) were
regularly measured in association with histological studies of structural changes in ovarian follicles. Regardless of
the photoperiod regime, the seasonal profile of plasma GTH I levels appeared to be multiphasic. The successive
transient elevations in GTH I levels appeared to be connected with the initiation of ovarian growth and vitellogene-
sis and also, with the synchronization of the late stages of maturation and ovulation. In contrast, the seasonal profile
of plasma GTH II levels was monophasic, with a single peak at ovulation, confirming that GTH II is not associated
with ovarian growth but promotes gamete maturation and release. Our results demonstrate that the reduction of
egg size cannot be due to a deficient secretion of GTH I in plasma, since GTH I levels were much higher during
vitellogenesis in both accelerated groups, but rather to an alteration of ovarian follicle growth during the late stages
of vitellogenesis. Finally, the early and middle stages of ovarian growth appeared to be photosensitive periods,
whereas the late stages were less so, and appeared to be controlled rather by an endogenous biological clock
synchronized by the photoperiod.

Introduction ponent, referred to as biological clock, underlying this

dependence on light (Duston and Bromage 1986). For
commercial trout farms, this natural seasonality of re-
In rainbow trout, as in many other teleost species,
production involves considerable production and man-
reproduction is an annual event primarily regulated
agement difficulties. To minimise these constraints,
by the seasonal waxing and waning of daylength
photoperiodic manipulation of the reproductive season
(Bromage and Cumaranatunga 1988). Long days are
has been developed over the last decade (Breton et al.
necessary for the initiation of vitellogenesis and sper-
1985; Bromage et al. 1992).
matogenesis, whereas short days, or the absence of
In teleost fishes, the influence of photoperiod on
long days, may cue the later dynamics of gametogene-
reproduction is mediated by the hormones of the cen-
sis and maturation (Bromage et al. 1984; Breton et al.
tral nervous system (Breton et al. 1985; Bromage and
1985). There is however, a strong endogenous com-
144

Cumaranatunga 1988). Among these, GTH I and GTH

II are the two pituitary gonadotropins involved in the
endocrine control of gametogenesis in salmonid fishes
(Suzuki et al. 1988a; Swanson et al. 1991; Govoroun
et al. 1997). The development of radio-immunoassays
(RIAs) to measure GTH levels in both plasma and
the pituitary gland (Suzuki et al. 1988b; Prat et al.
1996), of radioreceptor assays to characterize the go-
nadal GTH receptors (Breton and Sambroni 1989; Yan
et al. 1991), and also, of tests to investigate the bio-
logical activity of GTHs (Swanson et al. 1991), has
provided in the last decade new knowledge on the
physiology of both GTHs in salmonids. The emerg-
ing picture is that GTH I and GTH II are structurally
and probably biologically homologous to the tetrapod
Follicle-Stimulating Hormone (FSH) and Luteinizing Figure 1. Photoperiod regimes. N: Photoperiod regime simulat-
Hormone (LH) (Suzuki et al. 1988a). It has been sug- ing natural conditions (control group). S9 and S6: Accelerated
gested that as FSH, GTH I may regulate the initiation photoperiod regimes.
and the stimulation of gametogenesis, whereas as LH,
GTH II may only promote final maturation and gamete
that exposure of rainbow trout to accelerated seasonal
release (Slater et al. 1994; Prat et al. 1996).
light cycles induces precocious gonadal development,
However, the physiological role of both GTHs
but also produces smaller eggs (Duston and Bromage
in ovarian development and in vitellogenesis has re-
1986; Bon et al. 1997). Although neither egg quality
mained unclear in salmonid fishes, and particularly
nor the potential growth of fry are altered (Bromage
rainbow trout. Indeed, whereas a considerable amount
and Cumaranatunga 1988), the practice of buying
of data is available on plasma sex steroids in conjuc-
by volume and not by number of eggs makes them
tion with the structural changes in ovarian develop-
commercially unacceptable. To determine whether the
ment (van Bohemen and Lambert 1981; Duston et al.
smaller egg size under these breeding conditions may
1987; Bromage and Cumaranatunga 1988), there is a
result from a modification of the secretion process
paucity of information on plasma GTHs measured by
of GTHs in plasma, the effects of shortening the re-
sensitive homologous assays. Similarly, very little is
productive cycle by specific light regimes on plasma
known about the effects of photoperiodic manipula-
GTHs levels were also examined in combination with
tion on the secretion of both GTHs in plasma, and on
structural changes in ovarian development.
the consecutive structural changes in ovarian develop-
ment. To date, only preliminary results indicating that
constant long photoperiods could increase GTH I lev- Materials and methods
els during vitellogenesis have been reported (Davies
et al. 1995). At present no data are available concern- Maintenance of fish and photoperiod conditions
ing the effects of total or partial natural photoperiod
simulation on GTH I and II secretion in plasma, and Three hundred and sixty 2-year-old adult female rain-
on ovarian growth. bow trout (Oncorhynchus mykiss) with an average
In this study, a highly specific and sensitive ho- body weight of 1.46 ± 0.16 kg, were randomly se-
mologous RIA for rainbow trout GTH I and GTH II, lected the day of their first spawning. They were
recently developed by Govoroun et al. (1998), was divided into three groups of 120 fish, and 10 per
used to monitor changes in the circulating levels of group were individually identified by a number tag
these hormones in adult female rainbow trout exposed attached through the base of the dorsal fin. Fish were
to a natural-simulated photoperiod. To investigate the reared under constant temperature (8.5 ± 0.5 ◦ C) in
physiological role of both GTHs, histological observa- 4 m3 covered tanks continuously supplied with fresh
tions on structural changes in ovaries were performed, spring water. They were fed twice daily with a pel-
and correlated for the first time with the seasonal pro- leted broodstock diet (Trouvit, Trouw France), which
files of both GTHs in plasma. It has been demonstrated was distributed at a rate of 1% body-weight per day

over the first 3 months following spawning, then 0.7%
over the next 2 months, and finally 0.3% up to the next
spawning.
All groups were maintained under artificial pho-
toperiod by a 75-W incandescent bulb providing ap-
proximately 950 lux. Daylength was automatically
controled by 24 h electronic time clocks, and the
switch-off time was manually adjusted each week ac-
cording to the prescribed schedule (Figure 1). Females
from the control group (N) were subjected in February,
two months after spawning, to the photoperiod regime
they would normally receive in their natural environ-
ment. In the two other groups (S9 and S6), females
were subjected to accelerated photoperiod regimes
which were obtained by an abrupt increase in light
hours per day from the previous 8L:16D to 16L:8D,
in December, immediately after spawning in the S6
group, and in February, two months after spawning
in the S9 group. Afterwards, daily light was progres-
sively shortened from 16L:8D to 8L:16D, exactly as in
the controls (Figure 1).
Biological sample collection
Twice a month until spawning, the 10 tagged fe-
males from each group were anesthetized with 2-
phenoxyethanol (0.03%), weighed and blood sampled.
The blood was taken from a caudal vessel using a
heparinized syringe, and collected into heparinized
microtubes refrigerated on ice. Blood samples were
centrifuged (2000 g, 10 min, 4 ◦ C) and plasma was
collected, frozen at −80 ◦ C, then stored at −30 ◦ C.
As the spawning times of each group approached, the
tagged females were examined every two days for the
presence of ovulated eggs. The eggs were removed
from each mature female by stripping, and the di-
ameter of 20 to 30 eggs per female was immediately
measured under a binocular microscope to determine
mean egg size.
Also twice a month until spawning, 10 non-tagged
females per group were randomly selected, anes-
thetized, weighed and sacrificed. The ovaries were
removed from the body cavity and weighed to de-
termine the gonadosomatic index [GSI = (ovaries wt
/ body wt) × 100]. Portions of ovaries were taken,
weighed, and placed in Gilson’s fluid (Simpson 1951),
at 1 part ovary to 2 parts fixative (v/v), to separate fol-
licles from the ovarian stroma. After dissociation, the
diameters (D, expressed in mm) of approximately 100
follicles were measured under a binocular microscope,
considering that only the follicles measuring 0.4 mm
145
and above in diameter were retained. This critical size
indeed corresponds to the smallest follicles exhibiting
the first signs of vitellogenesis (Hyllner et al. 1994).
The mean follicle volume (V = 1/6 ×(πxD3 ), ex-
pressed in mm3 ), and the mean follicle growth rate
(GR = 1V /1T , expressed in mm3 per month) were
then estimated. All follicle diameters were multiplied
by 1.1 before calculations, to adjust for a 10% shrink-
age in size due to the fixing process as recommended
by Tyler et al. (1990).
Histological stages of ovarian follicle growth
Portions of gonads were taken from sacrificed fish and
placed for 72 h in Bouin-Hollande sublimate solution
for fixation. Gonad portions were embedded in paraf-
fin, serially sectioned (7 µm), and stained with Mas-
son’s trichrome (Martoja and Martoja-Pierson 1967).
Follicle diameters were determined using a micro-
scope equipped with an ocular micrometer. Follicle
growth was classified into different phases according
to structural changes:
– Oogonia (Figure 2a), follicles undergoing pre-
vitellogenesis, including those in the chromatin nucle-
olar stage (Figure 2b) and those in the Balbiani body
stage (Figure 2c), as well as follicles undergoing mat-
uration events (Figure 2h), were identified based on
Bromage and Cumaranatunga (1988).
– Based on our observations, follicles undergoing
vitellogenesis were classified into two physiological
stages which differ from the ’endogenous’ and ’exoge-
nous’ vitellogenesis stages reported in other studies
(Zohar et al. 1986; Bromage and Cumaranatunga
1988). Follicles undergoing Type I vitellogenesis (Fig-
ure 2d) were characterized by the presence of cortical
alveoli and lipid globules in the ooplasm, as well as
by numerous nucleoli located in the concavities of the
wrinkles of the nuclear envelope. Follicles undergo-
ing Type II vitellogenesis were characterized by not
only the presence of cortical alveoli and lipid glob-
ules in the ooplasm, but also by the concomittant
accumulation in the ooplasm periphery of yolk glob-
ules. The other change characterizing this stage was
the visualization of a glycoproteic matrix, the zona
radiata, located between the granulosa layer and the
oolemma (Figure 2f). This stage was sub-divided into
two sub-stages. During early Type II vitellogenesis
(Figure 2e), there is coincidence of endocytotic and
synthetic events. During intensive Type II vitellogen-
esis (Figure 2g), endocytotic events greatly predom-
inate compared with synthetic events, leading to the
146

Figure 2. Photomicrographs of 7 µm sections of rainbow trout ovarian follicles stained with Masson’s Trichrome. (a) Oogonial nest; (b) and
(c) follicles undergoing previtellogenesis; (d) follicle undergoing Type I vitellogenesis; (e) follicle undergoing early Type II vitellogenesis; (f)
details of membranes of follicles in early Type II vitellogenesis: thecal layer (Th), granulosa (Gr), zona radiata (Zr), yolk granule (Y) and lipid
globules (L); (g) follicle undergoing intensive Type II vitellogenesis; (h) follicle undergoing maturation events.
 147

accumulation of a central yolk mass, and to the dis-

placement to the ooplasm periphery of the cortical
alveoli and also of the lipid globules which fuse and
thus gain size.

Radio-immunoassays (RIA) for rainbow trout GTH I

and GTH II

Plasma GTH I and GTH II levels were evaluated using

the highly specific and sensitive RIAs recently devel-
oped in the rainbow trout by Govoroun et al. (1998).
In the GTH II assay, there was no cross-reactivity with
any other hormone preparation, and a sensitivity of
0.1 to 0.2 ng ml−1 . In the GTH I assay, there was a
cross-reactivity of only 3.7% with GTH II, and a sen-
sitivity of 0.5 to 1 ng ml−1 . Before being assayed,
plasma samples were half-diluted in the RIA buffer
(0.05 Tris-HCl buffer pH 7.6, 1% BSA, 0.1% sodium
azide, 0.1% Triton X-100). All assays were validated
for use by confirming that serial dilutions of rainbow
trout plasma were parallel to the standard curves.
Mode of data expression and statistical tests
All data are expressed as mean ±standard error (m
± SEM). A probability level of p ≤ 0.05 was taken
to indicate a statistically significant difference. Com-
parison of means within groups was made using the
Wilcoxon-Mann-Whitney test or the Wilcoxon test
(Scherrer 1984), depending on whether the data was
obtained on sacrificed fish (GSI, oocyte diameter)
or on regularly sampled fish (GTHs), respectively.
However, to determine the occurrence of significant
seasonal changes in plasma GTH I levels during ga-
metogenesis, the general mean (GM) of GTH I levels
was calculated as well as the associated intervals of
confidence (± IC). In this case, the hypothesis of a
significant difference was retained when bimonthly
GTH I values (m), also referred to as ‘snapshot’ mean,
were outside the area encompassed between the max-
imum (GM + IC) and minimum (GM − IC) curves
(Scherrer 1984). To examine the effects of photoperi-
odic treatments, comparison of means among groups
was analyzed using the Kruskal–Wallis test (Scherrer
1984). When significant differences between groups
were found, a multiple-comparison test was used to
identify which groups were statistically different.
Figure 3. Effects of accelerated photoperiod regimes on the sea-
sonal profile of the gonadosomatic index [GSI = (ovary wt /body
wt) × 100], and on the time of spawning ’S’. Each value is ex-
pressed as the mean (point) and the standard error (bar). ∗p <0.05,
∗∗p <0.01: significance of the difference from controls. Based
on the seasonnal profile of GSI in controls (N), distinct phases of
gonad development can be distinguished: VSD = very slow ovarian
development, SD = slow ovarian development, RD = rapid ovarian
development, M = final maturation, Pov = post-ovulatory period.
Results
I. Gonadal development
(a) Gonads growth
In the control group (N), the reproductive cycle was
divided into different phases based on the seasonal
profile of ovarian mass growth expressed through GSI
variations (Figure 3). From December to May, GSI
remained at a minimum (0.5 ± 0.07%) defining the
very slow ovarian development phase (VSD). During
this period, ovaries predominantly contained oogonia
undergoing mitotic division and follicles undergoing
previtellogenesis, but also some follicles undergoing
Type I and early Type II vitellogenesis. From May to
early September, GSI increased steadily to reach ap-
proximately 6.0% (i.e., the slow development phase,
SD), and ovaries contained mainly follicles in early
Type II vitellogenesis as well as several follicles in
previtellogenesis and in Type I vitellogenesis. After a
marked pause for two weeks at around 6%, GSI rose
sharply from mid-September to mid-November to el-
evated values (15.3 ±0.6%), characterizing the rapid
148

development phase (RD). During RD, ovaries not only

contained follicles undergoing intensive Type II vitel-
logenesis but also several oogonial nests. From mid-
November to late-November, GSI values increased
slightly, reaching a maximum value of 16.5 ± 0.5%
when fish entered the spawning period (i.e., matura-
tion phase, M). At this time, ovaries contained mainly
follicles undergoing maturation events as well as some
grouped oogonia. The post-ovulatory phase (Pov), in
December, was characterized by an abrupt decrease
in GSI which returned to basal values. Here, ovaries
contained mainly oogonial nests and previtellogeneic
follicles, but also some follicles in Type I and early
Type II vitellogenesis.
In the accelerated groups (S9 and S6), and regard-
less of the photoperiod regime, the seasonal profile of
GSI showed a similar pattern with a plateau occurring
when GSI reached approximately 6%, at the limit be-
tween SD and RD. However, at spawning, the GSI
only reached 14.4 ± 0.3% and 10.2 ± 0.6% in the S9
and S6 groups, respectively. This reduction in GSI was
significant in the S6 group (p = 0.0008) but at the limit
of significance (p > 0.05) in the S9 group, compared
to controls. In the S9 group, the duration of VSD was
around 30% shorter than in controls, whereas SD re-
mained nearly the same. In the S6 group, the duration
of VSD was around 60% shorter than in the controls,
and the duration of SD nearly 50% shorter. In contrast,
the duration RD and M remained unchanged in both
accelerated groups.

(b) Kinetics of growth of follicles undergoing

vitellogenesis
In controls (N), the mean follicle volume slightly in-
creased from around 0.03 to 0.20 mm3 (0.4 to 0.7 mm
in diameter) during VSD, whereas GSI remained at a
minimum. Subsequently, the variations of the mean
follicle size (Figure 4a) mirrored exactly those of GSI
(Figure 3). Volume then gradually increased up to
around 15 mm3 (3.05 ± 0.1 mm in diameter) during
SD, paused for two weeks at this value, correspond-
Figure 4. (a) Effects of accelerated photoperiod regimes on the sea-
ing to the same plateau observed for GSI values, and sonal profile of follicle volume and on egg size. Mean value (point)
rose sharply during RD to around 49.3 mm3 (4.55 ± and standard error (bar). ∗p <0.05, ∗∗p <0.01: significance of the
0.1 mm in diameter). At the same time, the growth difference from controls. S: time of spawning (for more details see
legend of Figure 3); (b) Effects of accelerated photoperiod regimes
rate (Figure 4b) also reached a maximum of around
on the growth rate of follicles undergoing vitellogenesis (0.4 mm in
23 mm3 per month. During M, the follicle diameter diameter and above). Mean follicle growth rate is expressed in mm3
slightly increased to a maximum of 4.66 ± 0.09 mm per month. SD = slow ovarian development, RD = rapid ovarian
at spawning. development.
In the accelerated groups (S9 and S6), the follicle
growth rate (Figure 4b) was higher (+20% and +60%,
respectively) than in controls during SD. Regardless
 149

of the photoperiod regime, a significant plateau al-

ways occurred at the limit between late SD and RD
(Figure 4a). At this time, the size reached by the de-
veloping follicles was similar to controls in the S9
group (p > 0.05), but significantly lower in the S6
group (at around 10 mm3 or 2.7 mm in diameter, p
= 0.01). Compared to controls, the follicle growth
rate was lower in the S9 and S6 groups (−30% and
−60%, respectively) during RD, leading to smaller
egg size (4.39 ± 0.06 mm and 3.76 ± 0.10 mm in
diameter, respectively) at spawning. A reduction in
the variability of mean vitellogenic follicle size was
observed between females in both accelerated groups
(i.e. a decrease in SEMs).

II. Seasonal profile of plasma gonadotropins

(a) GTH I
In the control group (N, Figure 5a), successive signifi-
cant (p < 0.05) transient elevations in GTH I levels,
termed waves, were observed all along the gonadal
development. A first elevation in GTH I levels (wave
1) occurred nine months before spawning, at the be-
ginning of VSD. During this period, plasma GTH
I levels rapidly increased from 3.9 ± 0.1 ng ml−1
in mid-January to 12.1 ± 0.14 ng ml−1 in Febru-
ary, whereas GSI remained at a minimum (Figure 3).
Thereafter, plasma GTH I decreased and stabilized
at around 7 ng/ml until the end of VSD in May. A
second elevation (wave 2) in GTH I levels was ob-
served five months before spawning, during early SD,
simultaneously to the first significant increase in GSI
values (Figure 3). At this time, GTH I levels slowly
but steadily increased up to 10.4 ± 0.29 ng ml−1
in June-July. Subsequently, GTH I levels returned to
basal levels (around 7 ng ml−1 ) even though the GSI
continued to increase. A third elevation in plasma
GTH I levels (wave 3) was also observed approx-
imately three months before spawning, at the limit
between late SD and RD. During this period, plasma
concentrations of GTH I increased markedly to peak at
around 15.4 ng ml−1 in late October, one month before
spawning, in parallel to the rapid increase in GSI (Fig- Figure 5. Seasonal changes in plasma GTH I levels of female rain-
ure 3). At this time, a great variability between females bow trout undergoing (a) a 12 month simulated natural reproductive
cycle (control group N), (b) a reproductive cycle shortened to 9
was observed with respect to the GTH I levels which, months (group S9), and (c) a reproductive cycle shortened to 6
depending on females, ranged from 2 to 37 ng ml−1 . months (group S6). Each GTH I value is expressed as the mean
Thereafter, GTH I levels rapidly decreased to stabilize (point) and the standard error (bar). The general mean of GTH I
at around 8 ng ml−1 until the end of RD. The last levels during gametogenesis (GM) and the associated confidence
intervals (IC) are estimated at a probability level of p < 0.05 (∗).
elevation in plasma GTH I levels (wave 4) occurred The significance of GTH I waves was retained when the monthly
around ovulation, in late November, when GSI was at values were outside the area between the GM ± IC curves (for more
a maximum. Wave 4 peaked at around 29 ng ml−1 but details see legend of Figure 3). The time of spawning is indicated
by an ’S’.
150

depending on fish, GTH I levels varied between 11 and

54 ng ml−1 . After spawning, during Pov, GTH I levels
rapidly returned to basal values.
In the accelerated S9 group (Figure 5b), the num-
ber of waves in plasma GTH I levels was the same
as controls observed during gonadal growth. How-
ever, their chronological occurrence was accelerated,
and several significant differences were observed with
respect to the GTH I levels reached. The general
mean of GTH I levels was significantly higher (GM =
17.3 ng ml−1 ) than controls (GM = 9.45 ng ml−1 ).
Wave 2 amplitude (19.5 ± 3.3 ng ml−1 ) was twice
that of controls, as was the case for wave 3 (32.3
± 3.9 ng ml−1 ) which was coupled with a reduc-
tion in the variability of individual GTH I levels (12
to 79 ng ml−1 ). Compared to controls, wave 4 re-
mained nearly unchanged both in its amplitude (28.2
± 2.3 ng ml−1 , p > 0.05) and its occurrence.
In the accelerated S6 group (Figure 5c), the general
mean of plasma GTH I levels was much higher (GM
Figure 6. Seasonal profile of plasma GTH II levels in both natural
= 22.7 ng ml−1 ) than in controls and S9 group. How- and accelerated groups. Each value is expressed as the mean (point)
ever, shortening the reproductive cycle to 6 months led and the standard error (bar). The time of spawning is indicated by
to the disappearence of the series of increasing plasma an ’S’ (for more details see legend of Figure 3).
GTH I waves 1 and 2, leaving only one wave over
the whole cycle with GTH I levels four times (63.6
controls (44.2 ± 3.5 ng ml−1 ), GTH II peak ampli-
± 9.0 ng ml−1 ) that of controls. Furthermore, this was
tude remained unchanged in S9 (49.2 ± 6.3 ng ml−1 ,
associated with a great reduction in the variability of
p > 0.05) but was significantly lower in S6 (22.1 ±
individual GTH I levels (30 to 97 ng ml−1 ). Compared
2.8 ng ml−1 , p = 0.001).
to controls, wave 4 amplitude was significantly lower
(p = 0.02) with GTH I levels down to a third (9.9 ±
1.5 ng ml−1 ).
Discussion
(b) GTH II Natural reproductive cycle
In the control group N (Figure 6), circulating levels
of GTH II were consistently detectable throughout Over the reproductive cycle, rainbow trout ovaries
most of the reproductive cycle (VSD, SD and RD grow from a threadlike structure (4 g) to a large struc-
phases) but quite low (between 0.14 ± 0.05 and 0.60 ± ture (over 300 g) representing around 18% of body
0.12 ng ml−1 ). They only started to increase in early weight. Until recently, most authors have used the
November and to around 5.9 ng/ml. During M, GTH stageing system for fish oocyte development, i.e. oo-
II levels rose sharply, reaching a maximum value of genesis, previtellogenesis, vitellogenesis, maturation
around 44.2 ± 3.5 ng ml−1 in late November around and ovulation, to characterize distinct physiological
ovulation. Thereafter, GTH II levels rapidly decreased periods of ovary development, considering that all
to basal levels which were reached less than one month follicles within an ovary were simultaneously at the
after spawning, at the end of Pov. same physiological stage of development (Bromage
In the accelerated groups (S9 and S6), the seasonal and Cumaranatunga 1988). Based on our observations
profile of GTH II was quite similar to that observed and on recent studies (Tyler et al. 1996), it is now
in controls. Regardless of the photoperiod regime, clear that different stages of follicle development co-
plasma GTH II remained at the limit of assay detection exist within ovaries during a same period in the course
and peaked only around ovulation. However, differ- of gonad growth. For this reason, an objective tempo-
ences were observed between groups with respect to ral division of the reproductive cycle was adopted in
the maximum GTH II levels reached. Compared to the present study. Exclusively based on the seasonal

profile of the gonadosomatic index (GSI) rather than
on the usual histological observation of follicle de-
velopment (Bromage and Cumaranatunga 1988), this
division reveals the existence of distinct periods such
as the very slow ovarian development phase (VSD),
the slow ovarian development phase (SD) and the
rapid ovarian development phase (RD). This proposed
temporal division of gametogenesis is not a new stage-
ing system for fish oocyte development, but simply
permits an objective and clear definition of the suc-
cessive physiological states of gonad development in
the female rainbow trout, as already reported by Bon
et al. (1997) and further discussed below.
This study is the first to relate the measurement
of both GTH levels in plasma, to histological obser-
vations of structural changes in ovaries. Taken as a
whole, the pattern of changes in plasma GTH levels
obtained in the present study is similar to those pre-
viously reported for these gonadotropin in the same
species (Prat et al. 1996) and in other salmonid species
(Haux et al. 1991; Swanson et al. 1991; Slater et al.
1994). However, the combination of very frequent
sampling, the particular mode of data analysis, and
above all, the enhanced GTH I assay sensitivity com-
pared to previous studies (Prat et al. 1996), provides
considerable new data on the mode of GTH I secretion,
as well as on the probable physiological role of this
hormone during gametogenesis. The data presented
demonstrates for the first time a multimodal seasonal
pattern for the plasma GTH I levels, with successive
transient elevations (GTH I waves), leading to a grad-
ual increase of the GTH I levels over the reproductive
cycle.
The first GTH I transient elevation (wave 1) was
registered 9 months prior to ovulation, during the
period of very slow ovarian growth (January–May,
VSD). Later in the period (i.e., April–May), whereas
ovarian growth was minor in terms of weight (GSI <
0.5%), several follicles (around 0.4 mm in diameter)
undergoing the first signs of Type I and early Type II
vitellogenesis were registered. This suggests that GTH
I wave 1 is responsible for the initiation of ovarian
recrudescence and for the selection of the season’s
batch of follicles undergoing vitellogenesis (i.e. the
initiation of the VTG uptake into follicles).
Successive GTH I waves of increasing amplitude
and frequency were detected later in the cycle, 5
months (wave 2) and 3 months (wave 3) prior to ovu-
lation, during the period of slow ovarian growth (SD,
May–September). At this time, ovarian growth was
due almost entirely to an increase in the size of the
151
follicles, and not to an increase in their number. This
resulted from active uptake of VTG, as can be seen
from the increasing proportion of follicles undergoing
Type II vitellogenesis. These observations suggest that
the prime function of GTH I waves 2 and 3 is to en-
hance vitellogenic growth. This is not surprising since
recent studies have demonstrated that GTH I is effi-
cient in stimulating the ovarian synthesis of E2 , the
main œstrogen involved in the hepatic synthesis of
VTG, as well as the uptake of VTG into follicles dur-
ing early to mid-vitellogenic development (Tyler et al.
1991).
Interestingly, the highest GTH I wave 3 lev-
els (around 15.4 ng ml−1 ) were registered in mid-
September, at the end of SD, when the seasonal
profile of both indices of ovarian growth exhibited a
two-week pause (GSI around 6.0% and follicle di-
ameter around 3.05 mm). This is the first time this
has been reported in fish. Although its significance
remains quite unclear, this phenomenon characterizes
a specific physiological state, such as a shift in the
dynamics of ovarian growth as well as in the hor-
monal and biochemical processes associated. Indeed,
whereas plasma levels of GTH I significantly declined
to a minimum during the next period (September to
early November), both indices of ovarian growth ex-
hibited a sharp increase (i.e., GSI around 15% and
follicle diameter around 4.5 mm), characterizing the
period of rapid ovarian growth (RD). Histological
studies demonstrated that most of this growth was due
to the very large amounts of VTG taken up by the
oocytes. According to Tyler et al. (1991), GTH I is
not involved in the enhancement of VTG uptake into
oocytes during the late stages of vitellogenesis. Our
observations confirm these results and permit us to as-
sume that GTH I is not involved in the enhancement of
gonad growth during the late stages of vitellogenesis.
Thereafter, plasma levels of GTH I rose again
(wave 4) during the peri-ovulatory period (early No-
vember to late November, M), simultaneously with
those of GTH II, leading to the highest plasma concen-
trations (ca. 30 and 44 ng ml−1 , respectively) around
ovulation, in late November. Histological studies in-
dicated during this period that ovaries predominantly
contained follicles undergoing maturation events, con-
firming that final maturation and ovulation are con-
nected with the periovulatory GTH II surge (Breton
et al. 1985; Prat et al. 1996). In contrast, the function
of GTH I wave 4 remains unclear. This is partly due to
the fact that the blood sampling around ovulation was
not frequent enough (every two days). Two hypotheses
152
were put forward: GTH I could potentiate the ovula-
tory action of GTH II; and/or GTH I wave 4 could be
involved in the initiation of the next cycle of ovarian
growth (i.e. ovarian recruitment). In the later stages of
gonad development, histological analyses have indeed
indicated the concomittant presence of both a group of
synchronously developing mature follicles which form
the season’s batch of eggs, and a population of primary
oocytes that have already begun to grow.
Previous articles have reported that ‘maturational’
GTH release in rainbow trout, consisted in daily
episodic pulses with varying amplitude and frequency
during the process of ovarian growth (Zohar et al.
1986). It now seems that they were more related to
GTH I, as the RIA for ‘maturational’ GTH used in
these earlier studies did not differentiate GTH I and
GTH II. Although our study was not designed to in-
vestigate the episodic nature of GTH I release, these
long-term variations (seasonal waves) in the GTH I
levels would seem to reflect significant modifications
in the pattern of possible short-term (daily pulse) pro-
files in GTH I levels. This may not be surprising since
daily cycles of various reproductive hormones have
already been reported in rainbow trout as well as in
other fish (Lamba et al. 1983; Zohar et al. 1986).
Accelerated reproductive cycles
In the present study, the length of the reproductive cy-
cle of female rainbow trout was shortened by 3 months
(S9 group) or 6 months (S6 group) in fish exposed
to accelerated photoperiod cycles, leading to earlier
spawning. These artificial photoperiod regimes were
obtained by modifying the increasing part of the daily
natural light cycle, and leaving the decreasing part un-
changed. This is different from the static light regimes
used in a preliminary study (Davies et al. 1995). As
mentioned in the introduction, one of the undesired
effects of shortening the reproductive cycle is a reduc-
tion in egg size. Our results confirm previous studies
(Duston and Bromage 1986) since egg size was re-
duced by around 0.3 mm (−6%) and 0.9 mm (−20%)
in the S9 and S6 groups, respectively. Measurement
of both indices of ovarian growth (i.e., GSI and folli-
cle diameter), and of circulating levels of both GTH I
and II provide a better understanding of the possible
physiological reason for smaller eggs.
The main effect of the photoperiodic manipulation
was to shorten the early and middle stages of gonad
development (i.e., VSD and SD). However, the data
presented clearly indicated that ovarian development
was much more rapid in both accelerated groups, due
to the fact that ovarian follicles grew at rates consid-
erably faster than normal. One possible explanation
is that follicles were exposed to considerably higher
plasma GTH I concentrations than would occur nor-
mally. Indeed, the chronological appearance of the
GTH I waves (1, 2, and 3) was accelerated, and their
amplitude increased, with plasma GTH I levels in the
S9 and S6 groups respectively twice and four times
higher, than in controls. This suggests a stimulatory
effect of accelerated light regimes on GTH I secretion
during the early and middle stages of ovarian growth.
The mechanism by which GTH I secretion influences
ovarian follicle growth remains unclear. The histolog-
ical analysis reveals an acceleration of the processes
of oogenesis and vitellogenesis, as well as an increase
in the number of follicles undergoing vitellogenesis,
in both accelerated groups. These observations sug-
gest a greater stimulation of vitellogenesis as well as
an enhancement of oocyte recruitment. In addition,
a better synchronisation of individual GTH I levels
occurred in both accelerated groups, coupled with a
better synchronization in follicle size. Collectively,
this data supports that GTH I probably plays a similar
role to that of FSH in mammals (Slater et al. 1994; Prat
et al. 1996), i.e., it affects recruitment of follicles into
vitellogenesis and their subsequent growth during the
early and middle stages of vitellogenic development.
However, several modifications of the seasonal profile
of plasma GTH I were registered in both accelerated
groups, notably the disappearence of the GTH I wave
1 and 2, and their presumable fusion with the GTH I
wave 3 to form a single GTH I wave. Recent studies
on the same species have shown that modifications in
photoperiod regimes may produce clear differences in
the patterns of secretion of lipids in plasma (Bon et al.
1997). All this data tends to demonstrate that during
the early and middle stages of ovarian growth, the
photoperiod is the main modulating factor acting on
the biochemical processes involved in reproduction.
In contrast, neither the duration of the late stages
of ovarian growth (i.e., RD and M), nor the general
profile of both indices of ovarian growth (GSI and
follicle diameter) nor that of the GTH levels, were
affected by photoperiod changes. This suggests that
at least during the late stages of ovarian growth, the
biochemical processes involved in reproduction are
controlled by an endogenous biological clock which is
synchronized by the seasonally changing photoperiod.
For example, the RD phase never took place before
the decreasing photoperiod reached 10L:14D. This is

not surprising since evidence has been provided in
the female rainbow trout for an endogenous biological
clock controlling the circannual variations of lipopro-
teins (Wallaert and Babin 1994), steroids (Duston and
Bromage 1987), lipids and vitellogenin (Bon et al.
1997). However, compared to controls, the dynam-
ics of ovarian growth seems to be somewhat altered
during the period of rapid ovarian growth (RD). Thus,
whereas GTH I levels remained considerably higher
than would occur normally at the same period, the
follicle diameter grew less rapidly in both accelerated
groups, leading to smaller follicle size and GSI value
two weeks before ovulation. This data confirms that
GTH I is not involved in the control of follicle growth
during the later stages of vitellogenesis (Tyler et al.
1991). In addition, whereas the pattern of secretion of
GTH I and II tended to be similar to controls in the S9
group at ovulation, it was significantly different in the
S6 group with plasma levels at only a third of controls.
This suggests an alteration of GTH I and II secretion
during the period of final maturation (M). From these
results, it is concluded that the endogenous biological
clock is not able to compensate for the biochemical
alterations induced by photoperiod changes during the
early stages of ovarian growth, leading to smaller egg
sizes at ovulation.
Conclusion
It is clear that GTH I, like FSH in tetrapods, regulates
the initiation and the stimulation of gametogenesis
during the early and middle stages of the cycle at least,
whereas GTH II, like LH, only promotes final matura-
tion and gamete release. However, the physiological
role of GTH I around ovulation remains quite unclear,
and further investigation is necessary. The shortening
of the reproductive cycle by partly modifying the daily
light regimes demonstrates that the early and middle
stages of the cycle are photosentive phases, whereas
the late stages of the cycle are governed by an endoge-
nous biological clock. It appears that the reduction in
egg size cannot be explained by a deficient secretion
of GTH I in plasma, but by an alteration of ovarian
follicle growth in the later stages of the reproductive
cycle. Additional studies have already been carried out
to verify the possible alteration of vitellogenin produc-
tion and/or an alteration of its incorporation into the
growing oocytes (to be published).
153
Acknowledgements
This work was financially supported by a scientific
research grant from the French government (ANRT:
Association Nationale de la Recherche et de la Tech-
nique, and ANVAR: Association Nationale pour la
Valorisation de la Recherche) and from the private
fishfarm ‘Salmonidés d’Aquitaine’. We wish to thank
U. Barbe, D. Brajat and A. Bawala from our labora-
tory at the University of Bordeaux I, for their technical
assistance. Thanks also to D. Florin from the Insti-
tut Universitaire de Technologie, Bordeaux, for her
valuable help in statistical analysis. We are also very
grateful to the staff of the private fishfarm ‘Pisciculture
des Fontaines’ for their assistance.
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