# Short-term profiles of plasma gonadotropin and estradiol-17β levels in the female rainbow trout, from early ovarian recrudescence and throughout vitellogenesis

Yonathan Zohar, Bernard Breton, Alexis Fostier

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Yonathan Zohar, Bernard Breton, Alexis Fostier. Short-term profiles of plasma gonadotropin and estradiol-17β levels in the female rainbow trout, from early ovarian recrudescence and throughout vitellogenesis. General and Comparative Endocrinology, 1986, 64 (2), pp.172-188. 10.1016/0016-6480(86)90002-X. hal-01600969

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# Short-Term Profiles of Plasma Gonadotropin and Estradiol-17β
Levels in the Female Rainbow Trout, from Early Ovarian
Recrudescence and throughout Vitellogenesis

Y. ZOHAR,<fnref n="1" /> B. BRETON, AND A. FOSTIER

Laboratoire de Physiologie de Poissons, INRA, Campus de Beaulieu, 35042 Rennes, France

Accepted May 12, 1986

At various stages throughout the annual reproductive cycle, female rainbow trout (*Salmo gairdneri*) were fitted with a catheter in the dorsal aorta. They were bled via the catheter at frequencies of once every 30, 60, or 240 min over periods of 5 to 24 hr. Gonadotropin (GtH), estradiol-17β (*E*₂₁₇β), and estrone levels were measured in the plasma samples. At early ovarian recrudescence (March), short-term (1-2 hr), high-amplitude (ΔGtH = up to 100 ng/ml), episodic pulses of GtH were recorded in the plasma of 12 of the 26 studied fish. In the others, GtH levels remained low and constant. No synchronization was found among the individual GtH profiles. *E*₂₁₇β levels in the same fish were low and constant while estrone was not detectable. At early stages of exogenous vitellogenesis (June), plasma GtH (1-3 ng/ml) and *E*₂₁₇β (0.5-1.5 ng/ml) levels were low and constant. At advanced stages of exogenous vitellogenesis (September-October), fluctuating GtH levels were found again in most of the females; basal GtH concentrations were only slightly higher than those recorded in June. The fluctuations consisted of short-term (1-3 hr) random GtH pulses of moderate amplitude (ΔGtH = up to 5 ng/ml), occurring at a relatively high frequency (up to 5 per 12 hr). Although no regular synchronous daily pattern of GtH was noted, most of the GtH pulses were observed during the photophase and early scotophase. The appearance of GtH pulsatility during exogenous vitellogenesis was accompanied by a large increase in plasma *E*₂₁₇β up to levels ranging from 6 to 30 ng/ml. In contrast to the GtH profiles, the *E*₂₁₇β profiles showed continuous and progressive variations, superimposed on the abrupt GtH pulses, and a high degree of synchronization. *E*₂₁₇β levels increased during the photophase and reached maxima toward and during early scotophase. © 1986 Academic Press, Inc.

The seasonal variations in plasma gonad-
otropin (GtH) and sex-steroid levels were
thoroughly studied in the female trout
(Salmo gairdneri). A careful examination
of the various studies reveals some discrep-
ancies amongst them. A transient increase
in GtH levels has been observed by some
authors at early stages of ovarian develop-
ment (spring) in the rainbow (Breton et al.,
1985) and brown (Billard et al., 1978;
Breton et al., 1983) trout, whereas others
have reported constant or only slightly
varying levels of the hormone (rainbow

trout: Scott and Sumpter, 1983; Sumpter *et al.*, 1984). At the same time, estradiol-17β (*E*₂₁₇β) levels have been reported as low and constant (rainbow trout: Lambert *et al.*, 1978; Whitehead *et al.*, 1978a, b; Scott *et al.*, 1980; Van Bohemen and Lambert, 1981) or increasing steadily (brown trout: Billard *et al.*, 1978; Breton *et al.*, 1983; rainbow trout: Sumpter *et al.*, 1984). During vitellogenesis plasma GtH levels have been reported as either (a) increasing moderately (brown trout: Breton *et al.*, 1983; Billard *et al.*, 1978; Crim and Idler, 1978), (b) remaining constant (rainbow trout: Scott and Sumpter, 1983), or (c) initially increasing and then declining continuously (rainbow trout: Bromage *et al.*, 1982a, b; Whitehead *et al.*, 1983). Plasma

<footnote n="1">¹ Present address for reprint requests: National Center for Mariculture, Israel Oceanographic and Limnological Research Institute, P.O. Box 1212, 88112 Elat, Israel.</footnote>
levels of E₂₁7β and testosterone rise considerably during vitellogenesis and decline progressively from its final stages (brown trout: Billard *et al.*, 1978; Breton *et al.*, 1983; rainbow trout: Fostier *et al.*, 1978; Scott and Sumpter, 1983; Scott *et al.*, 1980; Bromage *et al.*, 1982a, b).

One possible explanation for the above-mentioned discrepancies might be the fact that the long term (seasonal) profiles only partially reflect the hormonal function. In fact, in the majority of the mammalian species studied in this respect, the fundamental signals composing the hormonal message are generally of very short duration, i.e., from a few minutes up to a few hours (see review in Knobil, 1980; Lincoln and Short, 1980; Brinkley, 1981; Desjardins, 1981). Similarly, daily variations in the circulating levels of the reproductive hormones have been reported in different fish species. Daily cycles in plasma GtH levels have been demonstrated in the female goldfish, *Carassius auratus* (Breton *et al.*, 1972; Hontela and Peter, 1978, 1980a; Vodicnik *et al.*, 1978; Gillet and Billard, 1981; Gillet *et al.*, 1981) and in the male and female of the common carp, *Cyprinus carpio*; the silver carp, *Hypophthalmichthys molitrix*; and the grass carp, *Ctenopharyngodon idellus* (Pan *et al.*, 1980). Circulating levels of testosterone, E₂₁7β, and estrone have been shown to fluctuate during the day in the catfish, *Heteropneustes fossilis* (Lamba *et al.*, 1983). In salmonids, data on circadian changes in the levels of reproductive hormones are rare. Our previous reports (Zohar, 1980; Zohar *et al.*, 1982a) have presented some data showing that in the female rainbow trout, daily (short-term) fluctuations in plasma GtH levels do occur and are superimposed on the seasonal variations of the hormone.

The aim of the present work was to study, at some characteristic stages of the reproductive cycle of the female rainbow trout, the short-term (circadian and ultra-

dian) profiles of plasma GtH and main sex-steroid levels. Since our previous data (Zohar, 1980; Zohar *et al.*, 1982a) had indicated a lack of synchronization between fish in the GtH secretion pattern, we studied hormonal profiles of individual free-swimming trout, fitted with a catheter in the dorsal aorta. The present paper describes short-term profiles of GtH and E₂₁7β from early ovarian recrudescence and throughout vitellogenesis, whereas our following one (Zohar *et al.*, 1986) describes short-term profiles of GtH and 17α-hydroxy, 20β-dihydroprogesterone at the peri-ovulatory period.

## MATERIALS AND METHODS

### (a) Animals and Stocking Conditions

Female trout, aged 3 to 4 years and weighing 1.5 to 3 kg, were used in the present study. All of them had reached sexual maturity at least once before. About 2 to 3 months before the beginning of the acclimation to experimental conditions, fish were transferred from farm to indoor tanks supplied with recycled water. Then, 3 to 4 weeks before the initiation of the experiments the fish were transferred into tanks (70-150 liters) in which they were kept individually. Fish were exposed to a natural photoperiod. Water temperature was maintained at 10 ± 2° between December and May and at 15 ± 2° between June and November. Fish were fed with artificial food granules at a daily ratio of 1% of their body weight. Feeding time was between 1000 and 1200 hr.

### (b) Dorsal Aorta Catheterization

At various times of each year over a 3-year period, catheters were implanted in the dorsal aortas of 10-20 female trout. Only fish which had showed active feeding behavior for at least 1 week were catheterized. The procedure for implanting a catheter in the dorsal aorta has been described in detail by Zohar (1980). The same study (Zohar, 1980) and Bry and Zohar (1980) showed that the resumption of normal feeding behavior after catheterization reflected the recovery of fish from the stress situation related to the surgery. In such fish, the surgery and the presence of the catheter in the dorsal aorta did not affect gonadotropin levels in the blood or ovarian function (Zohar, 1980).

### (c) Blood Sampling

Before catheterization, 0.3-0.5 ml of blood was removed from the caudal vasculature of every fish. Hor-
mone levels in these samples were compared to those recorded in blood sampled successively via the dorsal aortic catheter during experimentation, as described by Zohar (1980). After catheterization, fish were allowed to recover for at least 3 days before further bleeding.

In agreement with our previous findings (Zohar, 1980; Bry and Zohar, 1980), only fish which resumed normal feeding behavior after surgery were selected for experimentation. Individual free-swimming cathe-terized trout were bled repeatedly via the catheter at frequencies of once ever ½, 1, or 4 hr over periods of 5 to 24 hr. In order to bleed the fish, the cannula, its stopper (a scaled syringe needle), and its cork floater were gently removed from the water. The cannula was unplugged and fitted with a 1-ml syringe. A volume of 250 µl, corresponding to 50 µl more than the dead volume of the catheter, was removed and discarded. The cannula tip was then fitted with a clean 1-ml syringe previously rinsed with lithium heparinate solu-tion (100-150 iu of heparine in 0.7% of saline). A volume of 300 to 400 µl of blood was withdrawn and transferred to ice-cold 3-ml tubes. At the end of each sampling, the blood filling the catheter was pushed back by 200 µl of lithium-heparinate saline solution (as above) and the cannula was plugged. Bleeding during the dark period was made using a dim red light to which wavelength salmonids adapted to dark are not sensitive (Hanyu and Tamura, 1978). Blood samples were centrifuged (15 min at 5000 rpm and at 4°), and the plasma was stored at -30° until further analysis.

(d) Determination of Ovarian
Developmental Stages

At the end of each experiment, the females were sacrificed and their ovaries submitted to histological analysis. We distinguished three major ovarian devel-opmental stages (based on Van den Hurk and Peute, 1979):

1. *Early ovarian recrudescence* (March). Ovaries contained isolated or grouped oogonia, oogonia un-dergoing mitotic divisions, oocytes at different stages of the prophase of the first meiotic division, and dif-ferent proportions of oocytes undergoing previtello-genic growth, endogenous vitellogenesis, and the very beginnings of exogenous vitellogenesis. Previtello-genic oocytes (50 and 500 µm in diameter) were char-acterized by the presence of Balbiany bodies. Their nuclei were relatively big and many nucleoli were scattered at its periphery. Their follicular layers were not completely organized. Oocytes which undergo en-dogenous vitellogenesis (400-900 µm in diameter) were characterized by the successive appearance of two structures: the vitellin vesicles (the future cortical alveoli) which appeared first in the ooplasm periphery and spread later toward its center, and the lipid glob-ules which appeared in the perinuclear area, fused,

and gained size. The zona radiata and the follicular layers were already organized in such oocytes. Oo-cytes undergoing early exogenous vitellogenesis are described below.

2. *Early exogenous vitellogenesis (June)*. Ovaries contained mainly oocytes undergoing initial stages of exogenous vitellogenesis. Such oocytes (which mea-sured 1 to 2 mm in diameter) were characterized by the progressive appearance, in the periphery of the ooplasm, of vitellin granules.

3. *Advanced exogenous vitellogenesis (September-October)*. Ovaries contained mainly vitello-genic oocytes with diameters of up to 4-5 mm. They were characterized by the massive appearance of vi-tellin granules all over the ooplasm. These granules coalesce to form spheric structures (vitellin spheres) which occupy most of the oocyte volume.

(e) Hormone Measurements

Gonadotropin was measured by radioimmunoassay (RIA) according to Breton and Billard (1977). The antibody used was anti-trout GtH at a final dilution of 1:130,000 to 1:200,000. A highly purified salmon GtH (Breton *et al.*, 1978) was used for the standard curve and for the radioactive labeling. Each unknown sample was measured in triplicate. All samples from a single experiment were measured in the same RIA run. GtH concentration in most of the samples was measured twice, in two different RIA runs, in order to confirm the existence of fluctuations in the hormonal concentration and to eliminate any error due to the measuring technique (see also Zohar, 1980).

The sensitivity of the RIA varied between 15 and 30 pg GtH per tube which corresponds to 0.3 and 0.6 ng GtH/ml of plasma. The intraassay variability (Table 1) was estimated by repetitive measurements, in each of the RIA runs, of the GtH concentrations in different pools of plasma. These pools were sampled from fish at different physiological stages and contained various GtH levels, covering the entire range of the standard curve.

The radioimmunoassays of estradiol-17β and e-strone were carried out according to Fostier *et al.* (1978), except that bound steroid was precipitated with polyethylene glycol (Fostier *et al.*, 1982). Both steroids were measured after being extracted from the plasma and separated from each other on an LH-20 column. Unknown samples were measured in tripli-cate. The estradiol-17β antibody was used at a final dilution of 1:50,000, and its degree of cross-reactivity with other steroids was 11% for estrone, 9% for 16-ke-toestradiol-17β, 8% for 16-epiestriol, and less than 0.5% for estriol, estradiol-17α, testosterone, 11-ke-totestosterone, and 17α-hydroxy,20β-dihydroprogesterone. The estrone antibody was used at a final dilu-tion of 1:4800, and its degree of cross-reactivity was less than 0.8% for estradiol-17β, estriol, testosterone,
<table><caption>TABLE 1
THE INTRAASSAY VARIABILITY OF THE RADIOIMMUNOASSAYS</caption><thead><tr><th>Hormone</th><th>Level (ng/mL)</th><th>Mean CV (%)</th><th>Range of CV (%)</th><th>Number of samples/assay</th><th>Number of assays</th></tr></thead><tbody><tr><td rowspan="6">GtH</td><td>1-3</td><td>9.3</td><td>6.0-10.9</td><td>5-7</td><td>7</td></tr><tr><td>4-6</td><td>7.7</td><td>4.1-12.3</td><td>5-7</td><td>7</td></tr><tr><td>7-12</td><td>9.9</td><td>7.3-11.9</td><td>5-7</td><td>9</td></tr><tr><td>14-20</td><td>6.6</td><td>4.0-9.9</td><td>5-7</td><td>8</td></tr><tr><td>40-50</td><td>6.8</td><td>2.3-8.8</td><td>5-6</td><td>5</td></tr><tr><td>105-125</td><td>4.9</td><td>3.6-6.9</td><td>5-7</td><td>5</td></tr><tr><td rowspan="4">Estradiol 17β</td><td>1-2</td><td>9.6</td><td>6.3-12.4</td><td>6</td><td>3</td></tr><tr><td>3-4</td><td>5.3</td><td>2.6-8.7</td><td>5</td><td>3</td></tr><tr><td>20-22</td><td>9.1</td><td>8.8-9.4</td><td>5</td><td>2</td></tr><tr><td>33-35</td><td>5.5</td><td>4.3-6.7</td><td>6-10</td><td>2</td></tr><tr><td>Estrone</td><td>1.5-2.0</td><td>10.5</td><td>9.6-11.4</td><td>5</td><td>2</td></tr></tbody></table>

11-ketotestosterone, and 17α-hydroxy,20β-dihydroprogesterone. The sensitivity of both radioimmunoassays was approximately 10 pg/tube, and the intraassay variability is given in Table 1.

### (f) Analytical Methods

The mean, coefficient of variation (CV) and variance were calculated for all the values composing each of the individual hormonal profiles. A second (hypothetical) variance was calculated for each profile, based on the highest CV obtained for a plasma pool (Table 1) having a hormone concentration close to the mean of the values composing the tested profile. This hypothetical variance (and CV) is thus the expected one, supposing that the observed hormonal fluctuations reflected only the variations of the RIA. The real and hypothetical variances were compared by $\chi^2$ test. On the basis of this comparison, all profiles having a real CV at least twice as large as the hypothetical one were considered as fluctuating significantly, due to physiological reasons. These fluctuating profiles showed a highly significant ($P < 0.005$) larger real CV than a hypothetical one. For the nonfluctuating profiles (real CV $\le 2 \times$ hypothetical CV), the mean of all values composing each of them was considered as the “basal level.”

We distinguished two types of fluctuating profiles: (1) Irregular profiles, showing random, episodic fluctuations of hormonal levels. In this group there was no synchronization between individual profiles of animals representing the same physiological stage; (2) Regular profiles, showing gradual and continuous changes in the hormonal levels. In this group there was a certain synchronization between profiles of individual animals. The two types of profiles were further analyzed by different methods.

(1) Irregular profiles. The hormonal concentrations composing each of the profiles were divided into two subgroups. The first one included the low concentra-

tions, representing the “baseline” levels or “inliers.” The second subgroup included the high hormonal concentrations, representing “pulsation” levels or “outliers.” The statistical detection of outliers was affected according to tests adapted for one (Dixon, 1953) or many (Grubbs, 1969) outliers. High hormonal levels which were characterized as “outliers” were then eliminated one by one, and the fluctuating nature of the resting values of the profile was tested each time as described above. This analysis was stopped when the CV of the resting values (“inliers”) became equal to or smaller than twice the hypothetical CV.

(2) Regular profiles. A global analysis of the profiles of all the animals representing a given ovarian developmental stage was realized by a two-factorial analysis of variance (the two factors analyzed were number of animals and sampling time). When significant differences were found, the analysis was continued by the Duncan test (Bliss, 1967). This enabled the identification of significant differences, during the experimental period, between groups of mean hormonal levels.

## RESULTS

### (a) Early Ovarian Recrudescence: March (Figs. 1 and 2)

In trout which were sampled every 4 hr for 24 hr, we observed in five of the nine fish studied significant short-term increases in plasma GtH levels (Fig. 1). In two of the females (Nos. 113 and 115), the amplitude of the increases reached around 100 and 50 ng/ml, respectively, whereas in the others the amplitude varied between 4 and 8 ng/ml. No synchronization was observed among the individual profiles. In
<figure><img src="image_2.png" /><figcaption>FIG. 1. Profiles of plasma levels of GtH (continuous lines) and E₂₁β (broken lines) in female trout at early ovarian recrudescence (March). Females were bled every 4 hr over a period of 24 hr. Individual GtH profiles with significant fluctuations in hormone levels, and the mean of profiles (n = 4 fish) with stable GtH levels, are presented. The number of each female is indicated. The statistical significance of the hormonal fluctuations is indicated by the following symbols: for GtH, Ω = nonsignificant and ⊥ = significant; for E₂₁β, ⧸ = nonsignificant and ⧹ = significant. The symbol * situated above a peak indicates that it is significantly higher than the basal level.</figcaption></figure>

four females, GtH levels remained constant. Four of the GtH peaks were observed in the middle or toward the end of the photophase, whereas another peak occurred during the scotophase. In the fish in which they were measured, estradiol-17β levels were found to be low and constant, whereas estrone levels were below the detection limits of the RIA (0.15 ng/ml).

Bleeding fish every hour for 12 hr, during the day (for one group) or throughout the night (for another), showed short-term increases (pulses) in plasma GtH levels in seven of the 17 sampled females (Fig. 2). In the other fish, GtH levels remained con-

stant. The amplitude of the GtH increases was relatively large in some of the cases, and ranged from 5 to about 100 ng/ml. In most of the females, a GtH pulse included only one point (sample) after which GtH returned to its basal level within 1 hr. In one female (No. 122), in which the GtH peak value was the highest (118 ng/ml), GtH returned to its basal level more gradually, through an intermediate value observed 1 h after the peak.

Although no synchronization was evident among individual profiles, six of eight significant GtH pulses occurred during the night, whereas the others occurred in the
<figure><img src="image_3.png" /><figcaption>FIG. 2. Profiles of plasma levels of GtH (continuous lines) and E₂₁₇β (broken lines) in female trout at early ovarian recrudescence (March). Females were bled every hour over a period of 12 hr, one group from 0900 to 2100 hr and a second group from 2100 to 0900 hr. For other details, see the legend of Fig. 1.</figcaption></figure>

late photophase (Fig. 2). In all the fish exhibiting GtH pulses, only one pulse was detected throughout the 12-hr sampling period, with the exception of one female, for which we observed two significant GtH elevations. In three females showing GtH pulses, we measured low and constant E₂₁₇β levels, whereas estrone was not detectable (Fig. 2, female Nos. 122-124).

(b) *Early Exogenous Vitellogenesis: June*
*(Fig. 3)*

At this stage of ovarian development, GtH levels were low (1-3 ng/ml) and relatively stable. When females were sampled once every 4 hr for 24 hr (Fig. 3a), we observed slight fluctuations in GtH levels in two of six fish, whereas in the others these
<figure><img src="image_4.png" /><figcaption>FIG. 3. Individual profiles of plasma levels of GtH (continuous lines) and E₂₁β (broken lines) in female trout at early exogenous vitellogenesis (June). Females were bled either every 4 hr over a period of 24 hr (a), or every hour over 9 hr (b). For other details, see the legend of Fig. 1.</figcaption></figure>

levels remained constant. In all females sampled every 1 hr for 10 hr (Fig. 3b), GtH levels were low and stable. When measured, E₂₁β levels were found to be low and constant (Fig. 3b).

### (c) Advanced Exogenous Vitellogenesis: September–October (Figs. 4–7)

Due to space limitations, we include here only a representative number of individual hormonal profiles out of nearly 50 made on fish at this stage of ovarian development.

Of the 47 analyzed individual GtH profiles of females undergoing advanced exogenous vitellogenesis, 29 exhibited significant daily fluctuations.

In Fig. 4 are shown individual GtH profiles of nine fish which were bled hourly for 9 hr. For two females (Nos. 145 and 147), two GtH profiles are shown which were determined in two separate RIA runs. Seven of the nine GtH profiles fluctuated significantly. In all these cases, one to two significant GtH pulses were found during the 9-hr sampling period.

In Fig. 5 are shown individual profiles of GtH, E₂₁β, and estrone in eight females which were bled once every hour for 12 hr. Six of the GtH profiles fluctuated significantly, whereas in another one (No. 155) fluctuations were very close to being significant. In four of the females, significant
<figure><img src="image_5.png" /><figcaption>FIG. 4. Individual profiles of plasma GtH levels in female trout at advanced exogenous vitellogenesis (September). Females were bled every hour over a period of 9 hr. The two curves in continuous and broken lines correspond, for the same female, to results obtained in two different runs of RIA. For other details, see the legend of Fig. 1.</figcaption></figure>

GtH pulses were recorded, whereas in another (No. 154) a long GtH elevation was observed. In both groups of profiles (Figs. 4 and 5), most of the GtH pulses included only one high GtH value (except in female No. 159).

No synchronization existed among the individual GtH profiles of fish bled hourly. However, we noted 13 significant GtH pulses in the eight females which were sampled between 0800 and 2000 hr (Fig. 5), and only six significant pulses in seven females sampled between 2000 and 0800 hr (individual curves not shown). The lack of synchronization between the individual GtH profiles is reflected by their mean profile (Fig. 6, low curve), which masks the existence of the GtH pulsatility and shows very constant GtH levels over the 24-hr sampling period. Figure 7 shows GtH profiles in eight females sampled once every 30 min for 5½ hours. In this case, significant fluctuations in GtH levels occurred in five of the females. Due to the more frequent

samplings, most of the elevations in GtH levels included at least two high GtH values.

The analysis of all GtH profiles which we recorded showed that when oocytes undergo advanced exogenous vitellogenesis, the dynamics of GtH secretion differs from that found at earlier stages of gonadal development. One to five GtH pulses occur over a period of 12 hr; their duration ranges from 1 to 3 hr and their amplitude reaches around 5 ng/ml.

As far as estradiol-17β is concerned, its mean level was much more elevated than at earlier stages of ovarian development (Fig. 6, Student *t* test, *P* < 0.01). The levels of this steroid fluctuated significantly during the day in eight of 11 studied females (Fig. 5 for the profiles of seven females sampled between 0800 and 2000 hr). The pattern of the individual E₂₁₇β profiles was different from that established for the GtH. E₂₁₇β levels did not change abruptly, but revealed continuous, relatively synchronized daily
<figure><img src="image_6.png" /><figcaption>FIG. 5. Individual profiles of plasma levels of GtH (continuous lines), E₂₁₇β (broken lines), and estrone (dotted lines) in female trout at advanced exogenous vitellogenesis (October). Females were bled every hour over a period of 12 hr. For other details, see the legend of Fig. 1.</figcaption></figure>

variations overriding the GtH pulses (Fig. 5). In the six fish showing fluctuating profiles out of the seven sampled from 0800 to 2000 hr, E₂₁₇β levels were lower in the first part of the photophase, increased progressively later on, and reached maxima during the later part of the photophase or at the beginning of the scotophase. In some of these females, E₂₁₇β tended to decrease after reaching maxima. This tendency continued in fish sampled hourly between 2000 and 0800 hr (curves not shown). The relative synchronization between the individual E₂₁₇β profiles is reflected in their mean profile (Fig. 6, upper curve), which shows a progressive, highly significant increase of E₂₁₇β during the day, from min-

imal levels at 0800 to 1000 hr up to maxima reached between 1500 and 1700 hr, followed by an initiation of a decline. Plasma E₂₁₇β profiles were recorded only in four females sampled between 2000 and 0800 hr. These profiles (not shown) exhibited relatively high individual variation, and the calculation of their average was thus meaningless. More fish should be studied in order to confirm the E₂₁₇β pattern during the nocturnal part of the 24 hr.

We measured estrone levels in five females (Fig. 5). Those levels were very low, close to the detection limit of the RIA. In some of the females (Nos. 156 and 160), the estrone profiles were parallel to those of E₂₁₇β.
<figure><img src="image_7.png" /></figure>

FIG. 6. Mean profiles (x ± tSE) of plasma levels of GtH (lower curves—24 hr) and of E₂₁₇β (upper curve—12 hr) in female trout at advanced exogenous vitellogenesis (October). The mean profiles were established from individual profiles of fish sampled every hour over a period of 12 hr (Fig. 5 for the group sampled from 0900 to 2100 hr). **Significantly different at *P* < 0.01.

DISCUSSION

Annual changes in GtH levels in the fe-
male rainbow trout have been described
previously (see introduction). The present
study together with our following one
(Zohar *et al.*, 1986) reveal that in addition
to the seasonal variations in the hormone
concentration, important modifications
occur in the pattern of the short-term pro-
files of GtH levels throughout oocyte de-
velopment.

A transient GtH elevation has previously
been observed in the circulation of the fe-
male rainbow and brown trout undergoing
ovarian recrudescence. This elevation oc-
curred either in spring (March) in fish ex-
posed to natural photoperiodic regime (Bil-
lard et al., 1978; Breton et al., 1983), or
earlier (between January and March) when
the photoperiodic regime was altered
(Breton et al., 1985). Our present data sug-
gest that this increase in GtH is the reflec-
tion of short-term, high-amplitude, episodic

elevations in the hormone level. The GtH
pulses observed in March were not
synchronized among the individual fish and
high GtH levels were recorded during no
more than 2 hr. Thus, a point sampling of a
group of fish undergoing early ovarian re-
crudescence (March), for the study of sea-
sonal GtH variations, might show a mean
elevated GtH level accompanied by high
individual variation, as was actually the
case (Breton *et al.*, 1983, 1985).

High-amplitude GtH pulses were ob-
served in only 50% of the fish studied in
March. This might be due to the short dura-
tion of the pulses and to their low fre-
quency (in most of the cases only one GtH
pulse was observed during the 12-hr sam-
pling period). However, the absence of
GtH pulses in half of the females should
also be considered together with the fact
that in June no more such GtH pulses were
observed, and with the findings of Breton
et al. (1983, 1985) concerning the transient
nature of the spring GtH surge. Such a con-
<figure><img src="image_8.png" /><figcaption>FIG. 7. Individual profiles of plasma GtH levels in female trout at advanced exogenous vitellogenesis (October). Females were bled every 30 min over a period of 5½ hr, between 1430 and 2000 hr. For other details, see the legend of Fig. 1.</figcaption></figure>

sideration might indicate that the period during which the high-amplitude GtH pulses occur in each female is short, and that they characterize a precise given physiological state. A low degree of physiological synchronization among the studied fish might explain the differences found in their individual GtH profiles.

According to Van den Hurk and Peute (1979) and Breton *et al.* (1983, 1985), exogenous vitellogenesis in the rainbow trout (as observed histologically) first occurs in May, 1 to 2 months following the spring GtH rise (March). However, a recent study of the same species (Sumpter *et al.*, 1984) has indicated that exogenous vitellogenesis can begin as early as March. Although Lambert *et al.* (1978), Whitehead *et al.* (1978a, b), Scott *et al.* (1980), and Van Bochemen and Lambert (1981) found very low and constant $E_{2}17\beta$ and vitellogenin levels in the female trout during spring, other studies have shown a slow increase in plasma $E_{2}17\beta$ levels starting in March (Billard *et al.*, 1978; Breton *et al.*, 1983) or

even earlier (Fostier and Le Bail, unpublished data; Sumpter *et al.*, 1984). Sumpter *et al.* (1984) reported an increase in circulating vitellogenin levels in the rainbow trout as early as January, and the beginning of its incorporation in late March. In the present study, the first signs of vitellogenin uptake were also histologically visible in the fish bled in March, in which the high-amplitude GtH pulses were found. These considerations indicate that the GtH pulses might play a role in the induction of vitellogenin uptake by the follicles. In fact, Breton and Derrien-Guimard (1983) have demonstrated that physiological GtH pulses stimulate vitellogenin incorporation into *in vitro* perfused oocytes of rainbow trout. However, our data, showing that $E_{2}17\beta$ levels in fish exhibiting high-amplitude GtH pulses were constant, do not necessarily mean that GtH pulses do not play a role in controlling the synthesis and/or secretion of $E_{2}17\beta$ from follicles at this time of the year. In a separate study, we have demonstrated that the administration of
physiological GtH pulses to in vitro peri-
fused ovarian fragments undergoing early
recrudescence stimulated their E<sub>2</sub>17β
output considerably (Zohar *et al.*, 1982b;
Zohar, Fostier, and Breton, unpublished
results). *In vivo*, the GtH pulses might
maintain constant E<sub>2</sub>17β levels over short
periods.

The exact role of the pituitary in the con-
trol of the different phases of oogenesis has
not yet been satisfactorily defined.
Whereas the previtellogenic growth of oot-
cytes seems to be independent of the pituita-
tary (Vivien, 1939; Barr, 1963; Yamazaki,
1965), it has been shown in various teleost
species that oogonial proliferation (Barr,
1963; Yamazaki, 1965) and vitellogenesis
are pituitary-dependent processes (Barr,
1963; Yamazaki, 1965; Sundararaj *et al*.,
1972; Khoo, 1979), the last one definitely
being controlled by gonadotropin (Hoar *et
al*., 1967; Hyder, 1972; Mackay, 1973;
Upadhyay *et al*., 1978). On the basis of
these observations, we cannot exclude the
possibility that the high-amplitude GtH
pulses found in March might also be related
to the numerous oogonial mitoses and oot-
cyte meioses, or to endogenous vitello-
genesis, which were observed in the
ovaries of the studied females.

In June, when most of the ovarian fol-
licles were in the early stages of exogenous
vitellogenesis, GtH (1-3 ng/ml) and E<sub>2</sub>17&beta;
(0.5-1.5 ng/ml) levels were low and con-
stant. In September-October, when exoge-
nous vitellogenesis was in its advanced
stages, plasma GtH levels fluctuated again.
In most of the studied females, episodic,
short-term GtH pulses of moderate ampli-
tude were recorded, while basal GtH levels
(2.4 &plusmn; 0.6 ng/ml) were only slightly higher
than those recorded in June (1.8 &plusmn; 0.4
ng/ml). The appearance of GtH pulsatility
during exogenous vitellogenesis was ac-
companied by a large increase of plasma
E<sub>2</sub>17&beta;, up to levels ranging from 6 to 30
ng/ml. At advanced exogenous vitello-

genesis, individual E<sub>2</sub>17β profiles showed
continuous and progressive variations su-
perimposed on the abrupt GtH fluctua-
tions. In contrast with the GtH profiles, a
relative synchronization existed among the
individual E<sub>2</sub>17β profiles. This was re-
flected by an average E<sub>2</sub>17β profile
showing regular significant daily fluctua-
tions (Fig. 6). E<sub>2</sub>17β levels increased during
the photophase and reached maxima to-
ward and during early scotophase; a de-
crease later on might occur, which should
be confirmed.

In the goldfish, Hontela and Peter (1978)
found constant plasma GtH levels in "re-
gressed" fish, in which oocytes undergoing
endogenous vitellogenesis dominated the
ovary. Daily fluctuations in GtH levels ap-
peared later on, in "maturing" and in "ma-
ture" females. In the ovaries of these fe-
males, the proportion of oocytes under-
going advanced stages of vitellogenesis was
high. In all groups, basal GtH levels were
the same. Based on these results, Hontela
and Peter (1978) and Peter (1981) suggested
that the daily fluctuations in GtH levels
might be more important for the stimula-
tion and the maintenance of the ovarian ac-
tivity than the progressive long-term
changes in the concentrations of the hor-
mone. On the basis of our present results, a
similar hypothesis might be proposed for
the female rainbow trout. Whereas pre-
vious studies suggested that the important
increase in circulating E<sub>2</sub>17β during exoge-
nous vitellogenesis is accompanied either
by a moderate increase or continuous de-
crease in the mean plasma GtH levels or by
constant concentrations of the hormone
(see introduction), our study suggests an
important change in the short-term GtH
profiles during the same processes. The
constant levels of GtH characterizing the
beginning of exogenous vitellogenesis be-
come pulsatile in its advanced stages, while
E<sub>2</sub>17β increases drastically. The basal GtH
levels remain low in both cases. This sug-
gests that in the female rainbow trout the appearance of GtH pulsatility during exogenous vitellogenesis, rather than a change in the hormone absolute levels, is responsible for the marked E₂₁₇β increase. However, the rainbow trout model might be more complicated than the goldfish one. The results obtained by Hontela and Peter (1978), and confirmed by Hontela and Peter (1980a, b) and Gillet *et al.* (1981), indicate that there is a relative synchronization between the individual GtH profiles of different females. This fact results in the detection of a significant daily cycle in GtH levels when different groups of fish are sampled every 4 hr. Our present data demonstrate that in the female rainbow trout, the individual GtH profiles are not synchronized since the short-term increases in GtH levels occur at different times in different females. Thus, the GtH fluctuations can be detected only if individual fish are sampled repeatedly. Averageing the individual GtH profiles masked the existence of GtH pulsatility, and resulted in stable mean GtH levels over the 24-hr sampling period (Fig. 6). A similar situation has been described by Pan *et al.* (1980) in three carp species; when individual GtH profiles were recorded during the spawning season, short-term GtH pulses were observed. As in the case of the rainbow trout at advanced exogenous vitellogenesis, those profiles were relatively nonsynchronized among individual fish.

Although it is difficult to determine the frequency of GtH pulses during advanced exogenous vitellogenesis, we observed up to 5 GtH pulses within a 12-hr period when the bleeding interval was 1 hr. However, the number of pulses varied between the females, and in some of them GtH levels were quite constant. This might reflect a lack of total physiological synchronization among the studied females or might be the result of not bleeding frequently enough in relation to the duration of the pulses. In

many of the profiles for which bleeding was done at 1-hr intervals, a pulse included only one value of high GtH level. The pulses were longer in a few cases, and in one female (Fig. 5, No. 154) the increase in GtH levels lasted a few hours. This last example might reflect the initiation of an increase in the basal GtH levels, related to a possible initiation of the germinal vesicle migration (see Zohar *et al.*, 1986). When the sampling interval was reduced to 30 min, most of the GtH pulses included more than one value of high GtH level. Considered together, all of the GtH profiles characterizing exogenous vitellogenesis indicate a pulse duration of 1 to 3 hr. Bleeding fish at even higher frequencies than that reported here is necessary for a precise analysis of the dynamics of the GtH pulses. Our present data do not demonstrate a clear circadian rhythmicity in GtH levels at advanced exogenous vitellogenesis. However, they indicate a possible decrease in the frequency of the GtH pulses during the night, which should be further confirmed.

The relationship between the GtH and E₂₁₇β secretion patterns in the female trout at advanced exogenous vitellogenesis cannot be precisely determined on the basis of the present *in vivo* study. They are considered in more detail elsewhere (Zohar, Fostier, and Breton, in preparation), taking into account the results of both *in vivo* and *in vitro* studies. However, the relationship between the GtH and E₂₁₇β secretion patterns differs from that described in most mammalian species, in which pulses are considered as basic signals at both the pituitary and the gonadal level. A pulsation of LH is immediately followed by a pulsation of a corresponding gonadal steroid in the mouse (Desjardins, 1981), the rabbit (Moor and Younglai, 1975), the goat (Muduuli *et al.*, 1979), the dog (De Palatis *et al.*, 1978), the ewe (Baird, 1978), the ram (Lincoln, 1976), the rhesus monkey (Steiner *et al.*, 1980), and
man (Backstrom *et al.*, 1982). In the female rainbow trout, however, the short-term GtH pulses are accompanied by regular E₂₁₇β variations of longer duration. The same phenomenon was also observed *in vitro* (Zohar *et al.*, 1982b and unpublished data). The greater abundance of GtH pulses during the day may indicate the importance of their frequency in the regulation of the progressive increase in E₂₁₇β levels during the photophase, as well as in the possible decrease of these levels later on.

Recently, the physiological function of fish gonadotropin(s) in relation to vitellogenesis has been a matter of discussion (e.g., Idler, 1982; Idler and Ng, 1983; Burzawa-Gerard, 1982). In this discussion, references have been made to results which are related to the circulating levels of the glycoproteic GtH. Our present study is the first to demonstrate that in the female rainbow trout, changes in the short-term secretion pattern of GtH occur from early ovarian recrudescence and throughout vitellogenesis, in addition to the seasonal evolution of the hormone level. Such short-term secretion patterns should be considered when the glycoproteic GtH role in vitellogenesis is discussed.

## ACKNOWLEDGMENTS

We thank Aline Solari for her valuable help in the statistical analysis of our data, and Elisabeth Sambrony and Odile Marcuzzi for their technical assistance. This work was supported by a grant from the "Yad Hanadiv" Foundation to Y.Z.

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