Growth Hormone (GH) - Science Direct

General and Comparative Endocrinology 113, 413–428 (1999) Article ID gcen.1998.7222, available online at http://www.idealibrary.com on

Growth Hormone (GH) and Gonadotropin Subunit Gene Expression and Pituitary and Plasma Changes during Spermatogenesis and Oogenesis in Rainbow Trout (Oncorhynchus mykiss) Jean Marc Gomez, Claudine Weil, Martine Ollitrault, Pierre-Yves Le Bail, Bernard Breton, and Florence Le Gac Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cedex, France Accepted November 2, 1998

In order to evaluate potential interactions between somatotropic and gonadotropic axes in rainbow trout (Oncorhynchus mykiss), changes in pituitary content of the specific messenger RNA of growth hormone (GH) and gonadotropin (GTH) ␣- and ␤-subunits were studied during gametogenesis with respect to pituitary and plasma hormone concentrations. Quantitative analyses of mRNA and hormones were performed by dot blot hybridization and homologous RIA on individual fish according to stage of spermatogenesis and oogenesis. All transcripts were detectable in 9-month-old immature fish. GH, GTH II␤, and GTH ␣ increased moderately throughout most of gametogenesis and then more dramatically at spermiation and during the periovulatory period. GTH I␤ mRNA increased first from stage I to V in males and more abruptly at spermiation, while in females GTH I␤ transcripts increased first during early vitellogenesis and again around ovulation. Pituitary GH absolute content (␮g/pituitary, not normalized with body weight) increased slowly during gametogenesis and more abruptly in males during spermiation. In the pituitary of previtellogenic females and immature males, GTH I ␤ peptide contents were 80- to 500-fold higher than GTH II ␤ peptide contents. GTH I contents rose regularly during the initial phases of vitellogenesis and spermatogenesis and then more abruptly in the final stages of gonadal maturation, while GTH II contents show a dramatic elevation during final oocyte growth and maturation, in 0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

postovulated females, and during spermiogenesis and spermiation in males. Blood plasma GTH II concentrations were undetectable in most gonadal stages, but were elevated during spermiogenesis and spermiation and during oocyte maturation and postovulation. In contrast, plasma GTH I was already high (E2 ng/ml) in fish with immature gonads, significantly increased at the beginning of spermatogonial proliferation, and then increased again between stages III and VI to reach maximal levels (E9 ng/ml) toward the end of sperm cell differentiation, but decreased at spermiation. In females, plasma GTH I rose strongly for the first time up to early exogenous vitellogenesis, decreased during most exogenous vitellogenesis, and increased again around ovulation. Our data revealed that patterns of relative abundance of GTH I␤ mRNA and pituitary and plasma GTH I were similar, but not the GTH II patterns, suggesting differential regulation between these two hormones at the transcriptional and posttranscriptional levels. Pituitary and plasma GH changes could not be related to sexual maturation, and only a weak relationship was observed between GH and gonadotropin patterns, demonstrating that no simple connection exists between somatotropic and gonadotropic axes at the pituitary level during gametogenesis. r 1999 Academic Press

An increasing number of reports indicate that somatotropic and gonadotropic axes could interact in 413

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vertebrates. Pubertal development delayed in isolated growth hormone deficiency syndrome in both human and animals can be restored by growth hormone (GH) treatment (Sheikholislam and Stempfel, 1972; Ramaley and Phares, 1980; Advis et al., 1981; Ovesen et al., 1992). Some of these studies suggest that GH and insulin-like growth factor I (IGFI) can directly modulate the gonadal function (reviews: Adashi et al., 1992; SpiteriGrech and Nieschlag, 1992; Katz et al., 1993; Le Gac et al., 1993). Another level of interaction may be located at the hypothalamic–pituitary levels. In GH-deficient dwarf mice, GH can modulate basal luteinizing hormone (LH) secretion (Chandrashekar and Bartke, 1993) and gonadotropin-releasing hormone (GnRH) action (Chandrashekar and Bartke, 1996), the major factor involved in gonadotropin control in vertebrates. Tang et al. (1993) demonstrated also that pituitary LH contents and LH␤ mRNA are elevated in transgenic mice expressing human GH. Systemic or/and pituitary IGFI probably exerts a positive role in the regulation of pituitary LH release: The addition of IGFI to rat anterior pituitary explants or dispersed cells augments basal and GnRH-stimulated in vitro LH release (Kanematsu et al., 1991; Soldani et al., 1994, 1995). Conversely, the sexually dimorphic pattern of GH secretion indicates that gonadal steroids may also be involved in the regulation of GH in mammals (Jansson et al., 1985). In teleost fish, although few studies were undertaken at the hypothalamic–pituitary level concerning possible interactions between somatotropic and gonadotropic axes, original data was obtained. GnRH was first discovered to stimulate GH release in goldfish (Carassius auratus) in vivo and in vitro (Marchant et al., 1989; Chang et al., 1990) and later in carp (Cyprinus carpio; Lin et al., 1993) and tilapia hybrid (Melamed et al., 1995), but not in African catfish (Clarias gariepinus; Lescroart et al., 1996; Bosma et al., 1997). In rainbow trout, the effect of GnRH on GH release by cultured pituitary cells depends on stage and incubation conditions (Le Gac et al., 1993; Blaise et al., 1995, 1997), but perifused pituitary fragments obtained from E2 primed rainbow trout strongly respond to sGnRH (Holloway and Leatherland, 1997). The gonadoliberin also stimulates GH mRNA content in the goldfish pituitary (Mahmoud et al., 1996), in accordance with the presence of GnRH receptors on somatotroph cells (Cook et al., 1991). Like in mammals, sex-steroid hormones can promote fish growth (Donaldson et al., 1979) directly or

Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Gomez et al.

indirectly via the modulation of endocrine factor involved in growth. In particular, GH secretion may be influenced by sex steroids since pharmacological treatments with estradiol changes somatotroph morphology (Olivereau and Olivereau, 1979) and physiological concentrations of androgens and estrogens are able to stimulate GH mRNA relative content (Huggard et al., 1996a) and pituitary GH protein content (Zou et al., 1997). Before further investigating physiological interactions between somatotroph and gonadotroph functions, more precise data on GH and gonadotropin production/release were needed. In many teleosts, two chemically distinct pituitary gonadotropins (GTH I and GTH II) have been reported (Suzuki et al., 1988; Kawauchi et al., 1989). They have distinct temporal pituitary and plasma patterns during the reproductive cycle (see Swanson, 1991; Swanson et al., 1989, 1991; Amano et al., 1992; Slater et al., 1994; Prat et al., 1996). Preliminary slot blot analysis (Weil et al., 1995) and in situ hybridization studies (Naito et al., 1991, 1995) also suggested differential changes in GTH subunit mRNA expressions in rainbow trout pituitary. However, most of these studies were limited to comparison of immature versus maturing animals or to description of seasonal variations of pituitary and/or plasma gonadotropin concentrations, and no data exist concerning concomitant changes in pituitary (mRNA and protein) and plasma gonadotropins as a function of the gametogenetic cycle. No data exist concerning changes in pituitary and plasma GH concentrations over the reproductive cycle in teleost fish. Only final stages of gametogenesis have been studied, and elevated plasma GH concentrations have been reported in several species (Stacey et al., 1984; Sumpter et al., 1991; Le Gac et al., 1991). In this work, changes in pituitary GH and gonadotropin I and II (mRNA and protein) in relation with plasma hormone levels were studied in individual male and female rainbow trout at precise stages of gametogenesis during a complete reproductive cycle.

MATERIALS AND METHODS Animals One-year-old rainbow trout (Oncorhynchus mykiss) of the Cornec Autumnal strain (spawns naturally

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GH and GTHs mRNA and Protein Changes during Fish Gametogenesis

around October–November) reared at the INRA experimental fish farm (Sizun, Finiste`re, France) were used. During the entire experimental period (January 1995– November 1995), fish were kept under natural temperature and photoperiod (48°N) in circulating fresh water tanks (capacity 1800 liters). The temperature increased regularly from January 8 (9°C) to September 21 (21°C) and then decreased rapidly until November (7.5°C). The fish were fed once daily (6 days/week, except for 2 days prior to sampling) with commercial pellets (Aqualife No. 17; Aqualim S.A., St. Estephe, France) at a rate ajusted for variations in weight and in water temperature as recommended by the manufacturer (0.7% in January to 1.4% in July). In August and September the feed rate was progressively decreased and feeding was stopped for 2 weeks due to the exceptionally high temperature (feeding was back to 1.4% at the end of September). In November we observed that most ovulating and spermiating fish had spontaneously stopped eating.

Experimental Design In order to describe changes in pituitary and plasma hormonal concentrations or mRNA contents during gametogenesis, 40 to 60 randomly caught rainbow trout (sexually immature, 508 ⫾ 27 g body wt in January; sexually mature, 1755 ⫾ 85 g body wt in November) were killed at approximately 30-day intervals (n ⫽ 11 samplings). At each sampling, after rapid anesthesia (3 to 4 min) in phenoxy-2-ethanol (0.5/1000, v/v), blood was collected quickly from the caudal vasculature with heparinized syringes. The samples were then centrifuged (4°C) at 3200g for 20 min, and the plasma was stored in aliquots at ⫺20°C until assayed. Fish were decapitated and pituitary glands were collected, frozen individually in liquid nitrogen, and stored at ⫺70°C until used. Gonads were dissected out and weighed in order to determine the gonadosomatic index (GSI, testicular or ovarian wt ⫻ 100/body wt). A transversal section from the middle part of the testis/ovary was fixed in Bouin’s solution for histological examination. The spermatogenetic stage of each male was determined according to a classification adapted from Billard and Escaffre (1975). The ovogenetic stage of each female was determined by histological examination as previously described by Breton et al. (1983) and by macroscopic observations for oocyte maturation (Jalabert, 1976).

Pituitary RNA Extraction Total RNA were extracted from individual pituitary according to Auffrey and Rougeon (1980). Pituitaries were homogenized in 500 µl of buffer containing urea (6 M), LiCl (3 M), heparin (0.2 mg/ml), and sodium acetate (0.05 M). The homogenate was supplemented with 0.05% (w/v) sodium dodecyl sulfate (SDS) and kept overnight at 4°C. After centrifugation (12,000g, 30 min, 4°C), the pellet was washed with 500 µl urea (8 M), LiCl (4 M), sodium acetate (0.05 M), and recentrifuged under the same conditions. The pellet was dissolved in 300 µl RNase-free water and the proteins were extracted three times with 300 µl phenol– chloroform–isoamyl alcohol. The RNA was then precipitated with 2.5 vol of absolute ethanol and 0.1 vol LiCl (4 M) for 1 h at ⫺70°C. After centrifugation (12,000g, 1 h, 4°C), the resulting pellet was desiccated twice with ethanol and redissolved in RNase-free water. Concentration and purity of the RNA were evaluated at 260 and 280 nm. RNA integrity was verified by electrophoresis on 0.8% (w/v) agarose. The amount of RNA recovered from an individual pituitary ranged from 1–4 µg to 50–60 µg, depending on the fish size and/or extraction yield of RNA, which was highly variable.

Probes and Hybridizations Trout GH cDNA (Rentier-Delrue et al., 1989), chum salmon GTH I␤, GTH II␤, common GTH ␣ cDNA (Sekine et al., 1989), and trout ␤ actin cDNA (Pakdel et al., 1989) were radiolabeled just prior to the hybridization with a megaprime DNA labeling system (Amersham, Les Ulis, France) by random priming (Feinberg and Vogelstein, 1983) with 58-[␣-32P]dCTP (3000 Ci/ mM, Amersham). The specificity of trout GH and chum salmon GTH probes for detection of rainbow trout transcript were previously verified by Northern blot hybridization by Le Gac et al. (unpublished data) and Weil et al. (1995). Dot blot hybridization was performed according to the stringent conditions of Thomas (1980), replacing salmon sperm DNA with calf thymus DNA. For each sexual stage, individual total RNA preparations were loaded at 0.5, 1, and 2 µg (preliminary experiments indicated a linear relationship between RNA blotted and the autoradiographic signal) onto nylon mem-


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branes (Amersham, Hybond N⫹ ). Rainbow trout liver total RNA was also included on every membrane as a negative control and in order to evaluate the background of nonspecific hybridization. The whole experiment was performed in duplicate on two separate nylon membranes. Each membrane was submitted to three successive hybridizations in the following order: GTH I␤, GH, and ␤-actin cDNA for the first membrane and GTH II␤, GTH ␣, and ␤-actin cDNA for the second one. Prehybridization (4 h) and hybridization (overnight) for dot blots were carried out at 42°C with gentle agitation in 50% (1/1, v/v) formamide, 5⫻ SSC (1⫻ SSC ⫽ 0.15 M NaCl, 0.015 M sodium citrate, pH 7), 5⫻ Denhardts (Denhardts, 1966), and calf thymus DNA (100 µg/ml). The membranes were then washed 4 ⫻ 5 min in 2⫻ SSC, 0.1% SDS at room temperature and 2 ⫻ 15 min in 0.1⫻ SSC, 0.1% SDS at 50°C. The radioactive signal was quantified using an InstantImager analyzer (Packard Instrument Co., Meriden, CT) linked to an image counter software. After data analysis, the probe was stripped by incubation in three successive baths (1 h in 50% formamide at 65°C, 15 min in 0.1⫻ SSC, 0.1% SDS and in 1 M Tris–HCl at room temperature). This procedure resulted in a complete loss of signal of the previous probe.

Hormone Measurements Pituitary content for each sexual stage. Pituitary glands were homogenized individually in 1 ml assay buffer (50 mM Tris–HCl, 10 mM MgCl2, 0.05% (w/v) NaN3, 0.5% (w/v) BSA (RIA Grade, A-7888; Sigma Chemical Co., St Louis, MO), 0.1% (v/v) Triton X-100). Pituitary and plasma GH concentrations were determined in duplicate by homologous RIA (Le Bail et al., 1991). The sensitivity of the assay (ED90 ) was 0.2 ng/ml for 50 µl of assayed plasma, and the ED50 was 1.0 ng/ml. No cross-reaction was observed with GTH I or GTH II. Pituitary and plasma GTH I and GTH II concentrations were measured in duplicate by a homologous RIA developed in our laboratory (Govoroun et al., 1998), using antibodies raised against the ␤-subunits and gonadotropins purified from rainbow trout pituitaries (Govoroun et al., 1997) as standard and labeled hormone. The sensitivity of the assay for GTH I and GTH II was 0.5 and 0.1 ng/ml for 50 µl of assayed plasma. The ED50 levels for GTH I and GTH II assay were 15 and 0.9 ng/ml. The GTH I assay recognized GTH I heterodimer and GTH I ␤-subunit equally and


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had a 6–8% cross-reaction with GTH II, whereas the GTH II assay was highly specific for GTH II (heterodimer and ␤-subunit), having no cross-reaction with GTH I.

Data Analysis Pituitary contents of ␤-actin transcript were not found significantly affected by the stage of gametogenesis (data not shown) and were used to correct differences in the amount of RNA applied to the membranes. Results of dot blot analysis are expressed (after correction for background values) as arbitrary units of GH mRNA/␤ actin mRNA and gonadotropin subunit mRNA/␤ actin mRNA ratios (the yield of total RNA extraction from a single pituitary is highly variable and the absolute mRNA content per pituitary could not be calculated). The GTH ␣, GTH I␤, and GTH II␤ cDNA probes, being of similar length, labeled to similar specific activity, and used under the same incubation conditions, allowed us to compare the data obtained for the three gonadotropin subunit transcripts. Pituitary DNA or total protein content were not available. The total hormone contents shown here are expressed in µg/pituitary. However, pituitary size changes with sexual development and with body growth. In an attempt to minimize/reduce the influence of general growth and to analyze the changes in pituitary hormone content more specifically linked to the reproductive stage, we calculated a relative content normalized to body weight (µg/kg body wt). The statistical difference during the reproductive stages for mRNA contents and pituitary and plasma hormone concentrations were analyzed by one-way analysis of variance (ANOVA) followed by multiple range test (Kruskal–Wallis). Differences were considered significant when P ⬍ 0.05. Linear regression analysis was used to determine the relationship between the different variables. All results are expressed as means ⫾ SEM of n individuals at the same stage of gametogenesis.

RESULTS Evolution of GSI and Gametogenesis The germ cell types and GSI observed at the various stages of spermatogenesis and oogenesis are reported

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TABLE 1 Rainbow Trout Spermatogenetic Stages as Defined by the Germ Cells Present and the Corresponding Gonadosomatic Index (GSI) Germ cells Stages I II III IV V VI VII VIIIa

A-gonia ⫹⫹⫹ ⫹⫹ ⫹ ⫹ (⫹) (⫹) (⫹) (⫹)

B-gonia

Spermatocyt

⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹

⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ (⫹)

Spermatid

(⫹) ⫹⫹ ⫹⫹⫹ ⫹⫹ (⫹)

Spermatozoa

(⫹) ⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹

Spermiation

GSI (%)

(⫹) ⫹ to ⫹⫹

0.06 ⫾ 0.003 0.08 ⫾ 0.003 0.10 ⫾ 0.007 0.3 ⫾ 0.03 1.3 ⫾ 0.1 4.3 ⫾ 0.2 5.5 ⫾ 0.3 3.8 ⫾ 0.2

Note. ⫹ to ⫹⫹⫹⫹ indicate the relative abundance of each germ cell type observed during histological examination. (⫹), rare. All males in stage VIII were at the beginning of spermiation.

a

in Tables 1 and 2, respectively. In autumn spawning rainbow trout, after 1 year of immaturity, the first sign of gametogenesis occurred in March–April as indicated by the presence of B spermatogonia (stage II) for males and vitellogenic follicles for females (stage 2). Testicular weight was low during the first stages of

TABLE 2 Rainbow Trout Ovarian Stages Based on Oocyte Histological and Macroscopic Characteristics and on Gonadosomatic Index (GSI) Stages

Characteristics

GSI

1

Immaturity/previtellogenesis

0.1 ⫾ 0.004

2

Early endogenous vtg

3

Endogenous vtg

4.1

Early exogenous vtg

4.2

Midexogenous vtg

4.3

Advanced exogenous vtg Preovulation Postovulationa

Previtellogenic oocytes ⫹ very rare oocytes with cortical alveoli Previtellogenic oocytes ⫹ oocytes with cortical alveoli Most oocytes with cortical alveoli and lipidic globules Scarce oocytes with yolk globules (Increasing number of oocytes with yolk globules and increasing amounts of yolk deposit) From GV⫾ to GVBD 1 day to 4 weeks postovulation

5 6

spermatogenesis (stages II–IV, mainly stages of spermatogonia proliferation and of meiosis), increased dramatically between stages IV and VII (mainly stages of spermiogenesis), and slightly decreased during spermiation (stage VIII). In females, ovarian growth was also slow in the early stages (early endogenous vitellogenesis to midexogenous vitellogenesis) and increased dramatically at the end of exogenous vitellogenesis (stages 4.3–5) to reach maximum value during final maturation (from subperipheral germinal vesicle (GV⫾) to germinal vesicle breakdown (GVBD)). Spermiation and ovulation occurred in October–November. In the present experiment, 100% of the animals went through their first gametogenetic cycle.

0.1 ⫾ 0.01

Changes in Specific mRNA and Pituitary and Plasma Hormone Concentrations during Spermatogenesis

0.2 ⫾ 0.02

Our study showed that the four transcripts were detectable at all reproductive stages, including in 9-month-old (data not shown) and 12-month-old immature male pituitaries (Fig. 1). GH mRNA relative content increased (4- to 5-fold) between immaturity and stage VI and then rose dramatically in spermiating fish (Fig. 1a). GTH I␤ mRNA relative content increased from stage I to stage VII (13-fold) and more rapidly at stage VIII (Fig. 1b). In contrast, GTH II␤ transcript appeared nearly constant during the entire spermatogenesis, increasing 8-fold at spermiation (Fig. 1c). GTH ␣ mRNA relative content was also constant from stage I to stage IV, increased weakly during spermiogenesis (stages V–VII), and increased more abruptly at spermia-

0.4 ⫾ 0.02 0.7 ⫾ 0.04 4.3 ⫾ 0.4 13.1 ⫾ 0.5 0.7 ⫾ 0.05

Note. vtg, vitellogenesis; GV⫾, germinal vesicle migration to the oocyte periphery; GVBD, germinal vesicle breakdown. a Eggs were maintained in the abdominal cavity of females used in stage 6.


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tion (Fig. 1d). The results shown here, normalized with ␤-actin mRNA level, were in agreement with the results expressed per microgram of total RNA (data not shown). Pituitary GH total content (expressed in µg hormone/pituitary, Fig. 2a) increased slowly and regularly from immaturity to stage VI and more rapidly between stages VI and VIII of spermatogenesis (P ⬍ 0.001). However, GH relative content normalized to the size of the fish (expressed in µg/pituitary/kg body wt) did not show these changes and even tended to decrease during spermatogenesis, except at spermiation, where an increase was still observed (data not shown). Pituitary GTH I and GTH II proteins (immunoreactive GTH ␤-subunits ⫹ heterodimers) were both detectable during the entire spermatogenetic development, although in stages I and II, GTH II contents were 80- and 30-fold lower than GTH I contents, respectively. GTH-I contents were already high in premature fish (stage I): 1.05 ⫾ 0.06 µg/pituitary in 9-month-old fish and 2.83 ⫾ 0.56 µg/pituitary in 11- to 15-monthold fish. They rose regularly during spermatogenesis (10-fold between stages I and VI) and more abruptly (3.5-fold between stages VI and VII) at the end of the cycle (Fig. 2b). GTH II contents remained low during most of the cycle (0.012 to 1.6 µg/pituitary from stage I to stage V) and dramatically increased after stage VI (Fig. 2c). Pituitary GTH I content was 30- to 40-fold lower in stage I than in stage VIII, while GTH II content was ⬇6000-fold lower in stage I than in stage VIII. For both gonadotropins, patterns of hormone contents normalized for body growth (expressed in µg/pituitary/kg body wt) were similar to those described above, although the changes were less pronounced (data not shown). Blood plasma mean GH concentrations in males were low and stable (0.6 ng/ml) during most of spermatogenesis, except in stage VIII, where an increase was observed (Fig. 3a). Plasma GTH I concentrations were already high before the beginning of spermatogenesis, showing a transient increase in stage II

FIG. 1. Changes of pituitary GH (a), GTH I␤ (b), GTH II␤ (c), and GTH ␣ (d) mRNA relative contents in male rainbow trout during spermatogenesis. Results are expressed as means ⫾ SEM for 6–10 individual pituitaries. Similar letters above two histograms indicate that the difference is not statistically significant.



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(spermatogonial proliferation). It further increased from stage IV to reach maximal values toward the end of spermatogenesis (stages VI–VII) (Fig. 3b). At spermiation, plasma GTH I returned to the level previously observed in the early stages. In contrast, plasma GTH II was undetectable at the beginning of the cycle and the mean GTH II concentration increased regularly

FIG. 3. Changes of plasma GH (a), GTH I (b), and GTH II (c) concentrations in male rainbow trout during spermatogenesis. Results are expressed as means ⫾ SEM for 12–33 fish. Similar letters above histograms indicate that the difference is not statistically significant. ND, non detectable.

(15-fold) between stages III and VIII (Fig. 3c), always remaining lower than GTH I.

FIG. 2. Changes in male rainbow trout of pituitary GH (a), GTH I (b), and GTH II (c) absolute contents (in µg/pituitary) during spermatogenesis. Results are expressed as means ⫾ SEM for 6–17 individual pituitaries. Similar letters above two histograms indicate that the difference is not statistically significant.

Changes in Specific mRNA and Pituitary and Plasma Hormone Concentrations during Oogenesis Figure 4 shows changes of pituitary GH and gonadotropin subunits mRNA relative contents during oogen-


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esis in rainbow trout. As previously shown for males, all four transcripts were detectable during the entire cycle, including 9-month-old (data not shown) and 12-month-old immature female pituitaries. Our study revealed that GH mRNA levels (Fig. 4a) appeared relatively constant throughout vitellogenesis (with an increase during early exogenous vitellogenesis). In ovulated fish, GH mRNA relative content was threefold higher than that during the rest of the cycle. Pituitary GTH I␤ transcript rose at first during early exogenous vitellogenesis and a second time during the final stages, reaching maximal values after ovulation (Fig. 4b). GTH II␤ mRNA relative abundance remained constant during the entire vitellogenesis and increased significantly in ovulated fish (Fig. 4c). GTH ␣ transcript, also low until the end of endogenous vitellogenesis, was more elevated (threefold) during exogenous vitellogenesis and then peaked dramatically in ovulated fish (Fig. 4d). The results normalized with ␤-actin mRNA contents were in agreement with the results expressed per microgram of total RNA (data not shown). In female rainbow trout, pituitary GH total content (expressed in µg hormone/pituitary) increased significantly from stage 3 to stage 5 (Fig. 5a). However, when normalized for body size, the pituitary GH relative content (µg/pituitary/kg body wt) decreased regularly throughout oogenesis (data not shown). Pituitary GTH I and GTH II proteins were both detectable during the entire female cycle (Figs. 5b and 5c). However, in previtellogenic fish, GTH I was present in large amounts (⬃6 µg/pituitary), while GTH II contents were about 500-fold lower. Pituitary total contents of GTH I increased regularly and significantly from the immature stage to the beginning of exogenous vitellogenesis (4-fold), and a second rise occurred at final oocyte maturation and after ovulation (Fig. 5b). GTH II contents were low during most of the cycle (ⱕ0.01 to 2 µg/pituitary from stage 1 to stage 4.2)

FIG. 4. Changes in female rainbow trout of pituitary GH (a), GTH I␤ (b), GTH II␤ (c), and GTH ␣ (d) mRNA relative contents during oogenesis. Results are expressed as means ⫾ SEM for six to nine individual pituitaries. Similar letters above histograms indicate that the difference is not statistically significant. GV⫾, migration of germinal vesicle; GV⫹, germinal vesicle at the periphery of the oocyte; GVBD, germinal vesicle breakdown.



421

two stages. For both gonadotropins, similar general patterns were observed when pituitary hormone contents were normalized for body weight changes (expressed in µg/pituitary/kg body wt), but of course the amplitude of the changes were less pronounced. Plasma GH concentrations in females were low from the beginning of this experiment (0.7 ng/ml) and tended to decrease further during oogenesis (Fig. 6a). In accordance with pituitary GTH I␤ transcripts, plasma GTH I concentrations clearly increased until the beginning of exogenous vitellogenesis, then decreased during the rest of exogenous vitellogenesis (Fig. 6b). GTH I rose a second time during final maturation to reach maximal concentrations after ovulation. Plasma GTH II was undetectable at the beginning of the cycle, including early endogenous vitellogenesis, and remained undetectable (⬍0.1 ng/ml) in most females in stages 3 and 4.1 (86 and 76%) as well as in 54 and 20%, respectively, of the females in stages 4.2 and 4.3 of rapid follicular growth. Then it increased abruptly during final maturation and after ovulation (Fig. 6c).

Relationship between Pituitary Contents and Plasma Hormone Concentrations

FIG. 5. Changes in female rainbow trout of pituitary GH (a), GTH I (b), and GTH II (c) absolute contents (in µg/pituitary) during oogenesis. Results are expressed as means ⫾ SEM for 6–22 individual pituitaries. Similar letters above histograms indicate that the difference is not statistically significant. See also the legend to Fig. 4.

and dramatically increased in the final stages of oocyte growth and maturation and after ovulation (200 µg/ pituitary) (Fig. 5c). GTH I total contents were 20-fold lower in stage 1 than in stage 5, while GTH II total contents increased 10,000- to 20,000-fold between these

With a view to analyzing the relative changes of pituitary peptide hormone content versus specific transcript abundance and of plasma hormone concentration versus pituitary hormone content during spermatogenesis and oogenesis, ratios of the mean values of these parameters were calculated for each hormone (arbitrary units) (Table 3). Pituitary GH/GH mRNA ratio tended to decrease slowly from stage I to stage V of spermatogenesis and between stage 1 and stage 4.1 of vitellogenesis (two- to threefold) and more abruptly at spermiation in males and in postovulated females. This tendency was considerably amplified when pituitary GH contents were normalized for body growth (data not shown). Plasma GH/total pituitary GH ratio also tended to decrease two- to threefold during active gametogenesis; however, this ratio remained quite stable when pituitary GH content was normalized for body growth (data not shown). This ratio decreased in final ovarian maturation. In comparison, the pituitary GTH II/GTH II␤ mRNA ratio increased greatly, mainly after meiosis and spermiogenesis had started in males (800-fold between stages III and VII) and during vitellogenesis in females


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tion. In contrast, in both sexes, pituitary GTH I/GTH I␤ mRNA and plasma GTH I/pituitary GTH I ratios show only minor changes during gametogenesis. However, for this hormone the ratio plasma to pituitary content was low in the late stages of gamete development, while in the pituitary, the ratio protein/mRNA was high (stage VII in male and stage 5 in female). When possible, correlations were looked for between individual values of the different parameters from stage I to stage VII for males and from stage 1 to stage 5 for females (the last stage was excluded from the analysis because the data were too different from the rest of the cycle and because stages of spermiation and postovulation are not strictly stages of gametogenesis). Messenger RNAs and pituitary hormone contents were not measured on the same pituitaries. In both sexes, GTH I␤ mRNA were significantly correlated with GTH ␣ mRNA (for males: r ⫽ 0.33, P ⬍ 0.04; for females: r ⫽ 0.53, P ⬍ 0.01). Plasma GTH I concentrations were significantly correlated with GTH I␤ mRNA in females (r ⫽ 0.55, P ⬍ 0.01), but not in males (r ⫽ 0.23). A significant correlation was observed in females between GTH I␤ mRNA and GTH II␤ mRNA (r ⫽ 0.43, P ⬍ 0.01) and between GTH I␤ mRNA and GH mRNA (r ⫽ 0.47, P ⬍ 0.01). No other significant correlations were observed between pituitary hormone contents (mRNA or proteins) and plasma concentrations or between different specific transcripts, different hormone pituitary contents, and different hormone plasma concentrations.

DISCUSSION

FIG. 6. Changes of plasma GH (a), GTH I (b), and GTH II (c) concentrations in female rainbow trout during oogenesis. Results are expressed as means ⫾ SEM for 23–82 fish from stage 1 to stage 4.3 and 9–14 fish from stage 5.1 to stage 6. Similar letters above histograms indicate that the difference is not statistically significant. ND, non detectable. See also the legend to Fig. 4.

(2000-fold between stages 3 and 5). While the plasma GTH II/pituitary GTH II ratio decreased during the last stages of spermatogenesis (50-fold between stage V and spermiation), in females it decreased in stage 4.3 of vitellogenesis but increased again in final matura-


With a view to investigating possible interactions between somatotropic and gonadotropic axes, growth hormone together with gonadotropin production and release were studied at precise stages of spermatogenesis and oogenesis in rainbow trout. This study describes, for the first time in fish, changes during gametogenesis in the relative abundance of mRNA encoding for GTH I and GTH II ␣- and ␤-subunits, with respect to pituitary and plasma levels of these hormones. The changes in pituitary and plasma levels described in this work are in agreement with seasonal variations already reported (see introduction) and enable us to give the following details.

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TABLE 3 Changes in the Following Ratiosa: Pituitary Protein (pit.pro) Contentb/Specific Transcript Contentc and Plasma Protein Concentrationd/Pituitary Protein Content for GH, GTH I, and GTH II during Spermatogenesis and Oogenesis in Rainbow Trout GH Stage

pit.pro/ mRNA

GTH I

plasma/ pit.pro ⫻100

pit.pro/ mRNA␤

GTH II


pit.pro/ mRNA␤


0.65 0.73 0.32 0.26 0.21 0.32 0.09 0.035

0.018 0.051 0.060 0.22 0.79 5.08 47.57 11.47

nd nd 27.6 23.3 22.9 3.8 0.5 0.3

0.59 0.64 0.97 0.61 0.45 0.20 0.08 0.15

0.009 0.001 0.012 0.498 0.620 12.39 30.09 17.38

nd nd nd to 100 nd to 15.0 13.0 1.6 5.7 6.9

Spermatogenesis I II III IV V VI VII Sperm.

35.2 27.1 14.8 13.4 12.3 12.8 18.3 2.87

1.45 1.11 1.17 0.99 0.55 0.90 0.56 1.06

2.80 2.01 1.56 2.93 3.75 3.51 7.69 2.51 Oogenesis

1 2 3 4.1 4.2 4.3 Mat. PO

22.8 15.6 15.4 10.5 16.1 22.1 24.8 6.4

1.61 1.82 1.42 0.73 0.86 0.78 0.25 0.52

6.01 6.64 3.76 2.42 5.65 8.90 11.23 4.96

Note. Sperm., spermiation; Mat., final maturation; PO, postovulation; nd, not detectable in plasma. a Ratios of the mean values of these parameters at each stage, in arbitrary units. b Pituitary hormone total content in µg/pituitary. c GH or GTH ␤-subunit mRNA relative abundance normalized to actin mRNA. d Plasma hormone content in ng/ml.

In the male there was a simultaneous increase of GTH I in the pituitary (mRNA and protein) and plasma, between stages IV and VII, suggesting a role of potential regulator for this hormone during the development of spermatogenesis/spermiogenesis. Moreover, the increased plasma concentrations of this gonadotropin at stage II could equally implicate GTH I in spermatogonial multiplication process, since in vitro the capacity of GTH I to stimulate the proliferation of premeiotic cells in total testicular cell cultures has been shown by Loir and collaborators from our laboratory (unpublished data). In the female, GTH I seems to be principally implicated in early phases of the initiation of vitellogenesis. We observed that throughout oocyte growth (stage 4.1 to stage 4.3–5) there was a decrease in the relative abundance of GTH I␤ mRNA coinciding with a stagnation in the pituitary contents and a very significant decrease in circulating levels of GTH I. This decrease confirms the plasma data of Prat et al. (1996) and poses the question of the role of GTH I during full

exogenous vitellogenesis. The decrease of the GTH I␤ mRNA that we observe during exogenous vitellogenesis designates this subunit as a possible regulatory factor in the production of GTH I protein. This result contrasts with the limiting role attributed to GTH ␣ at the end of exogenous vitellogenesis by Naito et al. (1991), following the observation of a decrease in the expression of this subunit in GTH I cells in rainbow trout. Our study did not reveal any significant modification in the GTH ␣-subunit relative content. However, changes of the latter are difficult to interpret as the observed signal can be the result of hybridization with subunit ␣ of GTH I, GTH II, and thyroidstimulating hormone. Nevertheless, it should be noted that in higher vertebrates, the limiting factor in the synthesis of gonadotropins is preferentially the specific hormone ␤-subunit (Leung et al., 1988). Recent data demonstrated that estrogens exert a negative feedback on the release of GTH I in immature or vitellogenic rainbow trout (Saligaut et al., 1998), and therefore the


424

physiological E2 increase that occurs during vitellogenesis may also be responsible for GTH I decrease in the female blood at that stage. Overall, this study demonstrates similar changes in expression of GTH I␤ mRNA and pituitary accumulation and plasma concentration of GTH I during spermatogenesis and oogenesis. This suggests that in vivo the plasma concentration of GTH I is by and large the reflection of the pituitary stock, this being itself the reflection of the levels of GTH I␤ mRNA (in agreement also with the constant ratios reported in Table 3). In sheep, the rate of FSH secretion was also found directly linked to the rate of synthesis of FSH␤ mRNA by Brooks et al. (1992). Whereas pituitary mRNA and protein content remain elevated, the decrease of GTH I plasma/pituitary ratios in females undergoing final maturation and in males at spermiation stage could signify that the secretion of this hormone is selectively inhibited at spawning time. Under our experimental conditions, GTH II␤ mRNA was detected in significant quantities at all stages of gametogenesis, including in immature rainbow trout, aged 9 months. This result agrees with the presence of GTH II␤ mRNA in juvenile male African catfish (Schulz et al., 1995), but is in contrast with the absence of in situ hybridization signal in the salmonid pituitary during previtellogenesis (Naito et al., 1991; Nagae et al., 1996). These divergences are probably the reflection of differences in the ages of animals and in the sensitivity of the methods used. The presence of GTH II␤ mRNA at the initial stages of spermatogenesis and of oogenesis, whereas pituitary synthesis and/or accumulation of the GTH II␤ protein are very low, suggests that this gonadotropin, beyond the positive regulation of the GTH II␤ gene by GnRH and sexual steroids (Xiong et al., 1994; Huggard et al., 1996b; Huang et al., 1997a), could be submitted to a negative translational or posttranslational regulation in the early stages of gametogenesis. Changes in pituitary GTH II/GTH II␤ mRNA and plasma GTH II/pituitary GTH II ratios (Table 3) illustrate an increased pituitary accumulation of this hormone during late stages of gametogenesis. Last, in the spermiating male and the ovulating female, our study shows a large increase in pituitary levels of GTH II in accordance with the intense increase in the GTH II␤ mRNA relative content. These results confirm previous data obtained by dot blot (Weil et al., 1995), those obtained from in situ hybridization (Naito et al.,


Gomez et al.

1991, 1995), and the descriptions of seasonal variations (see Introduction), all indicating an important role for the GTH II in final gonadal maturation. Surprisingly, the plasma GTH II concentration did not abruptly increase at spermiation, suggesting another level of regulation of this secretion (possibly linked to rearing conditions). It must be mentioned that, as stated under Materials and Methods, our immunoassays are highly specific for the homologous gonadotropin, but they potentially measure both the free and the dimerized ␤-subunits. Therefore, we cannot exclude that the values obtained in the blood plasma are not representative of the biologically active heterodimer concentrations and these results should be interpreted with precaution. The general increase of gonadotropin mRNA relative abundance during gametogenesis could be linked to increases in the proportion of gonadotroph cells during sexual maturation (Nozaki et al., 1990; Naito et al., 1991; Weil et al., 1994), in the transcription rate, and/or in the stability of these mRNA. The potential stimulatory effects of androgens or estrogens on the relative abundance of the GTH II␤ subunit (see Xiong et al., 1994; Huggard et al., 1996b; Huang et al., 1997a) suggest that the physiological increase in plasma sexual steroids during gametogenesis had an inducing role on the GTH subunits transcription. The rapid increase in the relative abundance of gonadotropin mRNA at spermiation and postovulation stages also supports the hypothesis of changes in the transcription rate or stability of these mRNA. Analysis of interactions between circulating levels of the principal sex steroids and the different levels of expression of gonadotropin subunits is being investigated at present in our laboratory. This work also describes, for the first time in fish, changes during gametogenesis in GH gene expression, in relation to pituitary and plasma levels of the hormone. In our study, plasma GH concentrations were low and show little variation (probably linked to the good metabolic state of the animals or to the strain of trout used), and this was probably not propicious to the observation of interactions/correlations between GH and the reproductive parameters. In fact, in higher vertebrates, such a relationship could only be suggested in individuals having abnormally high circulating GH concentrations or, conversely, deficiency syndromes. However, an increase in pituitary (mRNA and protein) and plasma concentration was observed in the


425

spermiating male, in accordance with the increase in plasma levels of GH described at the end of the cycle in other studies (Stacey et al., 1984; Sumpter et al., 1991; Le Gac et al., 1991). The increase in GH mRNA in ovulated females could equally entail a plasma increase (lower or later than in spermiating males), as reported by Sumpter et al. (1991). In salmonids, spontaneous decrease in eating at the time of reproduction could provoke these increases in plasma GH, in a way similar to that reported in fasting teleost (Barrett and McKeown, 1988; Niu et al., 1993). On the other hand, the very high sex steroid levels at the end of the reproductive cycle could imply a direct role of these steroids on production and secretion of GH. In effect, the administration of physiological concentrations of androgens and estrogens is capable of stimulating pituitary mRNA and protein GH content (Huggard et al., 1996a; Zou et al., 1997). Analysis of interactions between the circulating levels of the principal sex steroids and levels of pituitary/plasma GH during the gametogenetic cycle is also being investigated at present in our laboratory. Our descriptive study has not demonstrated major simple relationships between GH and gonadotropins at the pituitary level during the reproductive cycle in the trout. Nevertheless, a number of observations deserve to be mentioned. Pituitary size changes with sexual development but also with body growth, and we found a highly significant correlation between GH or GTH I total pituitary contents and body weight in immature fish (data not shown). In an attempt to minimize the influence of general growth (with the view to analyzing the changes specifically linked to the reproductive cycle) we calculated pituitary hormone relative contents normalized to body weight (µg/kg body wt⫺1; data not shown), a transformation used to analyze growth hormone in the pituitary as a function of age, diet, etc. (Marti-Palanca et al., 1996). In the male (stage I to stage VI) and in the female (stage 1 to stage 4.3) pituitary GH relative content decreases, whereas gonadotropin relative content increases. This observation may be brought together with data obtained in vitro, showing that IGF I inhibits the basal production of GH (trout: Perez-Sanchez et al., 1992; Blaise et al., 1995; eel: Huang et al., 1997b), but increases the pituitary content and secretion of GTH II (eel: Huang et al., 1997b), and promotes GTH I and GTH II response

to GnRH (trout: Weil et al., in press). The evolution of IGF I liver mRNA and IGF I plasma levels during gametogenesis is at present under investigation in our laboratory and preliminary results show a significant increase of IGF I plasma concentrations from January to July in males. This is in agreement with the only published data, by Moriyama et al. (1997), that describes increasing plasma IGF I between March and August in precocious salmons. However, that study shows similar seasonal variations in immature females, suggesting that the changes are not linked to gametogenetic stages. Another point of interest is the trend of the increase of GH mRNA simultaneously with an increase in GTH I (mRNA, protein) at the beginning of exogenous vitellogenesis (stage 4.1). Beside the fact that GnRH is capable of stimulating both GH and gonadotropin secretions (see Introduction), estradiol promotes the positive effects of neuromediators on gonadotroph and somatotroph production (Trudeau et al., 1992; Holloway and Leatherland, 1997). The role of these endocrine parameters (GnRH and E2) at the pituitary level should be further investigated at this particular stage. In conclusion, our results demonstrate the existence of a differential regulation in the expression of gonadotropin subunits mRNA and in the storage and secretion of GTH I and GTH II during sexual maturation in rainbow trout. We bring more detailed pituitary and plasma data, suggesting an implication of GTH I at precise stages during the initiation of spermatogenesis and during the very early events of vitellogenesis and an implication of GTH II in the final gonad maturation. Between these two ultimate stages, a role for each of the two gonadotropins remains conceivable during oocyte rapid growth and full meiosis/spermiogenesis. Except during the spawning period, under our experimental conditions pituitary and plasma GH concentrations show only small changes that could be related to the stage of sexual maturation. The potential roles of IGF I and sexual steroids in the coincident changes in pituitary GH and GTH should be further investigated.

ACKNOWLEDGMENTS We thank Drs. S. Itoh, S. Sekine, F. Rentier-Delrue, and F. Pakdel for providing us with chum salmon GTH subunits cDNA, trout


426 cDNA, and trout ␤ actin cDNA, respectively. We are grateful for helpful discussions with Drs. G. Boeuf, S. Dufour, and B. Que´rat. We extend our thanks to Ms. J. Hall for help in revising the English of the manuscript. This work was supported by funds from the ‘‘Institut National de la Recherche Agronomique’’ and from the ‘‘Region Bretagne’’ (BRITTA). The first author received a fellowship from the ‘‘Ministe`re de la Recherche et de l’Enseignement Supe´rieur.’’

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