Structural organization of pi conjugated highly luminescent molecular material.

We report on striking evidence for a room temperature structural phase instability in p-hexaphenyl, inducing a nonplanar conformation of the molecules. Solid state proton NMR and single crystal x-ray diffraction allow the analysis of the organization, the individual dynamics and the involved symmetry breaking. The analysis of Raman spectra above and below room temperature reveals a singular behavior suggesting a modification of the overlap between the electronic wave function induced by the nonplanarity. These results provide a new basis to answer fundamental issues related to molecular and electronic materials and, in particular, luminescent organic devices.

Transcriptional analysis of testis maturation using trout cDNA macroarrays.

The project seeks to identify genes involved in key stages of trout spermatogenesis and their regulation. Within the framework of the French project of farm animal genomics (AGENAE) we produced an original normalised trout testis cDNA library and obtained 1152 trout ESTs corresponding to 967 potential genes. To study the expression of those genes throughout first stages of spermatogenesis, we used nylon macroarray. Gonads in stage of immaturity (stage I), or at initiation of spermatogonial proliferation (stage II), meiosis (stage III) or spermiogenesis were selected by histological analysis. Total RNA was extracted and then used to produce complex targets labelled with [33P]dCTP and hybridised with cDNA arrays. After filtering and normalisation of hybridisation signals, genes presenting differential expression as revealed by ANOVA analysis were submitted to k-means clustering and hierarchical classification. Genes were separated into five clusters which presented distinct profiles. One cluster overexpressed in stage I could be involved in the initial events of spermatogenesis as seminiferous tubule organisation. The second cluster displays a transient increase at the beginning of testicular recrudescence (stage II). Three other clusters group several genes involved in cell proliferation and protein synthesis and modification. One is particularly down-expressed during stage I, the two others show increased expression during stages III and IV and appear to be involved in spermatogonial and meiotic proliferation and in protein metabolism linked to cellular growth. This allows us to plan further experiments to better understand the functional implication of some of the genes that are found to be significantly regulated like CDC2, hematological and neurological expressed gene 1-like protein, HCDI protein, Mago Nashi, a BMP-like, and a steroid receptor binding protein. These data demonstrate the applicability of the array based technology using our trout cDNA arrays and highlight genes that are potential targets for the control of puberty and fertility in farmed fish.

In vitro action of leptin on FSH and LH production in rainbow trout (Onchorynchus mykiss) at different stages of the sexual cycle.

The short-term effect of recombinant human leptin (rhleptin) on FSH and LH production (release+intracellular content) was studied in vitro using pituitary cells from male and female rainbow trout during the first gametogenesis cycle. In our rearing conditions, we found a direct action of rhleptin at the pituitary level, which depends on the sexual stage of the fish. No effect of rhleptin on FSH or LH release and cellular content could be detected in immature fish and post-ovulatory females. However, throughout the process of spermatogenesis and ovogenesis, high concentrations (0.5 and 1 x 10(-6)M) of rhleptin stimulated FSH and LH release, without observable action on intracellular content of gonadotropins. A relatively constant response to rhleptin for FSH was observed throughout gonad maturation, while LH response tended to be higher at the first stages of gametogenesis (beginning of spermatogenesis and endogenous vitellogenesis). Preliminary results on the potential interaction of rhleptin and salmon GnRH (sGnRH) suggest a possible synergistic effect of high concentration of rhleptin (10(-6)M) and sGnRH only at restricted phases of gonadal development when the gametogenetic process was already fully started (full spermatogenesis and early vitellogenesis). The direct action of leptin on FSH and LH release, evident only when gametogenesis had already started suggests that leptin is not the unique signal for the activation of the gonadotropic axis but requires a combined action with other promoting factors.

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis.

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO - Igepal(R) 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine (3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations > or =10 micromol/l. The presence of IGF-I (10-100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25-50 micromol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1-100 micromol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 micromol/l), NP2EO and NP (30 micromol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1-100 nmol/l 17beta-estradiol had no effect in our in vitro system, Triton(R) X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.

Synthesis of gill Na(+)-K(+)-ATPase in Atlantic salmon smolts: differences in alpha-mRNA and alpha-protein levels.

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na(+)-K(+)-ATPase activity of Atlantic salmon smolts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in alpha-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na(+)-K(+)-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abundance and had basal levels of alpha-protein and enzyme activity. Measurement of the binding of [(3)H]ouabain to Na(+)-K(+)-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20-23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in alpha-mRNA levels after 24 h with a rise in Na(+)-K(+)-ATPase activity occurring only after 11 days. No qualitative change in alpha-expression was revealed at either the mRNA or protein level. Immunological identification of the alpha-protein was performed with polyclonal antibodies directed against the rat alpha-specific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na(+)-K(+)-ATPase activity in smolt gills depends first on an increase in the alpha-mRNA expression and is followed by a slower rise in alpha-protein abundance that eventually leads to a higher synthesis of Na(+)-K(+) pumps.

Growth hormone receptors in ovary and liver during gametogenesis in female rainbow trout (Oncorhynchus mykiss).

Changes of growth hormone receptivity in the ovary during the reproductive cycle were studied in rainbow trout (Oncorhynchus mykiss). A method for characterizing growth hormone receptors in crude ovary homogenate was required for this. Binding of radiolabelled recombinant rainbow trout growth hormone (125I-labelled rtGH) to crude ovary preparation was dependent on ovarian tissue concentration. The sites were specific to growth hormone, with no affinity for prolactins and gonadotrophins. Similar high affinities for 125I-labelled rtGH were obtained with crude ovary (4.2 x 10(9) +/- 0.3 mol l-1) and crude liver preparations (4.9 x 10(9) +/- 0.1 mol l-1) at all stages of ovogenesis, and with ovarian membrane preparations (8.2 x 10(9) mol l-1) tested at the beginning of vitellogenesis. Ovarian growth hormone receptor concentration was highest during the early phases of follicular development (endogenous vitellogenesis: 315-310 fmol g-1 ovary) and decreased regularly during oocyte and follicular growth (exogenous vitellogenesis) to reach a minimal value at oocyte maturation (42 fmol g-1 ovary). In postovulated fish, binding was at a similar level (297 fmol g-1 ovary) to that found in endogenous vitellogenesis. Conversely, the absolute binding capacity of the whole ovary was low from immaturity to early exogenous vitellogenesis (0.1-0.6 pmol per pair of gonads), increased slowly during vitellogenesis and more markedly during rapid oocyte growth and at the time of final maturation (10.8 pmol per pair of gonads). In postovulated fish, the absolute binding capacity decreased partially (4.4 pmol per pair of gonads). Mean hepatic growth hormone receptor concentration did not vary with the reproductive stage for most of the cycle (3.0-4.5 pmol g-1 liver) except in endogenous vitellogenesis where significantly higher concentrations were observed (6.7 pmol g-1 liver). Individual ovarian growth hormone receptor concentrations were correlated with hepatic growth hormone receptor concentrations, indicating that they are regulated in a similar way. We conclude that growth hormone receptors are present in the ovary during the entire ovarian cycle in rainbow trout, probably mainly in somatic cells as indicated by the same concentration of binding sites in immature and in postovulated fish. Growth hormone is potentially important during oocyte recruitment in vitellogenesis and initiation of growth and during final follicular maturation.

Growth hormone (GH) and gonadotropin subunit gene expression and pituitary and plasma changes during spermatogenesis and oogenesis in rainbow trout (Oncorhynchus mykiss).

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) alpha- and beta-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 IIbeta, and GTH alpha increased moderately throughout most of gametogenesis and then more dramatically at spermiation and during the periovulatory period. GTH Ibeta mRNA increased first from stage I to V in males and more abruptly at spermiation, while in females GTH Ibeta transcripts increased first during early vitellogenesis and again around ovulation. Pituitary GH absolute content (microgram/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 beta peptide contents were 80- to 500-fold higher than GTH II beta 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 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 ( approximately 2 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 ( approximately 9 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 Ibeta 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.

Growth hormone receptors in testis and liver during the spermatogenetic cycle in rainbow trout (Oncorhynchus mykiss).

Changes in growth hormone (GH) receptivity in the testis during the reproductive cycle were studied in rainbow trout (Oncorhynchus mykiss). This necessitated setting up a method to characterize GH receptors (GH-R) in crude testis homogenate. Binding of radiolabeled recombinant rainbow trout GH (125I-rtGH) to crude testis preparation was dependent on testicular tissue concentration. The sites were specific to GH, with no affinity for prolactins, gonadotropins, or insulin. Similar high affinities for 125I-rtGH were obtained with crude testis (4.5 x 10(9) + 0.25 M(-1)) and crude liver preparations (5.1 x 10(9) +/- 0.06 M(-1)) at all stages of spermatogenesis, as well as with testicular membrane preparations (2.4 x 10(9) M(-1)). GH-R concentration in testis was high during the immature stage (590 fmol/g testis); it then decreased regularly from the beginning (stage II, spermatogonia proliferation: 706 fmol/g testis) to the end of spermatogenesis (stage VII: 158 fmol/g testis). Conversely, the absolute testicular capacity increased dramatically between stages IV and VI (1.2-16.2 pmol/2 gonads), when testicular growth is extremely rapid. GH-R concentration in the liver was highest in early stages (6.5-6.7 pmol/g liver); this value then significantly decreased during midspermatogenesis (stage VI: 3.5 pmol/g liver) and returned to immature levels at the end of the cycle. No significant correlation was found between GH-R in testis, GH-R in liver, and plasma GH concentration. Preliminary experiments performed on isolated populations of testicular cells revealed that binding of 125I-rtGH was detectable in Sertoli cell-enriched fractions but not in germ cell populations. We conclude that GH-R are present in the testis during the entire reproductive cycle in rainbow trout, probably in somatic cells and in particular in Sertoli cells.

Effect of 17 beta-estradiol, testosterone, and 11-ketotestosterone on 17,20 beta-dihydroxy-4-pregnen-3-one production in the rainbow trout testis.

The principal hormone related to spermiation in Oncorhynchus mykiss is 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP). In the present study we analyzed the possible paracrine/autocrine effects of three other testicular steroids (17 beta-estradiol, 11-ketotestosterone, and testosterone) on the synthesis and secretion of this progestin in male rainbow trout. Pieces of testis at various stages of spermatogenesis were incubated for 24 or 48 hr with one of these steroids (5 to 800 ng ml-1) either alone or with the gonadotropin GtH II. Following incubation, 17,20 beta OHP was measured by RIA in the culture media. In vitro, only 17 beta-estradiol (E2) decreased 17,20 beta OHP secretion repeatedly and significantly when doses higher than or equal to 50 ng ml-1 were used. This effect was observed mainly at the spermiating stage and under gonadotropic stimulation. In turn, E2 did not seem to modify the testicular capacity to convert 17-hydroxyprogesterone into 17,20 beta OHP. In vivo, the circulating levels of E2 decreased at the beginning of spermiation, concomitantly with an increase of 17,20 beta OHP in plasma. These in vitro and in vivo data suggest a possible role for E2 in the regulation of 17,20 beta OHP secretion by testes, in particular during the spermiating period.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II >>> insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish.

20 beta-hydroxysteroid dehydrogenase activity in nonflagellated germ cells of rainbow trout testis.

Nonflagellated germ cells were isolated from rainbow trout testis to determine their ability to synthesize 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP), a progestin involved in the control of the release of sperm. Germ cells were obtained by enzymatic dissociation (collagenase; 3 mg.ml-1, 4.5 h, 12 degrees C) from testes that were immature and at the beginning of spermatogenesis. Somatic cells were eliminated by adhesion to the culture plates. Dose-related amounts of 17,20 beta OHP were measured by RIA in culture media of germ cells incubated with increasing dosages of 17-hydroxyprogesterone (17OHP; 0.05-10 micrograms.ml-1) for 20 h at 12 degrees C. Furthermore, 3H-17,20 beta OHP was identified by chromatography and co-crystallization with a reference in incubating cells provided by 3H-17OHP (2.5 and 4 h, 12 degrees C). Other metabolites were detected but not identified. 11-Ketotestosterone (11KT) was either nondetectable by RIA in control cultures or, when detected, was found at very low levels. In no case was 11KT stimulated by addition of 17OHP or gonadotropin II (GtH II; 400 ng.ml-1); this indicated the absence of contamination by Leydig cells. Thus, to our knowledge, this report demonstrates steroidogenic activities in nonflagellated germ cells of fish testis for the first time. 20 beta-Hydroxysteroid dehydrogenase (20 beta HSD) activity was identified, showing that germ cells are able to synthesize 17,20 beta OHP at an early stage in rainbow trout testis.

Synthesis and regulation of 17α-hydroxy-20ß-dihydroprogesterone in immature males of Oncorhynchus mykiss.

Three experimental approaches were chosen to study the question if the progestin 17α-hydroxy-20ß-dihydroprogesterone (17α20ßOHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20ß-hydroxysteroid dehydrogenase activity (20ßHSD), testes homogenates were incubated with (3)H-labeled 17αOHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 17α20ßOHP-(3)H, indicating that already immature testes contain 20ßHSD activity and are able to produce 20ß-reduced steroids. Second, 17α20ßOHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 17α20ßOHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20ßHSD is present in testicular cells other than spermatozoa. Furthermore, 17α20ßOHP is indeed secreted at a very early stage of testicular development; 17α20ßOHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.

Insulin-like growth factor-I and -II binding and action on DNA synthesis in rainbow trout spermatogonia and spermatocytes.

Radio-labeled recombinant human insulin-like growth factor (125I-rhIGF-I) bound specifically to total testicular cells and to spermatogonia plus primary spermatocytes (Go+CI) that had been prepared from trout testes at various maturation stages by centrifugal elutriation and then cleared of somatic cells by preculture in the presence of 2% Ultroser G. Binding sites showed high affinity (Ka = 0.5 +/- 0.2 x 10(10) M-1) and low capacity (1.1 +/- 0.8 fmol/10(6) testicular cells) for 125I-IGF-I. (Gln3, Ala4, Tyr15, Leu16)IGF-I ([QAYL]IGF-I) was equipotent to IGF-I in competing with 125I-IGF-I for site occupancy on Go+CI. Insulin-like growth factor-II (IGF-II) was to 10-fold less potent than IGF-I or (QAYL)IGF-I, while bovine insulin competed only about 300-fold higher concentrations. Go+CI were cultured for 3 days in the presence or absence of mammalian IGF-I, IGF-II, (QAYL)IGF-I, and salmon or bovine insulin. All these molecules stimulated the incorporation of [3H]thymidine (added during the last 24 h in culture) by Go+CI in a dose-dependent manner. The mean ED50, independent of testicular maturation stage, was 5.9 +/- 4.9 ng/ml and 29.1 +/- 15.8 ng/ml for IGF-I and IGF-II, respectively. (QAYL)IGF-I was as potent as IGF-I. Concentrations of salmon or bovine insulin 100- to 300-fold higher were required to produce effects similar to those of IGF-I. While recombinant human IGF binding protein (IGFBP-3) had no effect by itself of basal [3H]thymidine incorporation, it inhibited the effect of IGF-I in a dose-dependent manner; however, it had no effect on the stimulation by (QAYL)IGF-F. Although combinations of low concentrations of IGF-I and IGF-II or salmon insulin had additive effects, combinations of maximum concentrations did not. We conclude that, in vitro, IGFs stimulate DNA synthesis of trout male germ cells by interacting directly with these cells through one IGF receptor.

[Insulin-like growth factors and gonadal regulation in fish]. / Facteurs de croissance de type insulinique et régulation gonadique chez les poissons.

Recent data showing the occurrence of IGF in fish, mainly teleostean fish, and their biological activity in gonads are reviewed. IGF-I does occur in all vertebrates and first evidence of IGF-II occurrence in fish has been reported. IGF-I is expressed in the testis and the ovary, and receptors for the peptide have been demonstrated in both gonads. Besides, IGF-I seems implied in spermatogonial proliferation and in oocyte maturation. Both germinal cells and somatic cells could be responsive to IGF-I stimulation.

Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals.

We describe here an in vitro technique to assess the estrogenic activity of chemicals. This technique is based on rainbow trout hepatocytes incubated in a basic medium free of any additional growth factors or estrogenic chemicals and uses the production of vitellogenin (VTG) as a marker for the estrogenic potency of the compounds tested. The system allows at least some of the metabolic transformations which are undertaken by the liver cells in vivo and could therefore be used for xenobiotic compounds which exhibit estrogenic activities after liver metabolic transformation. A dose-response curve was always consistently obtained using estradiol-17 beta (E2), with a mid point at around 100 nM E2 and a maximum response at around 1000 nM. Established estrogens such as 17 a 1 ethynylestradiol (EE2) or diethylstilboestrol (DES) were also tested. EE2 appeared to be equipotent with E2 and DES slightly less potent. E2 conjugates were, perhaps surprisingly, also very potent. Estradiol-3-sulfate was equipotent with E2 and estradiol-17 beta-glucuronide approx. 10% as potent. Other steroids such as androgens and progesterone, though active in the bioassay, were 3 orders of magnitude less potent than E2. Of the various steroids tested, only cortisol, at concentrations up to 50 microM, was completely inactive. Six different phytoestrogens were tested in the assay. All were weakly estrogenic, possessing approximately one thousandth the potency of E2 (they were as potent as the androgens and progesterone). All six phytoestrogens, as well as the androgens and progesterone, were tested in the presence of tamoxifen. In all cases tamoxifen reduced the production of VTG significantly, demonstrating that the estrogenic action of all of these compounds was most likely mediated by the E2 receptor. The potencies determined here may not reflect the situation in vivo but can provide complementary results about the activity of chemicals which need an hepatic metabolization to be estrogenic. Hepatocyte cultures would profitably be developed in other species to sustain these results.

Growth hormone (GH) and reproduction: a review.

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.

Evidence for binding and action of growth hormone in trout testis.

Growth hormone (GH) binding to testis tissue and GH action on trout testicular cells were studied in vitro. Labeled salmon GH (sGH) was able to bind to a trout testis membrane preparation. Binding sites showed high affinity (Ka = 1-2 x 10(9) M-1) and low capacity (11 fmol/g fresh tissue) for 125I-sGH. Salmon GH and bovine GH, but not salmon gonadotropin, could compete with 125I-sGH for site occupancy. The binding characteristics were similar to those of trout liver GH receptors that we previously described. Salmon GH (0.1 and 1 microgram/ml) and bovine GH (10 micrograms/ml) could modulate steroidogenesis in cultured testicular cells: 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha 20 beta OHP) accumulation in culture medium was stimulated by GH addition, and this effect increased with duration of culture and/or stimulation; 11-ketotestosterone accumulation tended to be inhibited in the presence of GH at the beginning of culture. These effects were dependent on GH concentration and were observed both in the absence and presence of gonadotropin. The amplitude of the sGH effect varied between experiments, probably according to the physiological state of the cells used. In vivo, GH and 17 alpha 20 beta OHP plasma levels increased at the beginning of spermiation (sperm production) and decreased at the end of spermiation. This relationship suggests that, at the end of the reproductive cycle, high GH levels are associated with the production of 17 alpha 20 beta OHP, a progestin necessary for efficient spawning in this species. We conclude that GH may play a role in testicular physiology, at least at certain stages of spermatogenesis.

Influence of hypophysectomy, castration, fasting, and spermiation on SBP concentration in male rainbow trout (Oncorhynchus mykiss).

The influence of different experimental and physiological conditions on sex steroid binding protein (SBP) concentrations in the blood (and in hepatic and testicular cytosols) has been studied on male rainbow trout. SBP was measured with a specific binding assay. The aim was to further understanding of regulation of the SBP and in particular to determine the respective influences of reproductive and metabolic status. Twelve days after hypophysectomy, pituitary and steroid hormones were dramatically decreased and SBP concentrations were significantly lowered in blood (-32%) and in hepatic cytosol (-46%) while the binding protein concentration remained constant in testicular cytosol. Castration of maturing animals did not influence SBP concentration in blood and liver cytosol. Toward the end of the reproductive cycle, a dramatic decrease (-80%) of plasma SBP concentration occurred that appears independent of androgen changes that take place during this period, but is concomitant with a rapid increase of plasma growth hormone (GH) levels (and possibly secondary to a natural arrest of food intake). Long-term fasting that increases endogenous GH but decreases plasma IGFs (insulin-like growth factors) concentrations also induces a limited but significant decrease in SBP concentration. Treatment of intact control trout with recombinant bovine GH (1 microgram/g wt, twice a week, for 6 weeks) increased plasma IGF concentrations but did not significantly increase SBP levels. These results suggest that in mature male trout, testicular androgens have little or no influence on SBP regulation. GH levels or liver GH responsiveness and IGF might be involved in SBP regulation. This would, in part, explain the large decrease in SBP around spermiation in trout.

In vivo and in vitro studies on sex steroid binding protein (SBP) regulation in rainbow trout (Oncorhynchus mykiss): influence of sex steroid hormones and of factors linked to growth and metabolism.

The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout. In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration. Testosterone or cortisol injections have no effect. In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP. Its production is increased by addition of E2 (maximum: +300%). This effect develops slowly over several days of culture and is dose dependent; as little as 1-10 nM E2 is effective. Recombinant rainbow trout GH (rtGH)--0.01 to 1 microgram/ml--also increases SBP accumulation as compared to control cells and seems to maintain SBP production over culture duration. In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF1 (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory. Other hormones tested in vitro: triiodothyronine (10-1000 nM), thyroxine (100 nM), 17 alpha, 20 beta-dihydroprogesterone (10-2000 nM), and testosterone (1-1000 nM) did not influence SBP concentration in hepatic cells culture media.

In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout.

We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.