Brigitte Mourot

Development and validation of a highly sensitive radioimmunoassay for Chinook salmon (Oncorhynchus tshawytscha) growth hormone

This study describes the development of a highly specific and very sensitive radioimmunoassay for salmonid growth hormone. Antiserum raised against chinook (Oncorhynchus tshawytscha) GH2, which did not recognize 125I-sPRL and 125I-sGTH (at 1:1000 initial dilution), was able to inhibit growth when injected into rainbow trout (Oncorhynchus mykiss). 125I-sGH2, used as tracer, was not recognized by anti-sGTH or by anti-sPRL. Mammalian GH and ACTH and salmonid GTH, TSH, and PRL did not cross-react in the sGH assay. The inhibition curves for pituitary extracts and plasma from salmonids were parallel to the salmon GH standard, whereas those from carp, tilapia, and catfish showed no significant cross reactivity. The RIA ED90 and ED50 values were 0.2 and 1.5 ng/ml, respectively. Using this RIA for measuring GH release by cultured pituitary cell we observed a strong inhibiting effect of SRIF (10(-6) M) and a stimulatory effect of hGRF (10(-6) M). This RIA allowed us also to detect daily fluctuations in the plasma GH concentration in cannulated rainbow trout.

Proteins of seminal fluid and spermatozoa in the trout (Oncorhynchus mykiss): partial characterization and variations.

The protein composition of seminal fluid, blood serum, sperm plasma membrane and flagellum of rainbow trout were analysed by SDS-polyacrylamide gel electrophoresis. Immunological identity between proteins of the 2 fluids and sperm components was studied using crossed immunoelectrophoresis, rocket immunoelectrophoresis and immunoblotting. Results indicate that many seminal proteins are antigenically-related to serum proteins, proteins of sperm origin are present in seminal fluid in varying amounts, depending on the animals and sampling time, and several serum-like seminal proteins are bound to spermatozoa.Lipoproteins were isolated from seminal fluid (mean level: 33 μg/ml) and characterized. They were identified as being HDL-like lipoproteins. A possible physiological role is proposed for these seminal lipoproteins.

Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

BackgroundIn lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish.MethodsIn the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary.ResultsOur results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones.ConclusionTogether, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

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.

Enzyme-linked immunosorbent assay (Elisa) for rainbow trout (Oncorhynchus mykiss) vitellogenin

1. A specific and simple enzyme-linked immunoassay for rainbow trout (Onchorynchus mykiss) vitellogenin (Vtg) is described. This assay is performed using a rabbit antiserum for Vtg purified from trout plasma. 2. This assay is based upon the competition between soluble Vtg and Vtg adsorbed on microtiter plates, for the rabbit anti-Vtg antibody binding sites. 3. The adsorbed Vtg-antibody complexes are revealed through the peroxidase-antiperoxidase antibody, which is colored by o-phenylendiamin. This assay can be performed in a day and a night. 4. Under our conditions, 90-20% of binding gave a sensibility range of 33-1473 ng/ml. With almost a 50% binding yield (335 ng/ml) the intra-assay coefficient of variation (CV) was 5.2% (n = 26) and the inter-assay CV was 12.5% (n = 5). 5. There was low immunological cross-reactivity with sera from other salmonids and with ovary extracts. Extracts of liver from oestrogenized male rainbow trout yielded displacements parallel to the vitellogenin standard and to mature female serum or oestrogenized male serum. 6. This enzyme immunoassay is simple and easy to use. Its great specificity allows its use only for the rainbow trout species.

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.

High temperature increases the masculinization rate of the all-female (XX) rainbow trout "Mal" population.

Salmonids are generally considered to have a robust genetic sex determination system with a simple male heterogamety (XX/XY). However, spontaneous masculinization of XX females has been found in a rainbow trout population of gynogenetic doubled haploid individuals. The analysis of this masculinization phenotype transmission supported the hypothesis of the involvement of a recessive mutation (termed mal). As temperature effect on sex differentiation has been reported in some salmonid species, in this study we investigated in detail the potential implication of temperature on masculinization in this XX mal-carrying population. Seven families issued from XX mal-carrying parents were exposed from the time of hatching to different rearing water temperatures ((8, 12 and 18°C), and the resulting sex-ratios were confirmed by histological analysis of both gonads. Our results demonstrate that masculinization rates are strongly increased (up to nearly two fold) at the highest temperature treatment (18°C). Interestingly, we also found clear differences between temperatures on the masculinization of the left versus the right gonads with the right gonad consistently more often masculinized than the left one at lower temperatures (8 and 12°C). However, the masculinization rate is also strongly dependent on the genetic background of the XX mal-carrying families. Thus, masculinization in XX mal-carrying rainbow trout is potentially triggered by an interaction between the temperature treatment and a complex genetic background potentially involving some part of the genetic sex differentiation regulatory cascade along with some minor sex-influencing loci. These results indicate that despite its rather strict genetic sex determinism system, rainbow trout sex differentiation can be modulated by temperature, as described in many other fish species.

Sex differentiation in an all-female (XX) rainbow trout population with a genetically governed masculinization phenotype.

Sex determination is known to be male heterogametic in the rainbow trout, Oncorhynchus mykiss; however, scattered observations that deviate from this rather strict genetic control have been reported. Here, we provide a detailed morphological and histological characterization of the gonadal differentiation and development (from 43 days postfertilization to 11 months of age) in an all-female (XX) population with a genetically governed masculinization phenotype. In comparison with control males and females, the gonadal differentiation in these animals was characterized by many perturbations, including significantly fewer germ cells. This decrease in germ cells was confirmed by the significantly decreased expression of 2 germ cell maker genes (vasa and sycp3) in the masculinized XX populations as compared with the control females and control males. Although only a proportion of the total adult population was partially or fully masculinized, this early differentiating phenotype affected nearly all the sampled animals. This suggests that the adult masculinization phenotype is the consequence of an early functional imbalance in ovarian differentiation in the entire population. We hypothesize that the lower number of germ cells that we observed in this population could be one cause of their masculinization.

Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements

BackgroundSex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination.ResultsWe now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal.ConclusionsOur work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.