Daniel Boujard

Fish Growth Hormone Receptor: Molecular Characterization of Two Membrane-Anchored Forms

Abstract A RT-PCR approach was used to clone and sequence the full-length growth hormone receptor (GHR) of a teleost fish, the turbot (Scophthalmus maximus). Total liver RNA was amplified by RT-PCR with degenerate primers designed in extracellular and cytoplasmic regions, and a single DNA fragment of 1100 bp was obtained. The entire coding region was obtained by 5’ and 3’ RACE assays, and comprises an open-reading frame of 633 amino acids. This sequence shows the characteristic motifs of the class I cytokine receptor superfamily, and its amino acid identity with mammalian, avian, reptilian and amphibian GHRs is 32–36%. The 3’ RACE also revealed the occurrence of an alternate messenger encoding a membrane-anchored truncated receptor, which could facilitate the production of GH-binding protein in fish species. This report represents the first data on fish GHR sequence, and it provides evidence for the conservation of this receptor throughout vertebrate evolution.

Localization of the estradiol receptor mRNA in the forebrain of the rainbow trout.

In situ hybridization was used to localize the cells that express the estradiol receptor gene (ER) in the forebrain (hypothalamus, preoptic area, telencephalon) of the rainbow trout (Oncorhynchus mykiss). Both sense and anti-sense [35S]UTP-labeled single-stranded RNA probes were generated from the estradiol binding domain of the ER cDNA. The sense probe was used to evaluate the background of the hybridization reaction. In the forebrain, specific signal appeared in three areas: the posterior hypothalamus, the preoptic area, and the ventral telencephalon. Our localization correlates with [3H]estradiol binding studies in other teleost species. In the pituitary, we observed a weak signal when compared to the signal observed in the forebrain (about ten grains/cell in the pituitary against 35 grains/cell in the posterior hypothalamus). A significant difference was also observed between the intensity of labeling per cell when different forebrain nuclei were compared. We provide here evidence for a tissue-specific regulation of the ER mRNA levels in the trout hypothalamo-pituitary axis.

Insulin-like system and growth regulation in the Pacific oyster Crassostrea gigas: hrIGF-1 effect on protein synthesis of mantle edge cells and expression of an homologous insulin receptor-related receptor

The involvement of molecules belonging to the insulin/IGF family in regulation of growth has been investigated in the Pacific oyster Crassostrea gigas. In vitro biological effects of human recombinant IGF-1 (hrIGF-1) on mantle edge cells, involved in oyster shell and soft body growth, were studied over an annual cycle. In mantle edge cells hrIGF-1 stimulates protein synthesis of 56+/-5.1% over basal for 10(-10) M in September with in addition a clear dose-effect corresponding to the highest shell growth period, and 57.5+/-3.45% over basal for 10(-11) M in March and 51+/-5.4% over basal for 10(-10) M in April corresponding to the period of mantle growth. These insulin-like effects were associated with the expression of a recently identified C. gigas insulin receptor-related receptor (CIR) in mantle edge cells as demonstrated by RT-PCR. Moreover, in situ hybridisation (ISH) confirmed this expression at the level of the inner and outer epithelia involved in mantle growth and shell formation.

Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species

The insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase which plays essential role in the regulation of growth and development. In this study, we have cloned cDNAs encoding the tyrosine kinase domain of the IGF-1R from two species of fish. The turbot and trout nucleotide sequences share 82% identity. Moreover, the deduced polypeptides are also highly conserved (> 90% identity) compared with the IGF-1R sequences described in other vertebrates, particularly within domains involved in the catalytic activity and in the transduction pathway. Northern blot analyses have revealed a unique 13-kb mRNA transcript. Using an RT-PCR approach, we have also shown that the polyadenylation status seems to vary according to the developmental stage in turbot: polyadenylated in oocytes and in the first larval stages, the mRNA becomes undetectable in the polyadenylated fraction in later stages or in adult somatic tissues. These results suggest that IGF-1R mRNA undergoes complex post-transcriptional regulation.

The dynamics of the steroidogenic response of perifused Xenopus ovarian explants to gonadotropins

Steroid release before and during maturation was studied in Xenopus ovarian follicles under various conditions of stimulation of LH in a perifusion system. Acute stimulation by 15 micrograms of LH induces a 10-fold increase in the androgen (testosterone and androstenedione) level which reaches a maximum 4 hr later and then slowly decreases until the 25th hour. Repeated stimulations every 2 or 4 hr are followed by the same androgen increase during the first 8 or 10 hr and then by a slow decrease in the secretion despite new LH injections. A significant increase in progesterone secretion is seen only after at least two stimulations (8 hr). Estradiol secretion slowly increases to a moderate level during the first 5 hr and then remains stable whatever the stimulation. During continuous stimulation (LH 0.5 microgram/ml) androgen levels reach an initial maximum after 4 hr and then fluctuate with a 2-hr period. Addition of theophyllin to the medium enhances these fluctuations. After 12 hr when the progesterone has increased, androgen secretion diminishes to reach a basal level without fluctuations. Germinal vesicle breakdown occurs only in follicles that have been appropriately stimulated to secrete androgens and progesterone during the required time.

Identification of the blood group Lewisa determinant in the oviducal mucins of Xenopus tropicalis

The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewisa epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of 1H‐nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewisa‐bearing O‐linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(β1–3)GlcNAc(β1–6)GalNAc‐ol. These structural data suggested the emergence of an α1,4‐fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis‐fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.

Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters

Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopusleavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.

Zellzyklus und Apoptose

Das Leben ist von der molekularen Ebene bis zur Ebene ganzer Populationen oder zu den Okosystemen durch die permanente Erneuerung alternder oder zerstorter Systeme charakterisiert. Auf der Zellebene manifestiert sich dies in der Zellteilung. Eine Zelle teilt sich nach einer Wachstumsphase in zwei Tochterzellen, die ihre gesamten Eigenschaften erben. Vor jeder Teilung findet eine Verdopplung des Genmaterials zu zwei identischen Kopien statt. Dieses Material wird wahrend der Zellteilung auf zwei verschiedene Zellkerne an zwei entgegengesetzten Zellpolen aufgeteilt, der Prozess wird als Mitose bezeichnet. Sie umfasst die Auflosung des Zellkerns, die Trennung und die Migration der Chromosomen sowie die Wiederherstellung der beiden Kerne. Nach der Mitose teilt sich die Zelle durch Einschnurung in zwei Tochterzellen. Jede enthalt neben dem Zellkern ungefahr die Halfte der Organellen der Mutterzelle. Die Trennung in zwei verschiedene Zellen verlauft bei den pflanzlichen und den tierischen Zellen uber unterschiedliche Mechanismen. In der tierischen Zelle erfolgt die Trennung durch eine Einschnurung in der Mitte, die zu einer Verschmelzung der Membranen fuhrt (Abb. 132.1). Diese Einschnurung wird durch die Kontraktion eines Rings aus Aktinfilamenten verursacht, der an Myosinmolekule assoziiert ist. Die Lage dieses Aktinrings in der Aquatorealebene der Zelle ergibt sich aufgrund seiner Wechselwirkung mit den Mikrotubuli des Spindelapparats.

Expression pattern of insulin receptor mRNA during Xenopus laevis embryogenesis.

We have identified a member of the insulin receptor (InsR)/insulin-like growth factor-1 receptor (IGF-1R) family. The Xenopus insulin receptor (Xe-InsR) is present as a maternal 6.6 kb transcript. Northern blot analysis reveals the presence of this transcript until the mid-blastula transition (MBT), when levels decrease. At neurulation, two distinct transcripts of 6.6 and 7.7 kb are detected, both of which persist throughout embryogenesis. In situ hybridization analysis shows that InsR expression is restricted to regions of ectodermal and mesodermal origin, notably the encephalon, otic vesicles, optic vesicles, gills, somites and the pronephros.

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF‐1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF‐1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52ShcA. Western blot analysis using homologous antibodies evidenced a 60‐kDa protein, p60XlShc, that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60XlShc, Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant‐negative form of Grb2 specifically inhibits insulin‐induced resumption of meiosis. We finally show that Grb2 binds to p60Shc in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2‐Sos are implicated in ras‐dependent Xenopus oocyte maturation induced by insulin/IGF‐1; they also indicate that inability of insulin/IGF‐1 to activate the Ras‐MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.