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Les IGF, leurs récepteurs et leurs protéines de liaison constituent une famille moléculaire qui joue un rôle essentiel dans la régulation de la croissance et du développement. Nous nous sommes intéressés à la caractérisation moléculaire de l'IGF-l et de son récepteur (IGF-1R) chez le turbot (Psetta maxima), un poisson plat téléostéen. Par RT-PCR, nous avons clone un ADNc codant pour les quatre domaines de l'IGF-l mature. La protéine présente 70 à 96 % d'identité avec les autres IGF-I de vertébrés. Le clonage de la séquence codante entière du récepteur a nécessité la construction d'une banque d'ADNc d'embryons de turbots. Le récepteur est organisé en domaines dont la taille et les caractéristiques sont semblables à celles des autres récepteurs aux IGF de type 1 décrits chez les mammifères et le poulet. La région catalytique est particulièrement conservée. Dans certains domaines, notamment dans la région C-terminale du précurseur, quelques différences qui pourraient avoir une importance fonctionnelle sont cependant observées. Enfin, par RT-PCR, nous avons mis en évidence une régulation ontogénique du statut de polyadénylation des ARNm IGF-1R.

In bacteria, trans -translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans -translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans -translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans -translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans -translation, we have created a cell-free assay adapted to the rapid evaluation of trans -translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans -translation in these pathogens., (© 2021 Thépaut et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

In contrast to mammals that have limited proliferation and neurogenesis capacities, the Xenopus frog exhibit a great potential regarding proliferation and production of new cells in the adult brain. This ability makes Xenopus a useful model for understanding the molecular programs required for adult neurogenesis. Transcriptional factors that control adult neurogenesis in vertebrate species undergoing widespread neurogenesis are unknown. NeuroD1 is a member of the family of proneural genes, which function during embryonic neurogenesis as a potent neuronal differentiation factor. Here, we study in detail the expression of NeuroD1 gene in the juvenile and adult Xenopus brains by in situ hybridization combined with immunodetections for proliferation markers (PCNA, BrdU) or in situ hybridizations for cell type markers (Vimentin, Sox2). We found NeuroD1 gene activity in many brain regions, including olfactory bulbs, pallial regions of cerebral hemispheres, preoptic area, habenula, hypothalamus, cerebellum and medulla oblongata. We also demonstrated by double staining NeuroD1/BrdU experiments, after long post-BrdU administration survival times, that NeuroD1 gene activity was turned on in new born neurons during post-metamorphic neurogenesis. Importantly, we provided evidence that NeuroD1-expressing cells at this brain developmental stage were post-mitotic (PCNA-) cells and not radial glial (Vimentin+) or progenitors (Sox2+) cells.

In contrast to mammals, the brain of adult non-mammalian vertebrates exhibits a higher proliferative and/or neurogenic activity. To provide new models on this issue, we have examined origin, distribution and fate of proliferating cells in the entire brain of juvenile and adult Xenopus laevis. Using immunohistochemistry for the Proliferation Cell Nuclear Antigen (PCNA), and/or the thymidine analog, 5-Bromo-2' deoxyUridine (BrdU), the labeled cells are located in ventricular zones of the olfactory bulbs, cerebral hemispheres, preoptic region, ventral hypothalamus and cerebellum. Qualitatively, the highest level of proliferative cells was found in the telencephalic ventricles. By using in situ hybridization/immunocytochemistry double-labeling techniques, we demonstrate for the first time in post-metamorphic frog brain that the proliferative cells are localized in very close vivinity to the radial glial cells, progenitor cells that we have also identified in the ventricular layer using classical molecular markers (BLBP, Vimentin). In addition, after long post-BrdU administration survival times ranging between 14 and 28days, BrdU labeling combined with immunohistochemistry for markers of cell migration (DoubleCortin) or radial glial cells (BLBP), reveals that the proliferative cells are able to migrate from the ventricular zone into the brain parenchyma, most likely by migrating along the radial processes. Finally, at survival time of 28days and by using a combination of BrdU labeling and in situ hybridization for markers of differentiation states (Neuro-β-tubulin, Proteolipid Protein), we demonstrate that newborn cells can differentiate in large portion into either neurons or oligodendrocytes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis 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.

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 Lewis(a) epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of (1)H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis(a)-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(beta 1-3)GlcNAc(beta 1-6)GalNAc-ol. These structural data suggested the emergence of an alpha 1,4-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.

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.

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 p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), 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 p60(Shc) 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.

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of beta-catenin. Thus, the IGF pathway is required for head formation during embryogenesis., (Copyright 2002 Elsevier Science (USA).)

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.

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 micron < diameter < 1000 micron) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mos synthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively active raf mRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras --> Raf --> MAPKK --> MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the "Mos synthesis machinery." The present results show that incompetence of Xenopus stage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mos synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.

During the last 20 years, numerous studies have been performed to improve culture conditions in order to allow in vitro growth and meiotic maturation of oocytes isolated from primordial to antral follicles. The main objective of these studies is to define optimal culture conditions which will allow to increase the recovery rate of oocytes that can be successfully fertilized. This paper reviews the more recent advances obtained when studying mouse oocyte development in vitro.

Xenopus laevis postvitellogenic ovarian follicles release into conditioned medium nonsteroidal factor(s) that potentiate progesterone-induced oocyte maturation. The molecular weight of this factor is between 1000 and 10,000 Da. The potentiating activity is suppressed when follicles are incubated with cyanoketone to inhibit steroidogenesis, suggesting that the secretion of the potentiating factor can be modulated in vitro. These results indicate that the steroid-independent pathway in vitro, previously reported in oocytes exposed to insulin or IGF-1 to induce meiotic resumption, may be of physiological importance.

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.

We examined the qualitative patterns of protein synthesis in fully grown prophase-blocked oocytes of Xenopus laevis and after meiosis reinitiation accompanying maturation of the oocytes. Newly synthesized proteins labelled with [35S]methionine were run on isoelectric focusing gels and further separated in the second dimension on SDS-polyacrylamide slab gels. Three types of maturation inducer were compared: progesterone, considered as the natural inducer of Xenopus oocyte maturation, hCG (human chorionic gonadotropin) and insulin. Three polypeptides with apparent molecular masses of 37 kDa (pI 4.7-4.8), 78 kDa (pI 4.7) and 138 kDa (pI 4.6-4.7) were found to be always synthesized in all three types of stimulation, while the synthesis of a fourth one (molecular mass 116 kDa, pI 4.7) was arrested during oocyte maturation. Moreover, when the follicular cells surrounding the oocytes were part of the stimulating pathway, which is the case during hCG-induced maturation, an additional polypeptide was synthesized by the oocytes (molecular mass 106 kDa, pI 6.0-6.2). This polypeptide was not synthesized during progesterone- or insulin-induced oocyte maturation, two types of stimulation which do not require the presence of the follicular cells. The biological significance of the hCG-induced polypeptide, not necessary for oocyte maturation, is discussed. On the other hand, the four other modifications in protein synthesis taking place during all three types of maturation-inducing stimulation appear to be necessary for oocyte maturation, since oocytes which failed to mature in response to stimulation always missed one or several of these four polypeptides.

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.

Human sperm are a heterogeneous population, particularly with respect to their morphology, motility, and degree of nuclear maturity. The characteristics of human sperm and the degree of nuclear condensation with variable sexual abstinence times (long, 7 days; short, 12 h) have been studied. Long abstinence led to an increase in the number of sperm and a decrease in their motility, but their morphology remained unchanged. The DNA-protein complex demonstrated by ethidium bromide uptake was unchanged, but there was a significant increase in nuclear stability upon treatment with SDS. The duration of abstinence hardly affected the degree of nuclear condensation or stability of human sperm. The heterogeneity observed is essentially of testicular origin.

The dynamics of androgen production by perfused Xenopus testis explants has been investigated under various gonadotropic stimulations. Androgens were measured in perfusate by radioimmunoassay. The behavior of hormones in the perfusion system was assessed. Pieces of testis were capable of producing androgens after more than 20 hr in perifusion. Amplitude and duration of the response to a gonadotropic stimulation were the same 3 and 10 hr after the beginning of the experiment. After stimulation by pituitary extract, hCG, or carp GTH, there was a rapid tenfold increase in androgen secretion with a maximum after 1.5 hr and then a slow decrease to a basal line level. A typical log dose-response curve was obtained with hCG and cGTH. After 6 hr continuous stimulation by pituitary extract, steroid secretion increased and reached a plateau 4 hr later. The decrease occurred only after the end of stimulation. The same amount of pituitary extract injected into the column at the rate of 4 min stimulations every hr gave the same type of response. As far as is known, the present data are the first on perifusion systems on the testes of lower vertebrates.