The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.

Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo.

Chicken embryonic stem (CES) cells are pluripotent cells derived from chicken early blastoderm. In order to identify new genes specifically expressed in these pluripotent cells, we have used a gene trap strategy and cloned a novel gene family called cENS for chicken Embryonic Normal Stem cell gene. The cENS genes expression decreases after induction of CES cells differentiation in culture and is restricted in vivo to the very early embryo. We have characterized three different cENS genes. One, cENS-1, is composed of an open reading frame inserted between two terminal direct repeats which are the common point of the cENS genes. cENS-1 encodes a protein identical to cERNI, a recently described protein. cENS-2 is a truncated form of cENS-1. cENS-3 presents two adjacent open reading frames coding respectively for env and pol related proteins. The presence of conserved direct repeats, of retrovirus related genes and the absence of introns argue in favor of a retroviral origin of the cENS genes. In the cENS we identified a promoter region whose activity is strong in CES cells and decreases after induced differentiation showing a highly specific transcriptional activity specific of undifferentiated chicken embryonic stem cells.

Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development.

The biological activities of thyroid hormones are thought to be mediated by receptors generated by the TRalpha and TRbeta loci. The existence of several receptor isoforms suggests that different functions are mediated by specific isoforms and raises the possibility of functional redundancies. We have inactivated both TRalpha and TRbeta genes by homologous recombination in the mouse and compared the phenotypes of wild-type, and single and double mutant mice. We show by this method that the TRbeta receptors are the most potent regulators of the production of thyroid stimulating hormone (TSH). However, in the absence of TRbeta, the products of the TRalpha gene can fulfill this function as, in the absence of any receptors, TSH and thyroid hormone concentrations reach very high levels. We also show that TRbeta, in contrast to TRalpha, is dispensable for the normal development of bone and intestine. In bone, the disruption of both TRalpha and TRbeta genes does not modify the maturation delay observed in TRalpha -/- mice. In the ileum, the absence of any receptor results in a much more severe impairment than that observed in TRalpha -/- animals. We conclude that each of the two families of proteins mediate specific functions of triiodothyronin (T3), and that redundancy is only partial and concerns a limited number of functions.

Manipulation of blastodermal cells.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.

Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities.

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185-189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

A novel mechanism of action for v-ErbA: abrogation of the inactivation of transcription factor AP-1 by retinoic acid and thyroid hormone receptors.

Ligand-activated retinoic acid receptor alpha (RAR alpha) and c-ErbA alpha repress the AP-1-mediated transcriptional activation of the interstitial collagenase gene promoter by specifically decreasing the activity of the AP-1 transcription factor. On the other hand, the v-ErbA oncoprotein fails to repress the AP-1 activity and acts as a dominant negative oncoprotein by overcoming the repression of the AP-1 activity induced by RAR alpha and c-ErbA alpha. This maintenance by v-ErbA of a fully active AP-1 complex is correlated with the abrogation by this same oncogene product of the growth-inhibitory response of chicken embryo fibroblasts to retinoic acid treatment. This new mechanism of action of v-ErbA together with its previously discovered dominant repressor effect on transcription of thyroid hormone-activated target genes may explain the contribution of the v-erbA oncogene to sarcomatogenic and leukemogenic transformation.

v-erbA oncogene abrogates growth inhibition of chicken embryo fibroblasts induced by retinoic acid.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

EGF-R as a hemopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.

The carbonic anhydrase II gene, a gene regulated by thyroid hormone and erythropoietin, is repressed by the v-erbA oncogene in erythrocytic cells.

The v-erbA oncogene encodes an altered form of the nuclear receptor of the thyroid hormone triiodothyronine (T3). This altered receptor is unable to bind T3, and blocks the differentiation of chicken erythrocyte progenitor cells. To identify the cellular target genes of v-ErbA in the transformed cells, we analysed the expression of several genes in normal erythrocytic cells exposed to T3, and found that the gene encoding carbonic anhydrase II is transcriptionally activated by the hormone. In contrast, this gene is repressed in erythroleukemic cells transformed by the v-erbA product. To investigate in more details the effects of v-ErbA, we constructed a mutant of v-ErbA in which we restored the ability to bind T3. This mutant developed its oncogenicity only in the absence of T3. Upon binding of T3, the transformed cells differentiated and immediately expressed the carbonic anhydrase II gene. These data show that v-ErbA directly inhibits the transcription of the carbonic anhydrase II gene, presumably by competing with normal T3 receptors. The carbonic anhydrase II gene is the first identified target gene of the v-ErbA oncoprotein in erythroleukemic cells.

Expression of the v-erbA product, an altered nuclear hormone receptor, is sufficient to transform erythrocytic cells in vitro.

We investigated the effect of the v-erbA oncogene product, an altered thyroid hormone receptor, in chicken erythrocyte progenitor cells. Bone marrow cells were infected with a retrovirus vector (XJ12) carrying the v-erbA gene in association with the neoR gene. XJ12-infected erythrocyte progenitor cells gave rise to G418-resistant clones. Some were composed of blast cells identified as transformed CFU-Es blocked in their differentiation. These cells could be grown in culture for at least 25 generations and required anemic chicken serum as a source of erythropoietic growth factors. XJ12 can infect erythrocyte progenitor cells in vivo but is not sufficient to induce erythroleukemia. These data suggest that the activation of a nuclear hormone receptor might represent one step toward the development of neoplasms.