Insulin receptor and IGF1R are not required for oocyte growth, differentiation, and maturation in mice.

In mammals, insulin and insulin-like growth factors (IGFs: IGF1 and IGF2) act through 2 structurally related receptors, the insulin receptor (INSR) and the type 1 IGF receptor (IGF1R), both of which are expressed in developing oocytes. IGF1 plays an important role in female reproduction, and female Igf1 knockout mice fail to ovulate and are infertile. On the other hand, little is known about the in vivo role of the insulin signaling pathway in oocytes during follicular development, although exposure to insulin or IGF1 in vitro improves oocyte maturation. To further address the significance of insulin/IGF signaling, we used conditional mutant mice and ablated the function of the genes encoding INSR, IGF1R, or both receptors specifically in developing mouse oocytes. Our genetic evidence showed unexpectedly that the female reproductive functions are not affected when Insr, Igf1r or both Insr;Igf1r are ablated in oocytes, as the female mice are fertile and exhibit normal estrous cyclicity, oocyte development and maturation, parturition frequency, and litter size. In view of these novel observations indicating that the insulin/IGF signaling is not essential in oocytes, the IGF1-dependent female fertility is re-evaluated and discussed.

[Of epigenetics and development]. / De l'épigénétique et du développement.

Epigenetics is defined as the study of the heritable changes in gene expression that are transmitted from a mother cell to a daughter cell or a parent to its progeny, that occur without modifications in DNA sequences. The epigenetic mechanisms of gene regulation include DNA methylation and post-translational modifications of histone tails such as acetylation. Generally speaking, the more methylated the DNA is, and the less acetylated the associated histones are, the less a gene will be expressed. Herein, we introduce the reader to the field of epigenetic reprogramming and its importance during embryonic development and gametogenesis. We also summarize the current litterature on environmental influences on epigenetic processes including the epigenetic transgenerational actions of endocrine disruptors and diet.

Neuronal overexpression of tissue-type plasminogen activator does not enhance sensory axon regeneration or locomotor recovery following dorsal hemisection of adult mouse thoracic spinal cord.

CNS axons rarely regenerate spontaneously back to original targets following spinal cord injury (SCI). Neuronal expression of the serine protease tissue-type plasminogen activator (tPA) enhances axon growth in vitro and following PNS injury. Here we test the hypothesis that neuronal overexpression of tPA in adult transgenic mice promotes CNS axon regeneration and functional recovery following SCI. Adult wild-type and transgenic mouse spinal cords were subjected to dorsal hemisection at the level of the T10/T11 vertebrae. PCR confirmed incorporation of the transgene. Immunolabeling revealed overexpression of tPA in transgenic mice in neurons, including large-diameter neurons in lumbar dorsal root ganglia that contribute axons to the dorsal columns. Immunolabeling also revealed the presence of tPA protein within axons juxtaposing the injury site in transgenics but not wild types. In situ zymography revealed abundant enzymatic activity of tPA in gray matter of thoracic spinal cords of transgenics but not wild types. Rotorod locomotor testing revealed no differences between groups in locomotor function up to 21 days postinjury. Transganglionic tracer was injected into the crushed right sciatic nerve 28 days postinjury, and mice were killed 3 days later. There was no evidence for regrowth of ascending dorsal column sensory axons through or beyond the injury site. In conclusion, despite neuronal overexpression of tPA in injured neurons of transgenics, neither locomotor recovery nor regeneration of ascending sensory axons was observed following thoracic dorsal hemisection.

Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice.

The serpin plasminogen activator inhibitor (PAI) type 2 is expressed in differentiated epidermal keratinocytes. To explore its role in this tissue, we studied the impact of PAI-2 overexpression on epidermal differentiation and skin carcinogenesis. A mouse PAI-2-encoding transgene was targeted to basal epidermis and hair follicles under the control of the bovine keratin type 5 gene promoter. Two mouse lines were established, one of which strongly expressed the transgene and produced elevated levels of PAI-2 in the epidermis. Although it had no manifest impact on cellularity or differentiation of skin or hair follicles, PAI-2 overexpression rendered the mice highly susceptible to skin carcinogenesis induced by a single application of 7,12-dimethylbenz(a)anthracene (initiation) followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate [TPA (promotion)]. In transgenic mice, papillomas could be observed after 3 weeks of promotion; after 8 weeks, 94% (31 of 33) of transgenic mice had developed readily visible papillomas, whereas only 35% (7 of 20) of control mice (transgene-negative littermates) had barely detectable lesions. After 11 weeks, all but 1 (32 of 33) of the transgenic mice had papillomas as compared with only 65% (13 of 20) of control mice. After 11 weeks of promotion, application of TPA was terminated. In control mice, papillomas regressed and eventually disappeared; in transgenic mice, there was continued growth of papillomas, some of which further progressed to carcinomas. In contrast to massive apoptosis in regressing papillomas of control mice, only a few apoptotic cells were detected in transgenic papillomas after the cessation of TPA application. The effect of PAI-2 on papilloma formation did not appear to involve inhibition of the secreted protease urokinase-type plasminogen activator (uPA): PAI-2 accumulated predominantly in cells, and PAI-2 overexpression failed to alleviate a phenotype induced by uPA secretion, as demonstrated by a double transgenic strategy. In addition, in situ hybridization revealed that uPA mRNA is not expressed concomitantly with PAI-2 in developing papillomas. We conclude that overexpression of PAI-2 promotes the development and progression of epidermal papillomas in a manner that does not involve inhibition of its extracellular target protease, uPA, but appears to be related to an inhibition of apoptosis.

Inducible and irreversible control of gene expression using a single transgene.

Experimental or therapeutic designs involving the conditional expression of genes often require the use of two different transgenes; this can represent a major undertaking. One of these systems takes advantage of inducible recombinases. Here we show a novel use of such enzymes, in that an inducible recombinase-encoding sequence can function to both block the transcription of a gene placed downstream and, subsequently, irreversibly activate transcription of this very same gene. This double function, which circumvents the need for two transgenes, can be achieved by flanking the inducible recombinase gene by two of its target sequences. In our design we used as the inducible recombinase gene the Cre-ER(T) gene flanked by two loxP sites. This cassette was placed between a mouse phosphoglycerate kinase promoter and the enhanced green fluorescent protein (EGFP) coding sequence. Massive EGFP gene expression in BHK cells bearing this transgene was observed upon administration of 4-hydroxytamoxifen (4-OHT), the inducer of the recombinant activity of Cre-ER(T). In the absence of 4-OHT EGFP production was prevented. Because of its simplicity (only a single transgene needs to be used) this strategy is of obvious interest in certain protocols of gene or cell therapy and in a variety of experimental designs in which conditional expression of genes is required.

Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis.

Urokinase-type plasminogen activator (uPA) is a potent catalyst of extracellular proteolysis, which also binds to a high-affinity plasma membrane receptor (uPAR). Binding of uPA may influence pericellular proteolysis and/or activate intracellular signal transduction. Transgenic mice overexpressing either uPA or uPAR in basal epidermis and hair follicles had no detectable cutaneous alterations. In contrast, bi-transgenic mice overexpressing both uPA and uPAR, obtained by crossing the two transgenic lines, developed extensive alopecia induced by involution of hair follicles, epidermal thickening and sub-epidermal blisters. The phenotype was due to uPA catalytic activity since combined overexpression of uPAR and uPAR-binding but catalytically inactive uPA in the same tissue was not detrimental in another bi-transgenic line. It was accompanied by increased plasmin-generating capacity, up-regulation and activation of matrix metalloproteinases type-2 and -9, and cleavage of uPAR. Thus, combined overexpression of uPA and uPAR acts in synergy to promote pathogenic extracellular proteolysis.

The 3' untranslated region of messenger RNA: A molecular 'hotspot' for pathology?

The role of the 3' untranslated region in posttranscriptional regulation of mRNA expression is being elucidated. Here we describe diseases arising from anomalies in this region, that affect the expression of one or more genes.

Plasminogen activation in human acute leukaemias.

Plasminogen activation is implicated in solid tumour growth, invasion and metatastic spread. However, little is known about its role in leukaemia. We investigated the production by leukaemic cells of plasminogen activators [urokinase (uPA) and tissue-type PA (tPA)], cell surface receptor for uPA (uPAR) and PA inhibitors (PAI-1 and PAI-2). Leukaemic cells from 37 patients [26 with acute myeloid leukaemia (AML) and 11 with acute lymphoid leukaemia (ALL)] were analysed for mRNA content and enzymatic activities. High levels of uPA mRNA were found in M1, M2, M3 and M4-M5 AMLs, whereas tPA mRNA was not detected in any of the analysed cases. uPAR mRNA was confined to subtypes M4-M5. PAI-1 mRNA was detected in M3 and M4-M5. PAI-2 mRNA was found predominantly in M2 and M4-M5. SDS-PAGE/zymography analyses of cell extracts and supernatants after 24 and 48 h of culture confirmed the production of active uPA by AML cells (mainly M4-M5). but not by ALL. The finding of uPA, uPAR, PAI-1 and PAI-2 synthesized by leukaemic cells suggests that plasminogen activation may contribute to the invasive behaviour of these cells, the fibrinolytic imbalance observed in leukaemic patients and the differentiation and proliferation of M4-M5 by interaction of uPA with uPAR.

Enhanced hippocampal long-term potentiation and learning by increased neuronal expression of tissue-type plasminogen activator in transgenic mice.

Adult cortical neurons can produce tissue-type plasminogen activator (tPA), an extracellular protease that plays a critical role in fibrinolysis and tissue remodelling processes. There is growing evidence that extracellular proteolysis may be involved in synaptic plasticity, axonal remodelling and neurotoxicity in the adult central nervous system. Here we show that transgenic mice overexpressing tPA in post-natal neurons have increased and prolonged hippocampal long-term potentiation (LTP), and improved performance in spatial orientation learning tasks. Extracellular proteolysis catalysed by tPA may facilitate synaptic micro-remodelling, and thereby play a role in activity-dependent neuronal plasticity and learning.

Extracellular proteolysis alters tooth development in transgenic mice expressing urokinase-type plasminogen activator in the enamel organ.

By catalyzing plasmin formation, the urokinase-type plasminogen activator (uPA) can generate widespread extracellular proteolysis and thereby play an important role in physiological and pathological processes. Dysregulated expression of uPA during organogenesis may be a cause of developmental defects. Targeted epithelial expression of a uPA-encoding transgene under the control of the keratin type-5 promoter resulted in enzyme production by the enamel epithelium, which does not normally express uPA, and altered tooth development. The incisors of transgenic mice were fragile, chalky-white and, by scanning electron microscopy, their labial surface appeared granular. This phenotype was attributed to a defect in enamel formation during incisor development, resulting from structural and functional alterations of the ameloblasts that differentiate from the labial enamel epithelium. Immunofluorescence revealed that disorganization of the ameloblast layer was associated with a loss of laminin-5, an extracellular matrix molecule mediating epithelial anchorage. Amelogenin, a key protein in enamel formation, was markedly decreased at the enamel-dentin junction in transgenics, presumably because of an apparent alteration in the polarity of its secretion. In addition, increased levels of active transforming growth factor-beta could be demonstrated in mandibles of transgenic mice. Since the alterations detected could be attributed to uPA catalytic activity, this model provides evidence as to how dysregulated proteolysis, involving uPA or other extracellular proteases, may have developmental consequences such as those leading to enamel defects.

Two transgenic approaches to define the cell lineages in endocrine pancreas development.

Ontogenic relationships between the different endocrine cell types of the islets of Langerhans were explored by generating transgenic mice, in which cells transcribing the glucagon, insulin, or pancreatic polypeptide genes were destroyed through the promoter-targeted expression of the diphtheria toxin A chain. In an alternate approach, to assess whether insulin cells are derived from precursors producing glucagon or PP, transgenic mice were generated bearing an insulin promoter-driven, and loxP-containing ('floxed') reporter transgene that can be irreversibly 'tagged' by recombination. They were crossed with mice expressing another transgene ('tagger') encoding Cre (cyclization recombination) recombinase in either glucagon or PP cells. The results obtained using both approaches indicate that neither glucagon nor insulin gene-expressing cells are the precursors to the other islet cells; also, they suggest that PP gene-expressing cells are necessary for the differentiation of islet insulin and somatostatin cells, through a cell lineage or a paracrine relationship.

Masking, unmasking, and regulated polyadenylation cooperate in the translational control of a dormant mRNA in mouse oocytes.

The mechanisms responsible for translational silencing of certain mRNAs in growing oocytes, and for their awakening during meiotic maturation, are not completely elucidated. We show that binding of a approximately 80-kD protein to a UA-rich element in the 3' UTR of tissue-type plasminogen activator mRNA, a mouse oocyte mRNA that is translated during meiotic maturation, silences the mRNA in primary oocytes. Translation can be triggered by injecting a competitor transcript that displaces this silencing factor, without elongation of a pre-existing short poly(A) tail, the presence of which is mandatory. During meiotic maturation, cytoplasmic polyadenylation is necessary to maintain a poly(A) tail, but the determining event for translational activation appears to be the modification or displacement of the silencing factor.

Targeted gene disruption reveals a leptin-independent role for the mouse beta3-adrenoceptor in the regulation of body composition.

Targeted disruption of mouse beta3-adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the beta3-adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null (-/-) mice. In brown adipose tissue, beta3-adrenoceptor disruption induced a 66% decrease (P < 0.005) in beta1-adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in -/- mice, body fat accumulation was favored (+41%, P < 0.01), with a slight increase in food intake (+6%, NS). These effects were accentuated by high fat feeding: -/- mice showed increased total body fat (+56%, P < 0.025) and food intake (+12%, P < 0.01), and a decrease in the fat-free dry mass (-10%, P < 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in -/- and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed -/- mice suggests that the threshold of body fat content inducing leptin secretion is higher in -/- than in control mice. Taken together, these studies demonstrate that beta3-adrenoceptor disruption creates conditions which predispose to the development of obesity.

The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis.

Urokinase-type plasminogen activator (uPA) binds to a plasma membrane receptor (uPAR) that localizes plasmin generation to the cell environment. Mouse spermatozoa have surface-bound uPA, which appears to be acquired from genital tract secretions at ejaculation. We determined the presence of uPAR mRNA in spermatogenic cells and their uPA-binding activity. Northern blot and in situ hybridization demonstrated the presence of uPAR mRNA in germ cells. Binding of uPA, but not of a mutant enzyme lacking the receptor-binding domain, indicated the presence of uPAR on spermatids and spermatozoa. The uPAR and/or receptor-bound uPA may be involved in spermatogenesis, spermatozoa maturation or fertilization.

Acquisition of meiotic competence in growing mouse oocytes is controlled at both translational and posttranslational levels.

Full-grown mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment. By contrast, growing oocytes are not competent to resume meiosis; the molecular basis of meiotic competence is not known. Entry into M phase of the eukaryotic cell cycle is controlled by MPF, a catalytically active complex comprising p34cdc2 kinase and cyclin B. Incompetent oocytes contain levels of cyclin B comparable to those in competent oocytes, while their level of p34cdc2 is markedly lower; p34cdc2 accumulates abruptly at the end of oocyte growth, at the time of meiotic competence acquisition. We show here that this change in p34cdc2 concentration is not secondary to a corresponding change in the concentration of the cognate mRNA, indicating that translational control may be involved. Microinjection of translatable p34cdc2 mRNA into incompetent oocytes yielded high levels of the protein, but it did not lead to resumption of meiosis. Similarly, microinjection of cyclin B1 mRNA resulted in accumulation of the protein, but not in the acquisition of meiotic competence. By contrast, the microinjection of both p34cdc2 and cyclin B1 mRNAs in incompetent oocytes induced histone H1 and MAP kinase activation, germinal vesicle breakdown, and entry into M-phase including the translational activation of a dormant mRNA. Thus, endogenous cyclin B1 in incompetent oocytes is not available for interaction with p34cdc2, suggesting that a posttranslational event must occur to achieve meiotic competence. Microinjection of either p34cdc2 or cyclin B1 mRNAs accelerated meiotic reinitiation of okadaic acid-treated incompetent oocytes. Taken together, these results suggest that acquisition of meiotic competence by mouse oocytes is regulated at both translational and posttranslational levels.

In vivo antisense oligodeoxynucleotide mapping reveals masked regulatory elements in an mRNA dormant in mouse oocytes.

In mouse oocytes, tissue-type plasminogen activator (tPA) mRNA is under translational control. The newly transcribed mRNA undergoes deadenylation and translational silencing in growing oocytes, while readenylation and translation occur during meiotic maturation. To localize regulatory elements controlling tPA mRNA expression, we identified regions of the endogenous transcript protected from hybridization with injected antisense oligodeoxynucleotides. Most of the targeted sequences in either the 5' untranslated region (5'UTR), coding region, or 3'UTR were accessible to hybridization, as revealed by inhibition of tPA synthesis and by RNase protection. Two protected regions were identified in the 3'UTR of tPA mRNA in primary oocytes: the adenylation control element (ACE) and the AAUAAA polyadenylation signal. These sequences were previously shown to be involved in the translational control of injected reporter transcripts. During the first hour of meiotic maturation, part of the ACE and the AAUAAA hexanucleotide became accessible to hybridization, suggesting a partial unmasking of the 3'UTR of this mRNA before it becomes translationally competent. Our results demonstrate that in vivo antisense oligodeoxynucleotide mapping can reveal the dynamics of regulatory features of a native mRNA in the context of the intact cell. They suggest that specific regions in the 3'UTR of tPA mRNA function as cis-acting masking determinants involved in the silencing of tPA mRNA in primary oocytes.

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.

Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies.

Protease nexin 1 (PN-1), a potent serpin-class antiprotease, is thought to be synthesized in the murine kidney. However, neither the cellular localization of PN-1 synthesis nor its role has yet been defined. To address these questions, we determined by in situ hybridizations RNase protection assay and immunoblotting, the sites of PN-1 mRNA accumulation in normal mouse kidneys and the modulation of PN-1 expression in several pathological conditions. In normal kidneys, PN-1 mRNA was detected primarily in glomeruli, most likely in mesangial cells. The glomerular expression of PN-1 was substantially enhanced not only in lupus-like glomerulonephritis (induced by IgG3 monoclonal rheumatoid factors or occurring spontaneously in lupus-prone mice), but also in mild glomerular lesions associated with intracapillary thrombi induced by IgG3 anti-trinitrophenyl monoclonal antibodies. In contrast, no modulation of PN-1 mRNA levels was observed during the course of lipopolysaccharide-induced acute tubular necrosis. A constitutive PN-1 gene expression and its up-regulation during glomerular injury suggest a possible role for PN-1 in glomerular biology. In view of its high inhibitory activity towards thrombin, mesangial PN-1 may be involved in the control of glomerular coagulation following initial glomerular injuries.

The kidney is a major site of alpha(2)-antiplasmin production.

The serpin alpha2-antiplasmin (alpha2-AP) is the major circulating inhibitor of plasmin; it plays a determining role in the regulation of intravascular fibrinolysis, In addition to blood plasma, plasmin formation occurs in various organs where it is thought to fulfill a spectrum of functions not restricted to clot lysis. Alpha2-AP is synthesized by hepatocytes, but other possible sites of production have not been investigated. To explore the potential extravascular contribution of alpha2-AP in the regulation of proteolysis, we have isolated the murine alpha2-AP cDNA and determined its mRNA distribution in adult tissues. In addition to liver, kidneys are major sites of alpha2-AP mRNA accumulation in the mouse. The transcript is present in epithelial cells lining the convoluted portion of proximal tubules, and its accumulation is under androgen control. Human kidneys also contain high levels of alpha2-AP mRNA. Moderate amounts Of alpha2-AP mRNA are detected in other murine tissues such as muscle, intestine, central nervous system, and placenta. Our observations indicate that alpha2-AP can be synthesized in a number of tissues, where it could function as a distal regulator of plasmin-mediated extracellular proteolysis.