Carl Séguin

Increased testicular 5α-androstane-3α, 17β-diol formation induced by treatment with [D-Ser(TBU)6, des-Gly-NH210]LHRH ethylamide in the rat

Abstract While the intact male adult rats respond to LH with a predominant increase of testicular and plasma testosterone levels, the response to LH stimulation in animals treated with the LHRH agoriist, [D-Ser(TBU) 6 , des-Gly-NH 2 10]LHRH ethylamide is characterized by a major production of 5α-androstane-3α, 17β-diol. The marked increase of 5α-androstane-3α, 17β-diol levels in the presence of a 90% decrease of testosterone concentration strongly suggests that 5α-reductase and 3α-hydroxysteroid oxidoreductase activities are increased during testicular desensitization induced by treatment with the LHRH agonist.

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs.

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non‐canonical Wnt genes, Wnt‐5a and Wnt‐5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt‐5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration‐competent cells of the blastema.

Additive inhibitory effects of treatment with an LHRH agonist and an antiandrogen on androgen-dependent issues in the rat

Combined treatment of adult male rats with the LHRH agonist, [D-Ser(TBU)6, des-Gly-NH2(10)]LHRH ethylamide, and a non-steroid antiandrogen, RU 23908, led to a rapid and marked atrophy of the ventral prostate and seminal vesicles. Treatment with the LHRH agonist decreased androgen secretion and thus facilitated the action of the antiandrogen in androgen-dependent tissues. Such a combined treatment could be useful in the treatment of androgen-dependent pathologies in man, particularly in prostatic adenocarcinoma and possibly benign prostatic hyperplasia.

A nuclear factor requires Zn24 to bind a regulatory MRE element of the mouse gene encoding metallothionein-1

The metal ion requirement of nuclear proteins for binding to the metal regulatory element d(MREd) of the mouse gene encoding metallothionein-1 was investigated using an in vitro exonuclease III footprinting assay. The specific DNA-binding activity of the factor was inactivated by the chelating agents, EDTA and 1,10-phenanthroline. Binding activity was restored by Zn2+, but not by Cd2+. These results show that Zn2+ ions are a required component for specific in vitro DNA binding of the MREd-binding protein.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions

Metal activation of metallothionein (MT) gene transcription is dependent on the presence of metal regulatory elements (MREs), which are present in five non-identical copies (MREa through MREe) in the promoter of the mouse MT-1 gene and on the capacity of metal transcription factor-1 (MTF-1) to bind to the MREs in the presence of zinc. We detected a protein, distinct from MTF-1, specifically binding to the MREc region. DNA binding competition experiments using synthetic oligonucleotides and specific anti-NF1 antibodies showed that this protein binds to an NF1 site overlapping the MREc element as well as to a second site upstream of the Sp1a site and corresponds to NF1 or a related protein. Transfection experiments showed that loss of the two NF1 sites decreased metal-induced MT promoter activity by 55–70% in transiently transfected cells and almost completely abrogated metal and tert-butylhydroquinone (tBHQ) induction in stably transfected cells. Similarly, expression of an inactive NF1 protein strongly inhibited MT-1 promoter activity. Using synthetic promoters containing NF1 and MRE sites fused to a minimal MT promoter, we showed that these NF1 sites did not confer metal induction but enhanced metal-induced promoter activity. Chromatin immunoprecipitation assays confirmed that NF1 binds to the mouse MT-1 promoter in vivo and showed that NF1 binding is zinc-inducible. In addition, zinc-induced NF1 DNA binding was MTF-1-dependent. Taken together, these studies show that NF1 acts synergistically with MTF-1 to activate the mouse MT-1 promoter in response to metal ions and tert-butylhydroquinone and contributes to maximal activation of the gene.

Impairment of pituitary and gonadal functions in alloxan-induced diabetic male rats

The inhibitory effect of treatment with a potent LHRH agonist on testicular gonadotropin-receptor levels was compared in intact and diabetic rats. Basal and LHRH-induced pituitary gonadotropin secretion as well as the testicular steroidogenic response to oLH were assessed. A single injection of alloxan (65 mg/kg) led, after 6 weeks, to a 40% decrease of testicular LH- and prolactin-receptor levels. Treatment for 2 weeks with [D-Ala6,des-Gly-NH 1/2 0] LHRH ethylamide (100 ng every second day) led to a 70% reduction of LH-receptor levels accompanied by decreased testicular weight, a similar inhibition being found in intact and diabetic animals. Seminal vesicle and ventral prostate weight were markedly reduced in diabetic animals, a further decrease being obtained after treatment with the LHRH agonist. The loss of accessory sex-organ weight in alloxan-diabetic rats was accompanied by a reduction in the basal testicular content of pregnenolone, progesterone, 17-OH-progesterone, androstenedione, testosterone and dihydrotestosterone whereas the steroid response to oLH was within normal limits. We next examined the possible changes of LH and FSH secretion which could be responsible for the reduced testicular function in diabetic animals. Basal plasma-LH levels were 30% reduced in rats 6 weeks after treatment with alloxan while basal plasma-FSH levels remained unchanged. When the pituitary gonadotropin response to LHRH was measured in chronically cannulated freely-moving intact and diabetic rats, an approx. 50% inhibition of the LH and FSH responses to LHRH was observed in diabetic animals.

Isolation of cDNAs for two closely related members of the axolotl Wnt family, Awnt-5A and Awnt-5B, and analysis of their expression during development

To characterize molecular interactions between cells in the early amphibian embryo, we have isolated cDNAs for two members of the axolotl (Ambystoma mexicanum) Wnt family, Awnt-5A and Awnt-5B. The encoded proteins share 83% amino acid identity. Using a reverse transcription-polymerase chain reaction (RT-PCR) assay, we find that Awnt-5A transcripts are abundant in the blastula until gastrulation, barely detectable during gastrulation, and increase again during neurulation. They are detected throughout the remaining development and in hatched larvae. In contrast, transcripts for Awnt-5B are undetectable in the blastula. They appear with gastrulation, are present throughout neurulation and organogenesis, and decrease to barely detectable levels in hatched larvae. PCR reactions performed using cDNA library-phage DNA templates derived from whole neurulae versus embryos with the neuroectoderm removed suggest that, in the neurula, Awnt-5A transcripts are present in neuroectodermal as well as non-neuroectodermal tissues while Awnt-5B mRNAs are predominantly localized in the neuroectoderm. To localize Awnt-5A expression in embryos before gastrulation, early gastrulae were dissected by cutting along the animal-vegetal and future dorso-ventral axes and analyzed by RT-PCR. At this early stage, Awnt-5A transcripts appear to be predominantly localized in the dorso-vegetal region of the embryo. These results suggest that the two closely related Awnt-5 genes participate in different morphogenetic processes during early axolotl development.

Hypoxia acts through multiple signaling pathways to induce metallothionein transactivation by the metal-responsive transcription factor-1 (MTF-1)

Metal-responsive transcription factor-1 (MTF-1) is essential for the induction of genes encoding metallothionein by metals and hypoxia. Here, we studied the mechanism controlling the activation of MTF-1 by hypoxia. Hypoxia activation of Mt gene transcription is dependent on the presence of metal regulatory elements (MREs) in the promoter of Mt genes. We showed that MREa and MREd are the main elements controlling mouse Mt-1 gene induction by hypoxia. Transfection experiments in Mtf-1-null cells showed that MTF-1 is essential for induction by hypoxia. Chromatin immunoprecipitation analysis showed that MTF-1 DNA-binding activity was strongly enhanced in the presence of zinc but not by hypoxia. Notably, hypoxia inducible factor- (HIF) 1α was recruited to the Mt-1 promoter in response to hypoxia but not to zinc. MTF-1 activation was inhibited by PKC, JNK, and PI3K inhibitors and by the electron transport chain inhibitors rotenone and myxothiazol, but not by the antioxidant N-acetylcysteine. We showed that prolyl-hydroxylase inhibitors can activate MTF-1, but this activation requires the presence of HIF-1α. Finally, HIF-dependent transcription is enhanced in the presence of MTF-1 and induction of an MRE promoter is stimulated by HIF-1α, thus indicating cooperation between these 2 factors. However, coimmunoprecipitation experiments did not suggest direct interaction between MTF-1 and HIF-1α.

Metallothionein Gene Regulation in Mouse Cells

Heavy metals (Cd, Cu, Zn, etc.) can affect the expression of many genes. The best-known proteins that bind these metal ions are the metallothioneins (MTs). The genes encoding MTs are inducible at the transcriptional level by the same metal ions that the MTs bind. Metal activation of MT gene transcription is dependent on the presence of cis-acting DNA elements termed Metal Response Elements (MREs), and involves trans-acting protein (factor(s) interacting with the MREs, present in six nonidentical copies (MREa through MREf) in the 5′ flanking region of the mouse MT-I gene. Different MREs have different transcriptional efficiencies, MREd being the strongest. In vitro, footprinting analyses have revealed that one or more nuclear factors can bind to the different MRE elements of the mouse MT-1 gene. Moreover, the MREd binding activity is inactivated by EDTA and can be restored by addition of Zn2+. Using a Southwestern procedure, we found that a nuclear protein of 108 kDa, termed MEP-1, specifically binds to the different MRE elements of the mouse MT-I gene promoter. MEP-1 has been purified, and footprinting studies demonstrated that purified MEP-1 specifically binds to MRE sequences. MEP-1 binding activity is also inhibited by EDTA and can be restored by Zn2+.