Thierry Madigou

Differential expression of three different prepro‐GnRH (gonadotrophin‐releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax)

The expression sites of three prepro‐gonadotrophin‐releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser8‐mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp7Leu8‐mGnRH), and chicken GnRH‐II (cGnRH‐II: His5Trp7Tyr8‐mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH‐associated peptide (GAP)‐coding regions of the prepro‐GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP‐II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti‐sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA‐expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP‐positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations. J. Comp. Neurol. 429:144–155, 2001.

Cloning and Expression of Gonadotropin-Releasing Hormone Receptor in the Brain and Pituitary of the European Sea Bass: An In Situ Hybridization Study

Abstract A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the pituitary of the European sea bass, Dicentrarchus labrax. The complete cDNA is 1814 base pairs (bp) in length and encodes a protein of 416 amino acids. The 5′ UTR and 3′ UTR are 239 bp and 324 bp in size, respectively. The expression sites of this GnRH-R were studied in the brain and pituitary of sea bass by means of in situ hybridization. A quantitative analysis of the expression of the GnRH-R gene along the reproductive cycle was also performed. The GnRH-R brain expression was especially relevant in the ventral telencephalon and rostral preoptic area. Some GnRH-R messenger-expressing cells were also evident in the dorsal telencephalon, caudal preoptic area, ventral thalamus, and periventricular hypothalamus. A conspicuous and specific GnRH-R expression was detected in the pineal gland. The highest expression of the GnRH-R gene was observed in the proximal pars distalis of the pituitary. This expression was evident in all LH cells and some FSH cells but not in somatotrophs. In the pituitary, the quantitative analysis revealed a higher expression of GnRH-R gene during late vitellogenesis in comparison with maturation, spawning, and postspawning/resting periods. However, in the brain, the highest GnRH-R expression was evident at spawning or postspawning/ resting periods. These results suggest that the expression of this GnRH-R is regulated in a different manner in the brain and the pituitary of sea bass.

THE SHEEP ESTROGEN RECEPTOR : CLONING AND REGULATION OF EXPRESSION IN THE HYPOTHALAMO-PITUITARY AXIS

We have prepared an ovine pituitary cDNA library, isolated a clone containing the full-coding sequence of estrogen receptor (ER) cDNA, and determined its primary structure. This cDNA encodes a protein of 596 amino acids which shows great homology to other mammalian ER sequences, the highest degree being 95% with the porcine receptor. Northern blot analysis of ovine pituitary RNA revealed a 6.3 kb transcript. This receptor was showed to bind a consensus ERE and to be transcriptionally activated by E2. Studies investigating the pattern of expression of the ovine ER mRNA were also carried out, using the reverse transcription/PCR technique. Expression of ER mRNA was analyzed in ram pituitary and hypothalamus after contrasted light regimen and castration. Results showed that the light regimen had no effect on ER mRNA expression whereas castration induced a slight (approximately 20%) but significant increase of ER mRNA expression at both the hypothalamic (P < 0.05) and pituitary (P < 0.01) levels, indicating a negative regulation of ER gene expression by testicular steroids. Since we have previously shown no variations in ER protein levels after castration, data suggest the activation of a complex pattern including both transcriptional and post-transcriptional regulatory mechanisms in the ram hypothalamo-pituitary axis.

Estrogen receptor protein and mRNA expression in the ovary of sheep

Using immunohistochemistry and in situ hybridization, we attempted to identify the estrogen receptor (ER) protein and messenger RNA (mRNA) in sheep ovaries during the follicular phase of the estrous cycle. Monoclonal anti‐ER antibodies H222 and 1D5 were used for localizing estrogen receptor on ovarian cryo‐sections. Labeling for ER was found over the nuclei of surface epithelium, interstitial tissue, and granulosa cells of small as well as large ovarian follicles. In the preantral and small antral follicles, intense nuclear ER labeling was observed in mural granulosa cells and particularly in cumulus/granulosa cells surrounding the oocyte. In the large healthy looking follicles, greater diversity in labeling for ER was observed, which is characterized by mixed populations of granulosa cells expressing positive and more or less negative nuclear labeling. Such a pattern of labeling was particularly evident in follicles showing the signs of atresia. Generally, more intense nuclear staining was localized in granulosa cells proximal to basal membrane. In situ hybridization studies revealed the presence of ER mRNA in ovarian tissue. Autoradiographic visualization localized ER mRNA expression over the granulosa cells of healthy follicles of all sizes. Level of hybridization signal was comparable in mural and cumulus granulosa cells. In atretic follicles, the level of hybridization signal in granulosa cells was comparable to that of healthy follicles. A relatively weaker level of labeling was observed in granulosa cells dispersed in follicular antrum in follicles with advanced atretic lesions. Theca cells expressed a lower level of labeling than granulosa cells. Specificity of labeling for both ER protein and mRNA in ovary was proved by parallel probing the ovine uterus. Ovine ER recognition by both H222 and 1D5 antibodies was also proved by immunoblotting. These studies demonstrate the presence of the estrogen receptor and its messenger RNA in the sheep ovary and suggest an autocrine/paracrine role of estradiol and its receptor in the regulation of ovarian follicle development in sheep. Mol. Reprod. Dev. 48:53–62, 1997.

Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies evolutionarily unconserved CpGs as TET targets

Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine (5hmC) rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling (SCL) of 5hmC in genomic DNA with exonuclease (exo) digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.

Fully Automated Microinjection System for Xenopus laevis Oocytes with Integrated Sorting and Collection

Microinjection is the most flexible transfection method in terms of choice of reagents to inject into cells. But this method lacks the high throughput to compete with less flexible methods like chemical- or viral-based approaches. Various approaches have been pursued to increase the throughput by automating the microinjection process. However, these approaches focused solely on the microinjection itself and disregarded the tasks before and after the injection, which also belong to the critical time path of the whole process, that is, sorting out viable cells from a cell suspension, placing the cell for injection, and collecting the cell after the injection. In the approach with our XenoFactor, we demonstrate a system capable of running the whole process automatically. By optimizing the XenoFactor for Xenopus laevis oocytes, we could demonstrate the successful automated injection. Starting from a suspension with a mixture of defolliculated oocytes at different stages and quality levels, the manual approach requires 1 day in total for the preparation of 400 microinjected oocytes. The XenoFactor takes only 4 h for the same amount and delivers injected oocytes of reproducible quality and without the fatigue symptoms experienced during the manual approach.

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.

Cytosine modifications modulate the chromatin architecture of transcriptional enhancers

Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.

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.

Size Heterogeneity of Affinity-Labeled Estrogen Receptor in the Ram Hypothalamo-Pituitary Axis

The presence of multiple monomeric forms of estrogen receptor (ER) has been described in different target tissues. Using [3H]tamoxifen aziridine (TA) to covalently label ER and SDS-PAGE to analyze labeled products, ER forms were investigated in ram pituitary and hypothalamus. A major labeled protein of M(r) 60,000-65,000 and a minor species of 50,000-55,000 were found in the pituitary cytosol covalently labeled with [3H]TA. In the hypothalamic cytosol, the major TA-labeled species was the M(r) 50,000 form while the 65,000 ER was difficult to detect. Comparison of ER forms after in vitro translocation of the ER complex in purified nuclei of ram pituitary or hypothalamus again showed major ER forms of M(r) 65,000 and 50,000 for the glandular and nervous tissue respectively, suggesting a biological significance for the M(r) 50,000 species. A similar heterogeneity was also observed in male rats used as controls. Moreover, covalent labeling of cytosol from the pars tuberalis/median eminence area showed the presence of ER in this part migrating with a pattern between those of the hypothalamus and the pituitary. The ER heterogeneity was thus demonstrated in the hypothalamo-pituitary axis. The source of this heterogeneity could be: (1) different ER mRNAs according to tissue type; (2) a specific posttranslational processing such as a specific proteolytic activity within the nervous tissue.