Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro-fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 mu l of fertilization medium (FM); (ii) 250 mu l of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 mu l of FM with 250 mu l of NLAUTF and 4 mu l of RCA-1 lectin; (iv) 250 mu l of FM with 250 mu l of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 mu l of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 mu g heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Vitrification with Glutamine Improves Maturation Rate of Vitrified/Warmed Immature Bovine Oocytes

Contents The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 mu g/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Physiological roles and regulation of mammalian sulfate transporters

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients; in cells and is the fourth most abundant anion in human plasma (300 muM). Sulfate is the major sulfur source in many organisms, and because it is a hydrophilic anion that cannot passively cross the lipid bilayer of cell membranes, all cells require a mechanism for sulfate influx and efflux to ensure an optimal supply of sulfate in the body. The class of proteins involved in moving sulfate into or out of cells is called sulfate transporters. To date, numerous sulfate transporters have been identified in tissues and cells from many origins. These include the renal sulfate transporters NaSi-1 and sat-1, the ubiquitously expressed diastrophic dysplasia sulfate transporter DTDST, the intestinal sulfate transporter DRA that is linked to congenital chloride diarrhea, and the erythrocyte anion exchanger AE1. These transporters have only been isolated in the last 10-15 years, and their physiological roles and contributions to body sulfate homeostasis are just now beginning to be determined. This review focuses on the structural and functional properties of mammalian sulfate transporters and highlights some of regulatory mechanisms that control their expression in vivo, under normal physiological and pathophysiological states.

neptune, a Kruppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Kruppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta -globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Dynamin-dependent endocytosis is necessary for convergent-extension movements in Xenopus animal cap explants

Cadherin cell-cell adhesion molecules are important determinants of morphogenesis and tissue patterning. C-cadherin plays a key role in the cell-upon-cell movements seen during Xenopus gastrulation. In particular, regulated changes in C-cadherin adhesion critically influence convergence-extension movements, thereby determining organization of the body plan. It is also predicted that remodelling of cadherin adhesive contacts is important for such cell-on-cell movements to occur. The recent demonstration that Epithelial (E-) cadherin is capable of undergoing endocytic trafficking to and from the cell surface presents a potential mechanism for rapid remodelling of such adhesive contacts. To test the potential role for C-cadherin endocytosis during convergence-extension, we expressed in early Xenopus embryos a dominantly-inhibitory mutant of the GTPase, dynamin, a key regulator of clathrin-mediated endocytosis. We report that this dynamin mutant significantly blocked the elongation of animal cap explants in response to activin, accompanied by inhibition of C-cadherin endocytosis. We propose that dynamin-dependent endocytosis of C-cadherin plays an important role in remodelling adhesive contacts during convergence-extension movements in the early Xenopus embryo.

Expression and role of roundabout-1 in embryonic Xenopus forebrain

The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain. (C) 2002 Wiley-Liss, Inc.

The Mechanism of Centriole Inactivation in Starfish Oocytes

The centrosome is the major organizing center in a cell, composed by two centrioles, one mother and one daughter, and surrounded by a pericentriolar material, which nucleates microtubules. Centriole duplication and segregation is tightly coupled to cell cycle, which guarantees that centriole number is maintained over generations. During the somatic cell cycle, a pair of centrioles duplicates, after which each daughter cell receives a pair, forming a closed cycle. However, during fertilization, if both cells were to contribute with their pair of centrioles, gamete fusion would result in the double of the normal centriole number.(...)

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

CTCF-T, a paralogue of CTCF, directs sequence specific DNA methylation of the imprinting control region of Igf2/H19

SUMMARY Genomic imprinting is an epigenetic mechanism of transcriptional regulation that ensures restriction of expression of a subset of mammalian genes to a single parental allele. The best studied example of imprinted gene regulation is the Igf2/H19 locus, which is also the most commonly altered by loss of imprinting (LOT) in cancer. LOT is associated with numerous hereditary diseases and several childhood, and adult cancers. Differential expression of reciprocal H19 and 1gf2 alleles in somatic cells depends on the methylation status of the imprinting control region (ICR) which regulates binding of CTCF, an ubiquitously expressed 11-zinc finger protein that binds specifically to non-methylated maternal ICR and thereby attenuates expression of Igf2, while it does not bind to methylated paternal ICR, which enables Igf2 expression. Initial ICR methylation occurs during gametogenesis by an as yet unknown mechanism. The accepted hypothesis is that the event of differential maternal and paternal DNA methylation depends on germ-line specific proteins. Our Laboratory identified a novel 11-zinc-finger protein CTCF-T (also known as CTCFL and BORIS) that is uniquely expressed in the male germ-line and is highly homologous within its zinc-finger region with CTCF. The amino-acid sequences flanking the zinc-finger regions of CTCF and CTCF-T have widely diverged, suggesting that though they could bind to the same DNA targets (ICRs) they are likely to have different functions. Interestingly, expression of CTCF-T and CTCF is mutually exclusive; CTCF-T-positive (CTCF-negative) cells occur in the stage of spermatogenesis that coincides with epigenetic reprogramming, including de novo DNA methylation. In our study we demonstrate the role that CTCF-T plays in genomic imprinting. Here we show that CTCF-T binds in vivo to the ICRs of Igf2/H19 and Dlk/Gt12 imprinted genes. In addition, we identified two novel proteins interacting with CTCF-T: a protein arginine methyltransferase PRMT7 and an arginine-rich histone H2A variant that we named trH2A. These interactions were confirmed and show that the two proteins interact with the amino-teiminal region of CTCF-T. Additionally, we show interaction of the amino- terminal region of CTCF-T with histones H1, H2A and H3. These results suggest that CTCF-T is a sequence-specific DNA (ICR) binding protein that associates with histones and recruits PRMT7. Interestingly, PRMT7 has a histone-methyltransferase activity. It has been shown that histone methylation can mark chromatin regions thereby directing DNA-methylation; thus, our hypothesis is that the CTCF-T protein-scaffold directs PRMT7 to methylate histone(s) assembled on ICRs, which marks chromatin for the recruitment of the de novo DNA methyltransferases to methylate DNA. To test this hypothesis, we developed an in vivo DNA-methylation assay using Xenopus laevis' oocytes, where H19 ICR and different expression cDNAs, including CTCF-T, PRMT7 and the de novo DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt3L) are microinjected into the nucleus. The methylation status of CpGs within the H19 ICR was analysed 48 or 72 hours after injection. Here we demonstrate that CpGs in the ICR are methylated in the presence of both CTCF-T and PRMT7, while control oocytes injected only with ICR did not show any methylation. Additionally, we showed for the first time that Dnmt3L is crucial for the establishment of the imprinting marks on H19 ICR. Moreover, we confirmed that Dnmt3a and Dnmt3b activities are complementary. Our data indicate that all three Dnmt3s are important for efficient de novo DNA methylation. In conclusion, we propose a mechanism for the establishment of de novo imprinting marks during spermatogenesis: the CTCF-T/PRMT7 protein complex directs histone methylation leading to sequence-specific de novo DNA methylation of H19 ICR. RESUME L'empreinte génomique parentale est un mécanisme épigénétique de régulation transcriptionelle qui se traduit par une expression différentielle des deux allèles de certains gènes, en fonction de leur origine parentale. L'exemple le mieux caractérisé de gènes soumis à l'empreinte génomique parentale est le locus Igf2/H19, qui est aussi le plus fréquemment altéré par relaxation d'empreinte (en anglais: loss of imprinting, LOI) dans les cancers. Cette relaxation d'empreinte est aussi associée à de nombreuses maladies héréditaires, ainsi qu'à de nombreux cancers chez l'enfant et l'adulte. Dans les cellules somatiques, les différences d'expression des allèles réciproques H19 et Ig12 est sous le contrôle d'une région ICR (Imprinting Control Region). La méthylation de cette région ICR régule l'ancrage de la protéine à douze doigts de zinc CTCF, qui se lie spécifiquement à l'ICR maternel non-méthylé, atténuant ainsi l'expression de Igf2, alors qu'elle ne s'ancre pas à l'ICR paternel méthyle. Le mécanisme qui accompagne la méthylation initiale de la région ICR durant la gamétogenèse n'a toujours pas été élucidé. L'hypothèse actuelle propose que la différence de méthylation entre l'ADN maternel et paternel résulte de l'expression de protéines propres aux zones germinales. Notre laboratoire a récemment identifié une nouvelle protéine à douze doigts de zinc, CTCF-T (aussi dénommée CTCFL et BORRIS), qui est exprimée uniquement dans les cellules germinales mâles, dont la partie à douze doigts de zinc est fortement homologue à la protéine CTCF. La séquence d'acides aminés de part et d'autre de cette région est quant à elle très divergente, ce qui implique que CTCF-T se lie sans doute au même ADN cible que CTCF, mais possède des fonctions différentes. De plus, l'expression de CTCF-T et de CTCF s'oppose mutuellement; l'expression de la protéine CTCF-T (cellules CTCF-T positives, CTCF negatives) qui a lieu pendant la spermatogenèse coïncide avec la reprogrammation épigénétique, notamment la méthylation de novo de l'ADN. La présente étude démontre le rôle essentiel joué par la protéine CTCF-T dans l'acquisition de l'empreinte génomique parentale. Nous montrons ici que CTCF-T s'associe in vivo avec les régions ICR des loci Igf2/H19 et Dlk/Gt12. Nous avons également identifié deux nouvelles protéines qui interagissent avec CTCF-T : une protéine arginine méthyl transférase PRMT7, et un variant de l'histone H2A, riche en arginine, que nous avons dénommé trH2A. Ces interactions ont été analysées plus en détail, et confinnent que ces deux protéines s'associent avec la région N-terminale de CTCF-T. Aussi, nous présentons une interaction de la région N-terminale de CTCF-T avec les histones H1, H2, et H3. Ces résultats suggèrent que CTCF-T est une protéine qui se lie spécifiquement aux régions ICR, qui s'associe avec différents histones et qui recrute PRMT7. PRMT7 possède une activité méthyl-tansférase envers les histones. Il a été montré que la méthylation des histones marque certains endroits de la chromatine, dirigeant ainsi la méthylation de l'ADN. Notre hypothèse est donc la suivante : la protéine CTCF-T sert de base qui dirige la méthylation des histones par PRMT7 dans les régions ICR, ce qui contribue à marquer la chromatine pour le recrutement de nouvelles méthyl transférases pour méthyler l'ADN. Afin de valider cette hypothèse, nous avons développé un système de méthylation de l'ADN in vivo, dans des oeufs de Xenopus laevis, dans le noyau desquels nous avons mico-injecté la région ICR du locus H19, ainsi que différents vecteurs d'expression pour CTCF-T, PRMT7, et les de novo méthyl transférases (Dnmt3a, Dnmt3b et Dnmt3L). Les CpGs méthyles de la région ICR du locus H19 ont été analysé 48 et 72 heures après l'injection. Cette technique nous a permis de démontrer que les CpGs de la région ICR sont méthyles en présence de CTCF-T et de PRMT7, tandis que les contrôles injectés seulement avec la région ICR ne présentent aucun signe de méthylation. De plus, nous démontrons pour la première fois que la protéine méthyl transférase Dnmt3L est déterminant pour l'établissement de l'empreinte génomique parentale au niveau de la région ICR du locus H19. Aussi, nous confirmons que les activités méthyl transférases de Dnmt3a et Dnmt3b sont complémentaires. Nos données indiquent que les trois protéines Dnmt3 sont impliquées dans la méthylation de l'ADN. En conclusion, nous proposons un mécanisme responsable de la mise en place de nouvelles empreintes génomiques pendant la spermatogenèse : le complexe protéique CTCF-T/PRMT7 dirige la méthylation des histones aboutissant à la méthylation de novo de l'ADN au locus H19.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.