The embryonic development and pregnancy rates are influenced by variations in embryo in vitro production (IVP) systems and protocols for bovine embryos, such as gaseous atmosphere, culture media and the protein supply adopted in the different steps of IVP. Two studies were performed to evaluate the effect of O2 tension on in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes, and the protein supply in the embryonic in vitro culture. Immature Cumulusoocytes complexes (COC) were recovered by transvaginal ultrasound guided follicular aspiration (OPU) from Bos taurus indicus donors from commercial programs and randomly assigned according to the number and quality of the COC. Standard bovine IVM procedure was carried out in modified TCM-199, added of FSH, LH, Oestradiol, EGF, insulin and 10% bovine fetal serum (FCS), for 24h. Frozen semen was selected by Percoll gradient (90, 60 and 30%). The insemination was performed with 2x106 spermatozoa/mL in Fert-Talp with heparin and PHE, for 18 to 22h. Presumptive zygotes were in vitro cultured (IVC) in SOFaaci during 6 to 9 days with 5%CO2, 5%O2 and 90% N2 in air. All steps of IVC were performed in an incubator at 39ºC with satured humidity. The cleavage and blastocyst rates were analysed by GLM procedure. Hatching, quality I blastocyst and pregnancy rates were compared by Chi-square Test with 5% of significance. In experiment I (11 replicates) IVM and IVF of COC (n=1092) were conducted under 5% CO2 (20% O2) in air or in 5%CO2, 5%O2 and 90% N2 (5%O2). Experiment II was divided in two steps (IIa and IIb). In experiment IIa (n=1745 COC), the IVM and IVF was followed by in vitro culture (IVC) in SOFaaci added of 4mg/mL bovine serum albumin (BSA) or 4mg/mL BSA+2% bovine fetal serum (BSA+FCS). In experiment IIb (n=704 COC), after standard IVM and IVF, IVC was performed in the two media of Experiment IIa (BSA and BSA+FCS) and in a third group the SOFaaci was added of 4mg/mL BSA+2% FCS (BSA+FCSD4) on day 4 of IVC. The viability was accessed by morphological evaluation in D2 (cleavage), D7 (blastocysts and quality categories), D9 (hatched blastocysts), D45 (experiment I) and D60 (experiment II; pregnancy rates), considering day zero (D0) as the fertilization day. In experiment I, no differences were found (P>0.05) in cleavage rates (69 and 70%), blastocyst in D7 (37 and 38%) and blastocyst quality I (79 and 74%) in an atmosphere of 5% or 20%O2, respectively. The hatching rate in D9 considering all COC submitted to IVM was higher (P<0.05) for the 5%O2 group (21%) compared to the IVM and IVF group under 20%O2 (11%). A pregnancy rate of transferred embryos at 45 days (n=278) was similar (P=0.15) between treatments (25.8 e 33.6% for 5%O2 e 20%O2 ,respectively ). In experiment IIa blastocyst rates (51%) and quality I blastocysts (41%) in D7, was higher (P<0.05) in BSA+FCS group compared to the BSA group (42 and 30%), respectively. In experiment IIb, blastocyst rates were higher (P<0.05) in group BSA+FCS (47%) and similar between BSA (34%) and BSA+FCSD4 (43%) groups. Blastocyst rates quality I were higher (P<0.05) for groups BSA+FCS (34%) and BSA+FCSD4 (32%), compared to the BSA group (19.9%). A pregnancy rate of the transferred embryos (n=820) was similar between treatments in experiments IIa and IIb. Considering pregnancy rates corresponding only to IVM COC (Experiment IIa), the rates were higher (P<0.05) for the BSA+FCS group (16%) than the BSA group (12%). There was no detrimental effect in IVP of bovine embryos and their pregnancy rates when IVM, IVF and IVC were performed in 5%O2, 5%CO2 and 90%N2. IVP of bovine embryos is increased by the addition of 2% FCS to the IVC medium without any detrimental effect on their morphological quality and pregnancy rates. On the other hand, pregnancy rates related to IVM of COC incremented by the addition of 2% de Fc to the IVC medium SOFaaci+BSA. / Diferenças nos protocolos e sistemas de produção in vitro (PIV) de embriões bovinos incluindo atmosfera gasosa, meios de cultivo e suplementação de proteína utilizados nas diferentes etapas da PIV, influenciam diretamente os resultados finais de desenvolvimento embrionário e prenhez. Dois experimentos foram conduzidos com o objetivo de avaliar a influência da concentração de oxigênio na maturação in vitro (MIV) e fecundação in vitro (FIV) de oócitos bovinos e da suplementação protéica no cultivo in vitro (CIV) de embriões bovinos. Complexos cumulus-oócitos imaturos (COC) obtidos de fêmeas Bos taurus indicus por aspiração folicular transvaginal guiada por ultra-sonografia (OPU) foram divididos homogeneamente quanto ao número e a qualidade entre os tratamentos, sendo maturados por 24h, em TCM-199 modificado, acrescido FSH, LH, Estradiol, EGF, Insulina e 10% de SFB. Sêmen congelado selecionado por gradientes de Percoll (2 x 106 células/mL) foi utilizado para a fecundação em Fert-TALP com heparina e PHE, por 18 a 22h. Os prováveis zigotos foram cultivados in vitro em SOFaaci por 6 a 9 dias em atmosfera de 5%CO2, 5%O2 e 90% N2. Todas as etapas foram conduzidas em estufa a 39ºC com umidade saturada. Os percentuais de clivagem e de blastocistos foram analisados pelo procedimento GLM. As taxas de eclosão dos blastocistos e de prenhez foram comparadas por Qui-quadrado com significância de 5%. No experimento I (11 repetições), a MIV e FIV dos COC (n=1092) foram realizadas sob atmosfera de 5% de CO2 em ar (20%O2) ou em 5%CO2, 5%O2 e 90% N2 (5%O2). O experimento II foi dividido em 2 etapas (IIa e IIb). No experimento IIa (n=1745 COC) após a MIV e FIV foi realizado o CIV em meio SOFaaci acrescido de 4mg/mL albumina sérica bovina (BSA) ou 4mg/mL de BSA + 2% de soro fetal bovino (BSA+SFB). No experimento IIb (n=704 COC), após a MIV e FIV, o CIV foi conduzido nos dois meios do experimento IIa (BSA e BSA+SFB) e um terceiro grupo onde o SOFaaci+BSA foi suplementado com 2% de SFB no quarto dia de cultivo (BSA+SFBD4). As avaliações morfológicas foram efetuadas no D2 (clivagem), D7 (blastocistos e grau de qualidade), D9 (blastocistos eclodidos) e taxa de prenhez aos 45 (experimento I) e 60 dias (experimento II), considerando-se o dia da fecundação como D0. No experimento I, não houve diferença (P>0,05) nas taxas de clivagem (69 e 70%), blastocistos em D7 (37 e 38%) e blastocistos qualidade I (79 e 74%) entre atmosfera de 5%O2 ou 20% de O2, respectivamente. A taxa de eclosão em D9 sob o total de oócitos MIV foi superior (P<0,05) no grupo 5%O2 (21%) comparado ao grupo MIV e FIV sob 20%O2 (11%). A taxa de prenhez aos 45 dias dos embriões transferidos (n=278) foi semelhante (P=0,15) entre os tratamentos (25,8 e 33,6% para 5%O2 e 20%O2 respectivamente). No experimento IIa, a taxa de blastocistos (51,5%) e de blastocistos qualidade I (41%) em D7, foi superior (P<0,05) para o grupo BSA+SFB em relação ao BSA (42 e 30%), respectivamente. No experimento IIb, a taxa de blastocistos foi superior (P<0,05) no grupo BSA+SFB e semelhante (P>0,05) entre o grupo BSA e BSA+SFBD4. A taxa de blastocistos qualidade I foi superior para os grupos BSA+SFB (34%) e BSA+SFBD4 (32,2%) em comparação ao grupo BSA (19,9%). A taxa de prenhez em relação aos embriões transferidos (n=820) foi semelhante entre os tratamentos nos experimentos IIa e IIb. A taxa de prenhez correspondente aos oócitos colocados para MIV foi superior (P<0,05) para o grupo BSA+SFB (16%) comparado ao grupo BSA (12%) no experimento IIa. Não há comprometimento da PIV de embriões bovinos bem como da taxa de prenhez quando a MIV, FIV e CIV são conduzidas com 5%O2, 5%CO2 e 90%N2. A produção in vitro de embriões é incrementada pela adição de 2% de SFB ao meio de CIV, sem prejudicar a qualidade morfológica dos mesmos e a taxa de prenhez. A porcentagem de prenhez produzida em relação aos COC colocados para MIV é aumentada pela adição de 2% de SFB ao meio de cultivo in vitro SOFaaci+BSA.

Abstract Apoptosis plays a crucial part in human ovarian function from fetal development to the end of reproductive potential. Failures in the regulation of ovarian apoptosis are associated with many pathological conditions such as premature ovarian insufficiency, infertility and cancer. The purpose of the present study was to analyze the factors regulating cell survival in human fetal and adult ovaries. The fetus is exposed to maternal- and placental-derived estrogens and insufficient estrogen action has destructive effects on rodent ovarian development. We detected estrogen receptors and estrogen-converting enzymes in human fetal ovaries after primordial follicle formation, indicating that estrogens participate in human fetal ovarian development, especially after folliculogenesis. The WNT4 gene is crucial for female sexual differentiation, follicle formation and oocyte survival. We detected WNT4 in follicular cells of fetal and adult human ovaries. In addition, Wnt4- knockout mice demonstrated a dramatic loss of oocytes before birth. However, no changes were detected in protein expression patterns of common apoptosis-related proteins. The results support the possible role of WNT4 in human ovarian function and strengthen previous knowledge on the antiapoptotic role of Wnt4. Apoptosis signaling is mediated by extracellular- and mitochondria-associated- pathways, ending in caspase cascade activation and fragmentation of cellular structures. In the present study we analyzed the expression of several apoptosis-related factors and detected TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries. In addition, TRAIL was found to be a potent and rapid inducer of human granulosa tumor cell (KGN) apoptosis. Lentiviral downregulation of Bok or Bcl-XL protein expression in KGN cells also resulted in significant changes in cell vulnerability to apoptosis. The results show for the first time the spatiotemporal expression patterns of TRAIL, TNF, Bcl-XL, Bok and caspase-3 in human ovaries and suggest an important functional role of TRAIL, Bok and Bcl-XL in regulation of human ovarian apoptosis. The present study offers novel information on the expression and function of cell survival factors in human ovaries. These new findings open possibilities for future clinical research in attempts to understand and treat ovarian diseases caused by imbalanced regulatory pathways of apoptosis.

Malgré le succès de l’Aide Médicale à la Procréation (AMP), des données récentes suggèrent un lien entre le recours à l’AMP et des pathologies liées à des erreurs épigénétiques. Afin d’évaluer l’impact des procédés in vitro de l’AMP sur la méthylation de l’ADN, nous avons analysé : 1) le profil de méthylation de 2 locus soumis à empreinte, H19DMR et KvDMR1, dans des ovocytes recueillis immatures, vitrifiés, puis mûris in vitro après réchauffement. Nous avons montré pour la première fois que le procédé vitrification/MIV pouvait constituer une alternative prometteuse pour préserver le potentiel procréatif des femmes avant un traitement anticancéreux, puisqu’il n’altérait pas l’empreinte parentale. 2) le profil de méthylation de H19DMR dans des embryons humains en retard de développement ainsi que dans les gamètes des géniteurs. Nous avons mis en évidence des anomalies de l’empreinte dans ces embryons, sans lien apparent avec les indications d’ICSI mais liées à des altérations survenues soit au cours de l’ovogenèse, soit au cours du développement précoce. 3) le profil de méthylation des promoteurs d’OCT4 et NANOG, gènes essentiels à l’expression de la pluripotence, dans des embryons bloqués après culture prolongée. Nous avons montré que le promoteur d’OCT4 était significativement hyperméthylé dans les embryons bloqués, en particulier dans la population de couples présentant uniquement une infertilité masculine, alors que le promoteur de NANOG était hypométhylé / Despite the success of Assisted Reproductive Technologies (ART), recent data suggest a link between the use of ART and diseases related to epigenetic errors. To evaluate the impact of ART on DNA methylation, we analyzed: 1) the methylation profile of 2 imprinted loci, H19DMR and KvDMR1, in immature oocytes which were vitrified and matured in vitro after warming. We have shown for the first time that vitrification/IVM process did not alter the imprinting. Then, this new technology could be a promising alternative to preserve the procreative potential of women before cancer treatment. 2) the methylation profile of H19DMR in human embryos with developmental anomalies and in the gametes of the parents. We have shown that imprinting errors in these embryos have no apparent link with ICSI indications, but were related to alterations in H19DMR occurring either during oogenesis or early development. 3) the methylation profile of the promoters of OCT4 and NANOG, essential genes for the expression of pluripotency in arrested embryos after prolonged culture. We have shown that the promoter of OCT4 was highly methylated in arrested embryos particularly in the population of couples with male infertility only, whereas the NANOG promoter was hypomethylated

Un prélèvement efficace de K+ à partir du sol est essentiel au développement des végétaux. Sur un sol riche en NaCl, le maintien d'un prélèvement sélectif et efficace de K+ à partir du sol et le contrôle de l'exportation de Na+ par la racine vers les feuilles constituent des fonctions essentielles pour la survie de la plante. Chez les plantes, les transporteurs HKT (High-affinity K+ Transporters) sont classés en deux sous-familles sur des bases phylogénétiques et de sélectivité ionique. Les membres de la sous-famille 1 transportent sélectivement Na+. Plusieurs d'entre eux ont été identifiés comme des acteurs majeurs de l'adaptation des plantes aux fortes salinités du sol en prévenant l'accumulation de Na+ dans les parties aériennes. Les membres de la sous-famille 2 co-transportent Na+ et K+. Leur rôle dans la plante, notamment dans le transport de K+, est encore mal compris. Je me suis intéressé à différents systèmes de transports de K+ et Na+, appartenant essentiellement à la famille HKT chez le riz. La caractérisation que j'ai effectuée a fait appel à plusieurs approches : électrophysiologie (voltage-clamp après expression en ovocyte de xénope), biologie cellulaire, génétique inverse et PCR en temps réel. L'analyse de l'expression par RT-PCR en temps réel de toute la famille HKT (4 membres dans chacune des deux sous-familles) a montré que ces transporteurs sont différemment exprimés au niveau des racines et des feuilles, et que leur niveau de transcrits est fortement et differentiellement régulé en conditions de stress salin ou osmotique et en présence d'hormones, ce qui suggère que ces différents systèmes jouent des rôles propres et diversifiés dans la plante. L'analyse plus détaillée d'OsHKT2;4, a montré par expression hétérologue dans l'ovocyte de xénope que ce système possède des propriétés fonctionnelles originales: il transporte sélectivement K+ à faibles concentrations de Na+, mais co-transporte Na+ et K+ à fortes concentrations de Na+ (>10 mM). L'analyse de l'expression d'OsHKT2;4 a révélé que ce transporteur est surexprimé en condition de carence en K+ et de stress salin, suggérant qu'OsHKT2;4 pourrait jouer un rôle important dans le transport de K+ dans ces deux conditions. Enfin, un patron d'expression nouveau pour un transporteur HKT a été révélé par l'analyse de plantes transgéniques exprimant le promoteur d'OsHKT2;4 fusionné aux gènes rapporteurs GUS ou GFP : en plus d'une localisation classique dans les tissus conducteurs, une forte expression est observée dans les stomates des gaines et des limbes foliaires, suggérant un rôle dans l'osmocontractilité de ces cellules.Mots clés: Oryza sativa, transport de potassium, transporteur HKT, Na+-K+ co-transporteur, électrophysiologie, ovocyte de xénope, localisation tissulaire, PCR quantitative, stress salin / Efficient uptake of K+ from the soil solution is essential for plant development. When plants are grown on a soil rich in NaCl, the maintenance of an efficient and selective uptake of K+ and the control of Na+ export from roots to shoots are crucial for plant survival. In plants, transporters belonging to the HKT (Highaffinity K+ Transport) family have been sorted in two subfamilies based on phylogenetic grounds and functional properties. Subfamily 1 members transport selectively Na+. Several of them have been shown to play major roles in plant adaptation to salt stress by preventing excessive accumulation of Na+ in shoots. Subfamily 2 members are thought to co-transport Na+ and K+, at least when expressed in heterologous systems. Their roles in planta, especially their potential role in K+ transport, are still largely unknown. I have been interested in different K+ and/or Na+ transport systems in rice, mostly belonging to the HKT family. For their characterization, different approaches have been used: electrophysiology (two-electrode voltage-clamp after expression in Xenopus oocytes), cell biology, reverse genetics and real-time PCR. Realtime RT-PCR analyses on the whole family of rice HKT transporters (4 members in both subfamilies) showed that the expression level in roots and leaves of these different systems is variable, and is differentially regulated by salt and osmotic stresses as well as by hormonal treatments, which suggests that these transporters have diverse and differentiated functions in the plant. A detailed analysis of OsHKT2;4 revealed original functional properties: this HKT transporter was indeed shown to be K+-selectively in the presence of low external Na+, but to switch to Na+ and K+ co-transport mode at high (>10 mM) Na+ concentrations. Expression analysis of OsHKT2;4 showed that this transporter is overexpressed upon salt stress and K+ shortage, which suggests that it could play an important role in K+ transport in these two conditions. At last, a new expression pattern for an HKT transporter was evidenced through the analysis of transgenic rice plants expressing OsHKT2;4 promoter fused to the GUS or GFP reporter genes: in addition to a classical localization in vascular tissues, expression of OsHKT2;4 was observed in stomata, suggesting a role for OsHKT2;4 in osmotic regulation in these cells

Post-transcriptional regulatory mechanisms are widely used to influence cell fate decisions in germ cells, early embryos, and neurons. Many conserved cytoplasmic RNA regulatory proteins associate with each other and assemble on target mRNAs, forming ribonucleoprotein (RNP) complexes, to control the mRNAs translational output. How these RNA regulatory networks are orchestrated during development to regulate cell fate decisions remains elusive. We addressed this problem by focusing on Caenorhabditis elegans germline development, an exemplar of post-transcriptional control mechanisms. Here, we report the discovery of GLS-1, a new factor required for many aspects of germline development, including the oocyte cell fate in hermaphrodites and germline survival. We find that GLS-1 is a cytoplasmic protein that localizes in germ cells dynamically to germplasm (P) granules. Furthermore, its functions depend on its ability to form a protein complex with the RNA-binding Bicaudal-C ortholog GLD-3, a translational activator and P granule component important for similar germ cell fate decisions. Based on genetic epistasis experiments and in vitro competition experiments, we suggest that GLS-1 releases FBF/Pumilio from GLD-3 repression. This facilitates the sperm-to-oocyte switch, as liberated FBF represses the translation of mRNAs encoding spermatogenesis-promoting factors. Our proposed molecular mechanism is based on the GLS-1 protein acting as a molecular mimic of FBF/Pumilio. Furthermore, we suggest that a maternal GLS-1/GLD-3 complex in early embryos promotes the expression of mRNAs encoding germline survival factors. Our work identifies GLS-1 as a fundamental regulator of germline development. GLS-1 directs germ cell fate decisions by modulating the availability and activity of a single translational network component, GLD-3. Hence, the elucidation of the mechanisms underlying GLS-1 functions provides a new example of how conserved machinery can be developmentally manipulated to influence cell fate decisions and tissue development.

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.

Fully grown oocytes' of the African clawed frog, Xenopus laevis, are arrested at the diplotene stage of meiotic prophase I, which resembles the G2 phase of the mitotic cell cycle. Re-entry into the meiotic divisions is initiated by hormonal signaling normally provided by progesterone. Progesterone signaling leads to the activation of maturation promoting factor (MPF), a heterodimer consisting of the protein kinase cdk1 and cyclin B1; this complex promotes the oocyte's entry into M phase of meiosis I. A crucial event required for MPF activation is cytoplasmic polyadenylation element (CPE)-mediated translation of specific dormant mRNAs such as c-mos and cyclin B1. The CPE, which resides in mRNA 3' untranslated region (UTR), is bound by the CPE binding protein (CPEB), which in turn is bound by Maskin. Maskin is bound to the 5' cap binding protein eIF4E. This type of closed-loop mRNA structure inhibits the recruitment and assembly of the translation initiation complex at the 5'UTR of CPE containing mRNAs. To alleviate this inhibition, CPEB undergoes phosphorylation on S174 by the serine/threonine kinase Aurora A. Phosphorylated CPEB promotes the recruitment of specific polyadenylation factors leading to the polyadenylation of the dormant mRNA, resulting in the disassociation of Maskin from eIF4E. eIF4E is subsequently bound by translation initiation factors leading to mRNA assembly into polysomes and synthesis of the encoded protein. Insulin signaling has also been shown to induce oocyte maturation. However, this signaling cascade uniquely requires the activation of two upstream components, PI3 kinase and PKC zeta. In this thesis, I show that insulin induced oocyte maturation requires the same CPE-mediated mRNA translation mechanism as had been described for progesterone signaling. I also show that Aurora A kinase activation and S174 phosphorylation play an essential role in insulin-induced CPE-mediated mRNA translation. Interestingly, inhibition of PI3 kinase and PKC zeta inhibits CPE-mediated polyadenylation only in the insulin-signaling pathway; the progesterone pathway is unaffected. These results clearly indicate that different upstream signaling components control CPE-mediated translation between progesterone and insulin signaling cascades. However, both pathways are antagonized by over expressed GSK-3, leading to inhibition of oocyte maturation. Furthermore, I found that GSK-3 inhibits Aurora A kinase activity by directly phosphorylating Aurora A on serine 290/291, promoting an inhibitory autophosphorylation event on serine 349. The importance of a GSK-3/Aurora A interaction is underscored by the finding that GSK-3, Axin, and Aurora A reside in a complex in immature oocytes. During progesterone or insulin signaling, GSK-3 dissociates from Aurora A allowing Aurora A to become active, leading to CPEB phosphorylation, CPE-mediated mRNA translation and oocyte maturation.

No description available.

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell physiology, including cell signaling. It is also known as a compatible osmolyte. Three secondary active MI cotransporters have been identified; two are Na+- coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this study was to characterize MI uptake throught SMIT2 as expressed in epithelial cells and in Xenopus laevis oocytes. In order to achieve the characterization of this transport system, we used purified brush border membrane vesicles (BBMv) isolated from rabbit kidney and rat intestine. We first performed a quantification of mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2. We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is mainly localized in the renal cortex. This result was confirmed on Western blots using an antibody raised against SMIT2. Through inhibition studies using selective substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol), we showed that SMIT2 seems to be responsible for all the apical transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By transport studies we established that rabbit intestine seems to lack apical transport of MI while rat intestine has a very active uptake of this molecule. qRT-PCR quantification confirmed the absence of MI transporters in rabbit intestine. As for kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2 displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these functional characteristics essentially confirmed those found in rat intestine, a iv discrepancy exists between the two systems studied. Indeed, the affinity constant for glucose was approximately 40-fold lower in vesicles than in oocytes. We also tested the ability of SGLT1 and GLUT5, other sugar transport systems present in enterocytes apical membranes, to perform MI uptake. Because the inhibition of these transporters did not alter radiolabeled MI uptake, we concluded that they had no significant contribution to MI transport in rat intestine. Finally, the basolateral efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this transporter was not able to transport MI.