Étude de l'activation de la transcription chez le jeune embryon bovin

Chez une multitude de métazoaires étudiés, une période de quiescence transcriptionnelle est observée chez le jeune embryon suite à la fécondation de l'ovocyte. La durée de cette période est spécifique à chaque espèce et chez le bovin, l'embryon n'active son propre génome qu'aux stades 8- à 16-cellules. Précédemment à cette activation de la transcription, l'embryon subsiste grâce à l'utilisation des ARNm et des protéines fournies par l'ovocyte. C'est également à partir de ces réserves que l'embryon doit puiser les différents facteurs impliqués dans l'activation de son génome au moment requis. Les expériences présentées dans cette thèse étaient destinées à améliorer nos connaissances de l'activation du génome embryonnaire chez le bovin. Dans un premier temps, la caractérisation de l'expression de plusieurs facteurs de transcription chez l'embryon a été effectuée et le rôle envisageable de ces facteurs dans l'activation du génome a aussi été démontré. Par la suite, nous avons établi une liste exhaustive de plus de 300 transcrits embryonnaires exprimés très tôt dès l'activation du génome. Cette étude du transcriptome a permis l'identification d'une multitude de gènes associés à la transcription et au maintient de la pluripotence que l'on retrouve chez les cellules embryonnaires. Afin de définir la fonction ou le rôle des différents joueurs identifiés lors de nos études, nous avons mis au point un procédé qui cible spécifiquement un transcrit donné et induit sa dégradation dans les ovocytes bovins sans toutefois induire des effets collatéraux dommageables sur la compétence au développement de ces ovocytes. Cette méthode utilise l'interférence ARN qui réduit à des niveaux très faibles la présence d'un transcrit ciblé, ce qui permet d'étudier les effets de sa perte de fonction. Cette méthode a permis d'établir le rôle crucial dans l'embryon d'un gène issu de nos premières études : MATRIN 3. La dégradation de l'ARN de MATRIN 3, une composante architecturale de la matrice nucléaire qui agit aussi au niveau de la transcription, s'est avérée avoir des effet néfastes sur la survie embryonnaire. Les informations fournies par la combinaison des études présentées dans cette thèse contribuent à dresser un portrait mieux défini de l'activation du génome chez le bovin

Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis

<p>Abstract</p> <p>Background</p> <p>Various histone acetylases (HATs) play a critical role in the regulation of gene expression, but the precise functions of many of those HATs are still unknown. Here we provide evidence that MYST4, a known HAT, may be involved in early mammalian gametogenesis.</p> <p>Results</p> <p>Although <it>MYST4 </it>mRNA transcripts are ubiquitous, protein expression was restricted to select extracts (including ovary and testis). Immunohistochemistry experiments performed on ovary sections revealed that the MYST4 protein is confined to oocytes, granulosa and theca cells, as well as to cells composing the blood vessels. The transcripts for <it>MYST4 </it>and all-<it>MYST4</it>-isoforms were present in oocytes and in <it>in vitro </it>produced embryos. In oocytes and embryos the MYST4 protein was localized in both the cytoplasm and nucleus. Within testis sections, the MYST4 protein was specific to only one cell type, the elongating spermatids, where it was exclusively nuclear.</p> <p>Conclusion</p> <p>We established that MYST4 is localized into specialized cells of the ovary and testis. Because the majority of these cells are involved in male and female gametogenesis, MYST4 may contribute to important and specific acetylation events occurring during gametes and embryo development.</p

Breed specific factors influence embryonic lipid composition : comparison between Jersey and Holstein

Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage

Truth in Consequentiality: Theory and Field Evidence on Discrete Choice Experiments

This paper explores methodological issues surrounding the use of discrete choice experiments to elicit values for public goods. We develop an explicit game-theoretic model of individual decisions to a series of choice sets, providing general conditions under which surveys with repeated binary choices are incentive compatible. We complement the theory with a framed field experiment, with treatments that span the spectrum from incentive compatible, financially binding decisions to decisions with no direct financial consequences. The results suggest truthful preference revelation is possible in surveys, provided that respondents view their decisions as having more than a weak chance of influencing policy. Cette étude s’intéresse à des aspects méthodologiques associés à l’utilisation d’expériences avec choix discrets pour évaluer des biens publics. Nous avons développé un modèle explicite de jeux théoriques pour des décisions individuelles à des séries de choix, avec conditions générales sous lesquelles un questionnaire avec des choix binaires répétés incite la révélation des valeurs. Ce développement théorique est suivi d’expériences terrains avec traitements qui couvrent le spectre des incitatifs de la révélation des valeurs, passant de la décision avec mise en place réelle du projet et paiements réels de la part des participants, à celle sans aucune conséquence financière directe et avec projets hypothétiques. Les résultats indiquent qu’il est possible d’obtenir une révélation des valeurs réelles en situation hypothétique, si les participants pensent que leurs décisions ont un potentiel d’impact significatif sur une éventuelle politique.discrete choice experiment, framed field experiment, mechanism design theory, stated preferences, consequentiality , expériences avec choix discrets; expérience terrain; préférences révélées; conséquences, biais hypothétique

Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.

One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation was performed by real-time PCR. Results show that the major changes in gene expression occurred from 20 to 44 h of coasting, when oocyte quality increases. Secondly, among upregulated genes (20–44 h), 25% were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified two patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, three major functional changes were identified: i) the end of follicle growth (BMPR1B, IGF2, and RELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2, TF, and VNN1) and ii) apoptosis (KCNJ8) followed by iii) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase in apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases

The use of adenosine to inhibit oocyte meiotic resumption in Bos taurus during pre-IVM and its potential to improve oocyte competence

One of the major challenges of artificial reproductive technologies is to develop new methods for pro-ducing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo beforeoocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), apurine nucleoside found in follicularfluid, on the inhibition of oocyte meiotic resumption and theproduction of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption.The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in asignificant increase (p<0.05) of blastocysts compared to control conditions with no pre-IVM cultureperiod. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yieldwas time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing theADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermalgrowth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptionalanalyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonicalpathway affected by ADO. This opens up new opportunities for further investigations

Cumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes

The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus–oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n = 3 cows, 12 oocyte collections) and qRT-PCR (n = 3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process

Financing repurposed drugs for rare diseases: a case study of Unravel Biosciences

Abstract Background We consider two key challenges that early-stage biotechnology firms face in developing a sustainable financing strategy and a sustainable business model: developing a valuation model for drug compounds, and choosing an appropriate operating model and corporate structure. We use the specific example of Unravel Biosciences—a therapeutics platform company that identifies novel drug targets through off-target mechanisms of existing drugs and then develops optimized new molecules—throughout the paper and explore a specific scenario of drug repurposing for rare genetic diseases. Results The first challenge consists of producing a realistic financial valuation of a potential rare disease repurposed drug compound, in this case targeting Rett syndrome. More generally, we develop a framework to value a portfolio of pairwise correlated rare disease compounds in early-stage development and quantify its risk profile. We estimate the probability of a negative return to be $$80.8\%$$ 80.8 % for a single compound and $$56.1\%$$ 56.1 % for a portfolio of 8 drugs. The probability of selling the project at a loss decreases from $$79.2\%$$ 79.2 % (phase 3) for a single compound to $$55.4\%$$ 55.4 % (phase 3) for the 8-drug portfolio. For the second challenge, we find that the choice of operating model and corporate structure is crucial for early-stage biotech startups and illustrate this point with three concrete examples. Conclusions Repurposing existing compounds offers important advantages that could help early-stage biotech startups better align their business and financing issues with their scientific and medical objectives, enter a space that is not occupied by large pharmaceutical companies, and accelerate the validation of their drug development platform