Gaël Cagnone

Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.

While most assisted reproductive technologies (ART) are considered routine for the reproduction of species of economical importance, such as the bovine, the impact of these manipulations on the developing embryo remains largely unknown. In an effort to obtain a comprehensive survey of the bovine embryo transcriptome and how it is modified by ART, resources were combined to design an embryo‐specific microarray. Close to one million high‐quality reads were produced from subtracted bovine embryo libraries using Roche 454 Titanium deep sequencing technology, which enabled the creation of an augmented bovine genome catalog. This catalog was enriched with bovine embryo transcripts, and included newly discovered indel type and 3′UTR variants. Using this augmented bovine genome catalog, the EmbryoGENE Bovine Microarray was designed and is composed of a total of 42,242 probes, including 21,139 known reference genes; 9,322 probes for novel transcribed regions (NTRs); 3,677 alternatively spliced exons; 3,353 3′‐tiling probes; and 3,723 controls. A suite of bioinformatics tools was also developed to facilitate microrarray data analysis and database creation; it includes a quality control module, a Laboratory Information Management System (LIMS) and microarray analysis software. Results obtained during this study have already led to the identification of differentially expressed blastocyst targets, NTRs, splice variants of the indel type, and 3′UTR variants. We were able to confirm microarray results by real‐time PCR, indicating that the EmbryoGENE bovine microarray has the power to detect physiologically relevant changes in gene expression. Mol. Reprod. Dev. 78:651–664, 2011.

Differential Gene Expression Profile in Bovine Blastocysts Resulting from Hyperglycemia Exposure During Early Cleavage Stages

ABSTRACT To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatments being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.

The impact of exposure to serum lipids during in vitro culture on the transcriptome of bovine blastocysts

In vitro culture has a detrimental impact on early embryonic development, and serum addition to IVC is recognized to compromise blastocyst quality. Particularly, serum fatty acids affect embryonic lipid composition and reduce cryopreservation survival. To understand the molecular pathways of serum-induced embryonic stress, this study examined the early development of bovine embryos produced in different protein- or lipid-supplemented culture media: BSA alone (control), BSA + serum lipid fraction (SELF), delipidated serum and total serum. These protein-lipid treatments were applied from the eight to 16 cell stages to the blastocyst stage. As planned, SELF treatment increased the fatty acid concentration in the medium compared with control medium but did not induce embryo toxicity. However, microarray comparison between blastocysts cultured in BSA without or with SELF revealed differential transcriptomic profile associated with ceramide-induced oxidative stress and inflammation. Moreover, the SELF treatment had a significant impact on genes involved in cholesterol metabolism (LDLR, HMGCS1), with the potential upstream control of the transcription factors SREBP and PPARA, two major regulators of cholesterol metabolism. In addition, the expression of pluripotence-related genes (APEX, CLDN6) was downregulated in blastocysts subjected to either SELF or total serum. Taken together, these results illustrate how the early embryonic transcriptome responds to increased lipid exposure through an inflammatory and metabolic signature.

The Adenosine Salvage Pathway as an Alternative to Mitochondrial Production of ATP in Maturing Mammalian Oocytes

ABSTRACT Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

Transcriptomic Signature to Oxidative Stress Exposure at the Time of Embryonic Genome Activation in Bovine Blastocysts

In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions. Two pro‐oxidant agents, one that acts extracellularly by promoting reactive oxygen species (ROS) production (0.01 mM 2,2′‐azobis (2‐amidinopropane) dihydrochloride [AAPH]) or another that acts intracellularly by inhibiting glutathione synthesis (0.4 mM buthionine sulfoximine [BSO]) were added separately to in vitro culture media from Day 3 (8–16‐cell stage) onward. Transcriptomic analysis was then performed on resulting Day‐7 blastocysts. In the literature, these two pro‐oxidant conditions were shown to induce delayed degeneration in a proportion of Day‐8 blastocysts. In our experiment, no morphological difference was visible, but AAPH tended to decrease the blastocyst rate while BSO significantly reduced it, indicating a differential impact on the surviving population. At the transcriptomic level, blastocysts that survived either pro‐oxidant exposure showed oxidative stress and an inflammatory response (ARRB2), although AAPH induced higher disturbances in cellular homeostasis (SERPINE1). Functional genomics of the BSO profile, however, identified differential expression of genes related to glycine metabolism and energy metabolism (TPI1). These differential features might be indicative of pre‐degenerative blastocysts (IGFBP7) in the AAPH population whereas BSO exposure would select the most viable individuals (TKDP1). Together, these results illustrate how oxidative disruption of pre‐attachment development is associated with systematic up‐regulation of several metabolic markers. Moreover, it indicates that a better capacity to survive anti‐oxidant depletion may allow for the survival of blastocysts with a quieter metabolism after compaction. Mol. Reprod. Dev. 9999:XX–XX.

The embryonic stress response to in vitro culture: insight from genomic analysis

Recent genomic studies have shed light on the impact of in vitro culture (IVC) on embryonic homeostasis and the differential gene expression profiles associated with lower developmental competence. Consistently, the embryonic stress responses to IVC conditions correlate with transcriptomic changes in pathways related to energetic metabolism, extracellular matrix remodelling and inflammatory signalling. These changes appear to result from a developmental adaptation that enhances a Warburg-like effect known to occur naturally during blastulation. First discovered in cancer cells, the Warburg effect (increased glycolysis under aerobic conditions) is thought to result from mitochondrial dysfunction. In the case of IVC embryos, culture conditions may interfere with mitochondrial maturation and oxidative phosphorylation, forcing cells to rely on glycolysis in order to maintain energetic homeostasis. While beneficial in the short term, such adaptations may lead to epigenetic changes with potential long-term effects on implantation, foetal growth and post-natal health. We conclude that lessening the detrimental effects of IVC on mitochondrial activity would lead to significantly improved embryo quality.