The development of a complex technology such as in vitro fertilization (IVF) requires years of experimentation, sometimes comparing several species to learn how to create the right in vitro environment for oocytes, spermatozoa and early embryos. At the same time, individual species characteristics such as gamete physiology and gamete interaction are recently evolved traits and must be analysed within the context of each species. In the last 40 years since the birth of Louise Brown, IVF techniques progressed and are now used in multiple domestic and non-domestic animal species around the world. This does not mean that the technology is completely matured or satisfactory; a number of problems remain to be solved and several procedures still need to be optimized. The development of IVF in cattle is particularly interesting since agriculture practices permitted the commercial development of the procedure and it is now used at a scale comparable to human IVF (millions of newborns). The genomic selection of young animals or even embryos combined with sexing and freezing technologies is driving a new era of IVF in the dairy sector. The time has come for a retrospective analysis of the success and pitfalls of the last 40 years of bovine IVF and for the description of the challenges to overcome in the years to come.
Atef Ali and Marc-André Sirard
The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus–oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 μM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1–0.5 μM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.
Gael Cagnone and Marc-André Sirard
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.
Serge McGraw, Christian Vigneault and Marc-André Sirard
Distinct epigenetic modification events regulate gene expression and chromatin structure during the period between the immature oocyte and the blastocyst. Throughout this developmental period, important methylation fluctuations occur on genomic DNA and histones. Finding single orcombinations offactors, which are at work during this period is essential to understand the entire epigenetic process. With this in mind, we assessed the precise temporal expression profile, during preimplantation embryo development, of 15 key regulators involved in RNA, DNA or histone methylation, chromatin modification or silencing and transcription regulation. To achieve this, real-time RT-PCR was used to quantify the mRNA levels of ATF7IP, DMAP1, EHMT1, EHMT2, HELLS, JARID1A, JARID1B, JMJD1A, JMJD2A, LSD1, MeCP2, METTL3, PRMT2, PRMT5 and RCOR2, in the oocyte and throughout in vitro bovine embryo development. Our results demonstrate that all the 15 key regulators were present to different degrees in the developmental stages tested, and they can be divided into three different groups depending on their respective mRNA profile.
Christian Vigneault, Serge McGraw and Marc-Andre Sirard
Cleavage-stage bovine embryos are transcriptionally quiescent until they reach the 8- to 16-cell stage, and thus rely on the reserves provided by the stored maternal mRNAs and proteins found in the oocytes to achieve their first cell divisions. The objective of this study was to characterize the expression and localization of the transcriptional and translational regulators, Y box binding protein 2 (YBX2), TATA box-binding protein (TBP), and activating transcription factor 2 (ATF2), during bovine early embryo development. Germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, as well as 2-, 4-, 8-, 16-cell-stage embryos, morula, and blastocysts, produced in vitro were analyzed for temporal and spatial protein expression. Using Q-PCR, ATF2 mRNA expression was shown to remain constant from the GV-stage oocyte to the four-cell embryo, and then decreased through to the blastocyst stage. By contrast, the protein levels of ATF2 remained constant throughout embryo development and were found in both the cytoplasm and the nucleus. Both TBP and YBX2 showed opposite protein expression patterns, as YBX2 protein levels decreased throughout development, while TBP levels increased through to the blastocyst stage. Immunolocalization studies revealed that TBP protein was localized in the nucleus of 8- to 16-cell-stage embryos, whereas the translational regulator YBX2 was exclusively cytoplasmic and disappeared from the 16-cell stage onward. This study shows that YBX2, TBP, and ATF2 are differentially regulated through embryo development, and provides insight into the molecular events occurring during the activation of the bovine genome during embryo development in vitro.
Anne-Laure Nivet, Christian Vigneault, Patrick Blondin and Marc-André Sirard
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.
Mourad Assidi, Steph J Dieleman and Marc-André Sirard
Cumulus cells (CCs) are essential for oocytes to reach full development competency and become fertilized. Many major functional properties of CCs are triggered by gonadotropins and governed by the oocyte. Consequently, cumulus may reflect oocyte quality and is often used for oocyte selection. The most visible function of CCs is their ability for rapid extracellular matrix expansion after the LH surge. Although unexplained, LH induces the final maturation and improves oocyte quality. To study the LH signaling and gene expression cascade patterns close to the germinal vesicle breakdown, bovine CCs collected at 2 h before and 6 h after the LH surge were hybridized to a custom-made microarray to better understand the LH genomic action and find differentially expressed genes associated with the LH-induced oocyte final maturation. Functional genomic analysis of the 141 overexpressed and 161 underexpressed clones was performed according to their molecular functions, gene networks, and cell compartments. Following real-time PCR validation of our gene lists, some interesting pathways associated with the LH genomic action on CCs and their possible roles in oocyte final maturation, ovulation, and fertilization are discussed. A list of early potential markers of oocyte competency in vivo and in vitro is thereafter suggested. These early biomarkers are a preamble to understand the LH molecular pathways that trigger the final oocyte competence acquisition process in bovine.
Maud Vallée, Kazuhiro Aiba, Yulan Piao, Marie-France Palin, Minoru S H Ko and Marc-André Sirard
Cross-species comparison of gene expression is a powerful approach for discovering genes that have been conserved throughout evolution. Conserved genes are presumably very important in the mechanisms related to the unique molecular functions in oocytes. The objective of this study was to identify genes expressed in the oocyte and conserved across three diverse vertebrate species. We report the global gene expression profiles of Bos taurus and Xenopus laevis oocytes on an NIA mouse development microarray that consists of 60-mer oligonucleotide probes representing more than 20 000 mouse transcripts derived from stem cell, oocyte, and early embryo cDNA libraries. Analysis based on intensity values revealed that 9853 and 10 046 genes are expressed in bovine and Xenopus oocytes respectively. Furthermore, previously published microarray data on preimplantation development in the mouse were used for a comparative analysis of global oocyte gene expression profiles. Interestingly, a substantial proportion of the genes expressed in mouse oocytes is conserved between the three species (74%, 7275 genes). Moreover, functional annotation of these conserved oocyte-expressed genes confirmed that certain functions are conserved among the three species. RNA metabolism and cell cycle were among the over-represented Gene Ontology terms in the biological process category. Finally, a pattern-matching analysis identified 208 conserved maternally expressed genes. Results from these cross-species hybridizations allowed numerous genes expressed in oocytes and conserved between Mus musculus, B. taurus, and X. laevis to be identified. This comparative analysis of oocyte transcript profiles revealed a high degree of conservation among species.
David A Landry, Lia Rossi-Perazza, Simon Lafontaine and Marc-André Sirard
The use of younger gamete donors in dairy cattle genetic selection programs significantly accelerates genetic gains by decreasing the interval between generations. Ovarian stimulation (OS) and the practice of follicle-stimulating hormone (FSH) withdrawal, also known as coasting, are intensively used in pre-pubertal heifers without detrimental effects on subsequent reproductive performance but generally with lower embryo yields. However, recent data from embryo transfer programs showed similar embryo yields in younger and sexually mature animals but with a significant difference in the coasting period. The aim of the present study was to identify a set of granulosa cell biomarkers capable of distinguishing optimal follicle differentiation from late differentiation and atresia in order to assess the differences in coasting dynamics between pre- and post-pubertal donors. We integrated transcriptomic data sets from a public depository and used vote counting meta-analysis in order to elucidate the molecular changes occurring in granulosa cells during late follicle differentiation and atresia. The meta-analysis revealed the gene expression associated with follicle demise, and most importantly, identified potential biomarkers of that status in bovine granulosa cells. The comparison of the expression of six biomarkers between pre- and post-pubertal donors revealed that younger donors had more signs of atresia after the same period of coasting. We found different follicular dynamics following coasting in younger donors. It is possible that younger donors are less capable to sustain follicular survival most likely due to insufficient luteinizing hormone signaling. In summary, the pre-pubertal status influences follicular dynamics and reduces the oocyte developmental competence curve following OS and FSH withdrawal in heifers.
Anne-Laure Nivet, Audrey Bunel, Rémi Labrecque, Josée Belanger, Christian Vigneault, Patrick Blondin and Marc-André Sirard
Combinations of genetic, environmental, and management factors are suspected to explain the loss in fertility observed for over 20 years in dairy cows. In some cases, IVF is used. When compared with in vivo embryo production, IVF resulted in low success rates until the FSH coasting process (FSH starvation after superstimulation) was introduced in 2002. Increased competence associated with FSH withdrawal of aspirated oocyte for in vitro maturation and IVF has not been optimized nor explained yet. The goal here was to determine and characterize the optimal oocyte competence acquisition window during the coasting period by determining blastocyst rates and follicular cohort development. Commercial milking cycling cows (n=6) were stimulated with 3 days of FSH (6×40 mg NIH Folltropin-V given at 12 h intervals) followed by a coasting period of 20, 44, 68, or 92 h. Each animal was exposed to the four conditions and served as its own control. At the scheduled time, transvaginal aspirations of immature oocytes were performed followed by IVF of half the oocytes. The outcomes were as follows: i) FSH coasting was optimal at a defined period: between 44 and 68 h of coasting; ii) The best estimated coasting duration was ∼54±7 h; iii) Under these conditions, the best statistical blastocyst rate estimation was ∼70%; iv) Between 44 and 68 h of coasting, follicle size group proportions were similar; v) Follicle diameter was not linearly associated with competence. In conclusion, coasting duration is critical to harvest the oocytes at the right moment of follicular differentiation.