Christian Vigneault

Transcription Factor Expression Patterns in Bovine In Vitro-Derived Embryos Prior to Maternal-Zygotic Transition

Abstract Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

FSH withdrawal improves developmental competence of oocytes in the bovine model

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.

Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte.

Abstract Oocytes can synthesize and store maternal mRNA in an inactive translational state until the start of in vitro maturation. Cytoplasmic polyadenylation, driven by 3′-untranslated region (UTR) cis-acting cytoplasmic polyadenylation element (CPE), is associated with translational activation of cyclin B1 mRNA during maturation. The main aim of the present study was to investigate if bovine oocyte cyclin B1 mRNA undergoes cytoplasmic polyadenylation/translation during in vitro maturation, as in other species. We have found that cyclin B1 mRNA is present in two isoforms, consisting of the same open reading frame but with different 3′-UTR lengths. Only the longest isoform (cyclin B1L) has a putative CPE sequence and other regulatory sequences, and its mRNA level decreases during early embryo development. The polyadenylation state of cyclin B1L during in vitro maturation was studied. Results demonstrated that cyclin B1L bears a relatively long poly(A) tail in germinal vesicle-stage oocytes, which is further lengthened at 10 h of maturation, before metaphase I. Interestingly, cyclin B1L bears a short poly(A) tail when the ovaries and the oocytes are transported and manipulated on ice to stop the polyadenylation process. Cytoplasmic polyadenylation most probably occurs during ovary transport in warm saline, when oocytes are still in their follicular environment. Our results also show a link between cytoplasmic polyadenylation of cyclin B1 and translation/appearance of cyclin B1 protein before in vitro maturation.

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.

Unveiling the bovine embryo transcriptome during the maternal-to-embryonic transition.

Bovine early embryos are transcriptionally inactive and subsist through the initial developmental stages by the consumption of the maternal supplies provided by the oocyte until its own genome activation. In bovine, the activation of transcription occurs during the 8- to 16-cell stages and is associated with a phase called the maternal-to-embryonic transition (MET) where maternal mRNA are replaced by embryonic ones. Although the importance of the MET is well accepted, since its inhibition blocks embryonic development, very little is known about the transcripts expressed at this crucial step in embryogenesis. In this study, we generated and characterized a cDNA library enriched in embryonic transcripts expressed at the MET in bovine. Suppression subtractive hybridization followed by microarray hybridization was used to isolate more than 300 different transcripts overexpressed in untreated late eight-cell embryos compared with those treated with the transcriptional inhibitor, alpha-amanitin. Validation by quantitative RT-PCR of 15 genes from this library revealed that they had remarkable consistency with the microarray data. The transcripts isolated in this cDNA library have an interesting composition in terms of molecular functions; the majority is involved in gene transcription, RNA processing, or protein biosynthesis, and some are potentially involved in the maintenance of pluripotency observed in embryos. This collection of genes associated with the MET is a novel and potent tool that will be helpful in the understanding of particular events such as the reprogramming of somatic cells by nuclear transfer or for the improvement of embryonic culture conditions.

Temporal expression of factors involved in chromatin remodeling and in gene regulation during early bovine in vitro embryo development

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 or combinations of factors, 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.

Gene expression analysis of bovine oocytes with high developmental competence obtained from FSH-stimulated animals.

Recent progress in the ovarian stimulation protocol used for bovine in vitro maturation and fertilization, especially through optimization of the follicle‐stimulating hormone (FSH) withdrawal period (“coasting”) after ovarian pre‐treatment with FSH, has significantly improved blastocyst outcome. Despite this important success, the underlying factors leading to improved oocyte quality have not yet been identified. The aim of this project was to compare the transcriptome of germinal vesicle‐stage oocytes collected from FSH‐stimulated cows after various coasting periods (20, 44, 68, and 92 hr) to determine which transcripts were accumulated or depleted during the rise and fall of competence. Oocytes from each coasting period were compared to the three other times (optimal conditions, 44 and 68 hr; under‐matured, 20 hr; and over‐matured, 92 hr) per animal, allowing each cow to be its own control (24 collections). Microarray analysis revealed that between 5 and 338 transcripts were significantly different across the six comparisons, with an important longitudinal modulation in terms of gene expression profile. Not surprisingly, as the transcriptional activity decreased in these oocytes, several transcripts that are significantly modulated during coasting are related to RNA processing functions, as shown by functional analysis. Ingenuity Pathway Analysis also highlighted another important function: the control of chromosome segregation. The results presented here indicate that the quality gained with the optimal coasting time does not last, and also suggests a possible mechanism of control by transcript degradation that could be implicated if the oocyte is not ovulated at the right time. Mol. Reprod. Dev. 80: 428–440, 2013.

Investigation of MYST4 histone acetyltransferase and its involvement in mammalian gametogenesis

BackgroundVarious 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.ResultsAlthough MYST4 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 MYST4 and all-MYST4-isoforms were present in oocytes and in in vitro 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.ConclusionWe 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.

Impact of the LH surge on granulosa cell transcript levels as markers of oocyte developmental competence in cattle

In the case of in vitro embryonic production, it is known that not all oocytes detain the developmental capacity to form an embryo. This capacity appears to be acquired through completion of folliculogenesis, during which the oocyte and follicular cells influence their respective destinies. The differentiation status of granulosa cells (GCs) could therefore offer an indicator of oocyte quality. The aim of this study was to compare mRNA transcript abundance in GCs associated with oocytes that subsequently reach or not the blastocyst stage. GCs were collected from cattle following an ovarian stimulation protocol that did or did not include the administration of LH. GCs were classified according to the developmental stage achieved by the associated oocytes. Transcript abundance was measured by microarray. Follicles (n=189) obtained from cows before and after the LH surge were essentially similar and the rates of oocytes reaching the blastocyst stage were not significantly different (52 vs 41%), but blastocyst quality was significantly better in the post-LH-surge group. In GCs from the pre-LH-surge group and associated with developmentally competent oocytes, 18 overexpressed and 22 underexpressed transcripts were found, including novel uncharacterized transcripts, whereas no differentially expressed transcripts were associated with developmentally different oocytes in the post-LH-surge group. The novel transcriptomic response associated with LH appeared to mask the difference. Based on oocyte developmental competence, the period prior to the LH surge appears best suited for studying competence-associated mRNA transcripts in bovine follicle cells.

Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts.

Bovine embryo production is practiced worldwide for commercial purposes. A major concern of embryo suppliers is the impact of in vitro production systems on embryo quality. In the present study, we compared Buffalo Rat Liver cell coculture with semidefined, medium-based culture, oocytes recovered postmortem with those obtained from live animals, and in vitro with in vivo embryo development. Gene expression levels in expanded blastocysts were measured using microarray and quantitative RT-PCR. The systems were similar in terms of blastocyst yield and rate of development, whereas embryo productivity was greater for immature oocytes collected in vivo. Although immature oocytes collected in vivo had greater developmental competence, they yielded blastocysts that were indistinguishable (in terms of level of gene expression) from embryos derived from immature oocytes recovered postmortem. Culture conditions had a significant impact on gene expression, particularly among genes involved in lipid metabolism. Numerous uncharacterized novel transcript regions were also influenced by in vitro treatments. In conclusion, ovum pick-up combined with in vitro culture in semidefined medium provided a high blastocyst yield, without the deleterious effects associated with coculture.