Oocytes and early embryos contain minute amounts of DNA, RNA and proteins, making the study of early mammalian development highly challenging. The study of the embryo epigenome, in particular the DNA methylome, has been made accessible thanks to the possibility of amplifying specific sequences according to their initial methylation status. This paper describes a novel platform dedicated to the genome-wide study of bovine DNA methylation, including a complete pipeline for data analysis and visualization. The platform allows processing and integrating of DNA methylome and transcriptome data from the same sample. Procedures were optimized for genome-wide analysis of 10 ng of DNA (10 bovine blastocysts). Bovine sperm and blastocysts were compared as a test of platform capability.

The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed.

Assisted reproduction technologies (ART) and high selection pressure in the dairy industry are leading towards the use of younger females for reproduction, thereby reducing the interval between generations. This situation may have a negative impact on embryo quality, thus reducing the success rate of the procedures. This study aimed to document the effects of oocyte donor age on embryo quality, at the transcriptomic level, in order to characterize the effects of using young females for reproduction purpose. Young Holstein heifers (n = 10) were used at three different ages for ovarian stimulation protocols and oocyte collections (at 8, 11 and 14 months). All of the oocytes were fertilized in vitro with the semen of one adult bull, generating three lots of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used for the assessment of gene expression patterns at the blastocyst stage. Embryos from animals at 8 vs 14 months and at 11 vs 14 months were used for microarray hybridization. Validation was done by performing RT-qPCR on seven candidate genes. Age-related contrast analysis (8 vs 14 mo and 11 vs 14 mo) identified 242 differentially expressed genes (DEGs) for the first contrast, and 296 for the second. The analysis of the molecular and biological functions of the DEGs suggests a metabolic cause to explain the differences that are observed between embryos from immature and adult subjects. The mTOR and PPAR signaling pathways, as well as the NRF2-mediated oxidative stress response pathways were among the gene expression pathways affected by donor age. In conclusion, the main differences between embryos produced at peri-pubertal ages are related to metabolic conditions resulting in a higher impact of in vitro conditions on blastocyts from younger heifers.

Genetic selection for the best suited offspring drives the dairy industry to use young genitors and assisted reproductive technologies (ART) to reduce generation intervals. However, sperm samples collected from peri-pubertal bulls have lower counts and quality compared to samples from adult bulls. Moreover, our previous study identified differentially methylated regions (DMRs) in sperms from early-, peri- and post-pubertal bulls. The aim of this study was to further investigate the impacts of paternal age on early embryos. To achieve this, we evaluated the transcriptome and the epigenome of bovine blastocysts generated from spermatozoa of bulls at 10, 12, and 16 months of age and used in vitro fertilization (IVF) of oocytes recovered from the same adult cows. A total of 259 probes were differentially expressed and 6953 probes were differentially methylated in the 10- vs 16-month and the 12- vs 16-month groups. Ingenuity Pathway Analysis (IPA) of transcriptomic data demonstrated that energy-related pathways such as oxidative phosphorylation, EIF2 signaling, and mitochondrial dysfunction were affected the most by the age of the bull. Meanwhile, IPA analysis of the epigenome revealed that protein kinase A signaling, RAR activation, and other pathways were influenced by paternal age. Overall, we showed that the bull's age mainly influenced metabolism-related pathways in blastocysts, and this could therefore impact subsequent development.

The present study analyzed the changes in gene expression induced by the Cryotop vitrification technique in bovine blastocyst-stage embryos, using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos were vitrified and compared with nonvitrified (control) embryos. After vitrification, embryos were warmed and cultured for an additional 4 hours. Survived embryos were used for microarray analysis and quantitative polymerase chain reaction (qPCR) quantification. Survival rates were higher (P < 0.05) in the control embryos (100%) than in the vitrified embryos (87%). Global gene expression analysis revealed that only 43 out of 21,139 genes exhibited significantly altered expression in the vitrified embryos compared to the control embryos, with a very limited fold change (P < 0.05). A total of 10 genes were assessed by qPCR. Only the FOS-like antigen 1 (FOSL1) gene presented differential expression (P < 0.05) according to both the array and qPCR methods, and it was overexpressed in vitrified embryos. Although, the major consequence of vitrification seems to be the activation of the apoptosis pathway in some cells. Indeed, FOSL1 is part of the activating protein 1 transcription factor complex and is implicated in a variety of cellular processes, including proliferation, differentiation, and apoptosis. Therefore, our results suggest that a limited increase in the rate of apoptosis was the only detectable response of the embryos to vitrification stress.

One of the goals of the EmbryoGENE network was to gather information on the conditions leading to competent oocytes. Using a combination of transcriptomic analyses we are building the foundation of the folliculome, which will take the form of a virtual follicle with gene expression profiling data spanning small to ovulatory or atretic follicles. The different models currently being established not only provide information on the follicular conditions leading to good outcome but also intermediary steps, including evolution towards atresia. The physiology of very few species has been covered to the extent of our database, which is the only one for mono-ovulatory species. The first interesting observation extracted from our data is related to the plateau phase of follicular development, which is not a linear intermediate between growth and ovulation but rather an important modification step of tissue ontogenesis during which growth switches to differentiation or atresia. The markers of cell division, matrix rearrangement, mesenchymal differentiation, oxidation, steroidogenesis and ovulatory changes identified confirm known changes but also several others are now hinting to a more complex picture of this dynamic tissue. In addition to biomarkers, we have insight into the multiple pathways involved during the last few days before ovulation. Our new ability to validate these networks in vitro using primary granulosa cells culture also contributes to the construction of a follicular blueprint. The amazing list of gene responding to FSH alone is a good start but a complete meta-analysis will provide the foundation of the bovine folliculome.

Sperm miRNAs were reported to regulate spermatogenesis and early embryonic development in some mammals including bovine. The dairy cattle breeding industry now tends to collect semen from younger bulls under high selection pressure at a time when semen quality may be suboptimal compared to adult bulls. Whether the patterns of spermatic miRNAs are affected by paternal age and/or impact early embryogenesis is not clear. Hence, we generated small non-coding RNA libraries of sperm collected from same bulls at 10, 12, and 16 months of age, using 16 months as control for differential expression and functional analysis.

The physiological state of the dominant follicle is important as it may be linked to the competence of the oocyte within. The objective of this study was to analyze, by transcriptomic analysis, the changes occurring in granulosa cells from dominant follicles at different phases of follicular growth.

Gene expression analysis performed through comparative abundance of transcripts is facing a new challenge with the increasing need to compare samples of known cell number, such as early embryos or laser microbiopsies, where the RNA contents of identical cellular inputs can by nature be variable. When working with scarce tissues, the success of microarray profiling largely depends on the efficiency of the amplification step as determined by its ability to preserve the relative abundance of transcripts in the resulting amplified sample. Maintaining this initial relative abundance across samples is paramount to the generation of physiologically relevant data when comparing samples of different RNA content. The T7 RNA polymerase (T7-IVT) amplification is widely used for microarray sample preparation. Characterization of the reaction's kinetics has clearly indicated that its true linear phase is of short duration and is followed by a nonlinear phase. This second phase leads to modifications in transcript abundance that biases comparison between samples of different types. The impact assessment performed in this study has shown that the standard amplification protocol significantly lowers the quality of microarray data, rendering more than half of differentially expressed candidates undetected and distorting the true proportional differences of all candidates analyzed.

The objective of this study was to build the transcriptomic profile of granulosa cells originating from follicles 6 to 9 mm in diameter in dairy cattle using microarrays.

Environmental exposure of human populations to organochlorines is still widespread despite several international regulations banning or restricting their use. This study tested the hypothesis that an environmentally relevant complex mixture of organochlorines comprising polychlorinated biphenyls (PCBs), technical chlordane, dichlorodiphenyldichloroethylene and 12 other components is toxic for porcine embryos (at relative concentrations of 1-10000-fold the environmental organochlorine levels of contamination or 4.2 microg/l total PCBs). We also tested the embryotoxicity of a metabolised organochlorine mixture (relative concentrations of 0.9, 1.8, 2.7, 3.6 and 4.5 microg/l hydroxy-PCBs (OH-PCBs)) obtained by extracting plasma samples from sows treated with the native mixture. Embryos produced in vitro were exposed to either the organochlorine mixture or the metabolised extract for 9 days. The organochlorine mixture reduced embryonic development at the 10000x concentration (relative concentration of 42 mg/l PCBs; p=0.05). The organochlorine mixture also reduced the mean number of blastomeres per expanded blastocyst in a dose-dependent manner (p=0.038) but did not induce blastomere apoptosis (p>0.05). In contrast, the metabolised extract did not affect development or blastomere number at the concentrations tested, although the highest level of this mixture (4.5 microg/l OH-PCBs) was still very low (i.e. similar to the 1x concentration of the organochlorine mixture, which also did not alter embryo parameters). These data lead to the conclusion that while high concentrations of the native organochlorine mixture are toxic for porcine embryos, concentrations of either the native or the metabolised mixture that bear some relevance to exposure of human populations in the Arctic were without observable effect.

Bromodichloromethane (BDCM) is one of the trihalomethanes present in chlorinated water. Humans are thus daily exposed. Previous contradictory results failed to clearly establish the adverse effects of low concentrations of BDCM. By using the porcine preimplantation embryo as a sensitive model, we showed that exposure to low concentrations of BDCM (10 and 100ppb) during the first week of embryo development induced adverse effect on the blastocyst rate and alteration of the estradiol pathway. Our results also suggest that blastocysts exposed to BDCM present transcriptomic and epigenomic adaptive modifications compatible with the cardiac anomalies observed by previous studies of newborns exposed to BDCM during gestation. Thus, phenotypic observations and toxicogenomic adaptations of embryo to low concentration of BDCM provide insights for BDCM risk assessment. Indeed, our results support the use of sensitive toxicogenomic models using environmentally relevant concentrations to which humans are exposed in order to conduct the risk assessment.

One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production 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 a significant increase (p < 0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses 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 canonical pathway affected by ADO. This opens up new opportunities for further investigations.

In the dairy industry, using semen as soon as the bull is mature enough to produce it is advantageous for breeding purposes. Mammalian spermatogenesis is a hormone-dependent developmental program in which a complex cascade of events must take place to ensure that germ cells reach the proper stage of development at the proper time. Conventional indicators of semen quality such as sperm cell motility and viability usually improve as bulls mature, meeting quality criteria satisfactorily at around 16 months. Using semen before that age may affect embryo viability, but other changes occurring during the peripubertal period should be considered. Although it is known that establishment of these patterns begins during foetal life, the extent to which sperm cell DNA methylation changes during puberty has not been studied. The aim of this study is to correlate the age of a young bull with the overall DNA methylation pattern of its spermatozoa. Spermatozoa were collected from bulls at the ages of 10 months (early pubertal), 12 months (late pubertal) and 16 months (pubertal). Each animal (n = 4) was compared to itself with 16 months as control. Genome-wide DNA methylation was analyzed by microarray using the EmbryoGENE DNA Methylation Analysis platform. Using a fold change over 1.5 and a 5% FDR p-value correction, a total of 2602 differently methylated regions were found in common between 10 months of age and 16 months of age. No differently methylated regions between 12 months and 16 months of age were found at the same level of statistical significance. We conclude that spermatozoa from bulls aged 10 months have a different epigenetic profile, which could compromise their value.

Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences.

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL-1 and 0.1 µg mL-1), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation (ATP5D; CYP11A1; NDUFB7; NDUFB10; NDUFS8). Taken together, we hypothesise that the increased proliferation in the insulin-treated groups might impair the developmental potential of the embryos by inducing metabolic stress on the molecular level, which could be detrimental for the survival of the embryo.

MicroRNAs are potent regulators of gene expression that have been widely implicated in reproduction and embryo development. Recent studies have demonstrated that miR-21, a microRNA extensively studied in the context of disease, is important in multiple facets of reproductive biology including folliculogenesis, ovulation, oocyte maturation and early mammalian development. Surprisingly, little is known about the mechanisms that regulate miR-21 and no studies have characterized these regulatory pathways in cumulus-oocyte complexes (COCs). We therefore investigated miR-21 in an in vitro model of bovine oocyte maturation. Levels of the primary transcript of miR-21 (pri-miR-21) and mature miR-21 increased markedly in COCs over the maturation period. Cloning of the bovine pri-miR-21 gene and promoter by 5'3'RACE (rapid amplification of cDNA ends) revealed a highly conserved region immediately upstream of the transcription start site and two alternatively-spliced variants of pri-miR-21. The promoter region contained several putative transcription factor binding sites, including two for signal transducer and activator of transcription 3 (STAT3). Mutation of these sites significantly decreased both the intrinsic activity of pri-miR-21 promoter-luciferase constructs and the response to leukemia inhibitory factor (LIF) (a STAT3 activator) in cultured MCF7 cells. In COCs, treatment with a STAT3 pathway inhibitor markedly decreased pri-miR-21 expression and prevented cumulus expansion. Pri-miR-21 expression was also inhibited by the protein synthesis inhibitor cycloheximide, suggesting that a protein ligand or signaling cofactor synthesized during maturation is necessary for transcription. Together these studies represent the first investigation of signaling pathways that directly influence miR-21 expression in bovine oocytes and cumulus cells.

It was recently established that changes in methylation during development are dynamic and involve both methylation and demethylation processes. Yet, which genomic sites are changing and what are the contributions of methylation (5mC) and hydroxymethylation (5hmC) to this epigenetic remodeling is still unknown. When studying early development, options for methylation profiling are limited by the unavailability of sufficient DNA material from these scarce samples and limitations are aggravated in non-model species due to the lack of technological platforms. We therefore sought to obtain a representation of differentially 5mC or 5hmC loci during bovine early embryo stages through the use of three complementary methods, based on selective methyl-sensitive restriction and enrichment by ligation-mediated PCR or on subtractive hybridization. Using these strategies, libraries of putative methylation and hydroxymethylated sites were generated from Day-7 and Day-12 bovine embryos.

The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected.

Selection of the best oocyte for subsequent steps of fertilization and embryo transfer was shown to be the crucial step in human infertility treatment procedure. Oocyte selection using morphological criteria mainly Zona pellucida (ZP) has been the gold standard method in assisted reproductive technologies (ART) clinics, but this selection approach has limitations in terms of accuracy, objectivity and constancy. Recent studies using OMICs-based approaches have allowed the identification of key molecular markers that quantitatively and non-invasively predict the oocyte quality for higher pregnancy rates and efficient infertility treatment. These biomarkers are a valuable reinforcement of the morphological selection criteria widely used in in vitro fertilization (IVF) clinics. In this context, this study was designed to investigate the relationship between transcriptomic predictors of oocyte quality found by our group and the conventional morphological parameters of oocyte quality mainly the ZP birefringence.