Different pre-implantation phenotypes of bovine blastocysts produced in vitro.

Embryo transfer in cattle is performed with blastocysts produced in vivo or in vitro using defined media. However, outdated systems such as those that use serum and co-culture remain of interest for research purposes. Here, we investigated the effect of additional culture time on in vitro-produced embryos. Specifically, we compared embryos that formed a blastocoel at different times after fertilisation to those that stayed in culture for up to two additional days with respect to their development in vivo after temporary transfer to oestrus-synchronised recipients. A pre-transfer set (D6, D6+1, D6+2, D7, D7+1, D8) was examined using microarray analyses and correlated with a post-transfer set that included two different days of transfer (D6-T6, D6+2-T8, D7+1-T8, D8-T8). All surviving conceptuses reached primitive-streak stages and filamentous sizes similarly to in vivo (D18) or in vitro controls (D7/T7). The recovery rate differed between D6 and D8 embryos that were immediately transferred (58 vs 25%). With an intermediate survival rate (33%), the D6 embryos with two additional days in culture produced nine times more IFN-tau (IFNT) at D18 than the D6 embryos that were immediately transferred. At the end of culture, D6 and D6+2 embryos displayed the highest number of gene expression differences. Despite a mortality of 40­60%, no signature was detectable in any of the transferred groups that would account for the embryos' fates. Initially reputed to be beneficial in producing more blastocysts, our culture system of B2 medium plus serum and co-culture generated blastocysts that were distinct from those developed in vivo (D7).

STAT3 signaling stimulates miR-21 expression in bovine cumulus cells during in vitro oocyte maturation.

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.

Short-term effect of FSH on gene expression in bovine granulosa cells in vitro.

In reproduction, FSH is one of the most important hormones, especially in females, because it controls the number of follicles and the rate of follicular growth. Although several studies have examined the follicular response at the transcriptome level, it is difficult to obtain a clear and complete picture of the genes responding to an increase in FSH in an in vivo context because follicles undergo rapid morphological and physical changes during their growth. To help define the transcriptome downstream response to FSH, an in vitro model was used in the present study to observe the short-term (4h) cellular response. Gene expression analysis highlighted a set of novel transcripts that had not been reported previously as being part of the FSH response. Moreover, the results of the present study indicate that the epithelial to mesenchymal transition pathway is inhibited by short-term FSH stimuli, maintaining follicles in a growth phase and preventing differentiation. Modulating gene expression in vitro has physiological limitations, but it can help assess the potential downstream response and begin the mapping of the granulosa cell transcriptome in relation to FSH. This information is a key feature to help discriminate between the effects of FSH and LH, or to elucidate the overlapping of insulin-like growth factor 1 and FSH in the granulosa mitogenic response.

Metabolic stress induces modifications in the epigenetic program of preimplantation bovine embryos.

The mammalian embryo is sensitive to and adapts to its metabolic environment. The mother's metabolic health and nutrient availability, for example, can modulate the oviductal fluid composition and thus embryo development. In this project, we induced energetic stress in bovine embryos during early culture to observe the epigenetic responses associated with metabolic stress, using a treatment paradigm known to decrease blastocyst rates. Embryos were generated using oocytes from slaughtered cows, and then exposed to an elevated glucose concentration (5 vs. 0.2 mM in control conditions) for the first 3 days post-fertilization, followed by normal media until the blastocyst stage. The EmbryoGENE platform was then used to identify DNA methylation differences between the two treatments. Probes (450,000) were then analyzed based on their genome location and methylation differences. Our results revealed that elevated glucose led to hypomethylation close to telomeric regions and methylation changes on genomic regions associated with energy metabolism.

Spermatozoa DNA methylation patterns differ due to peripubertal age in bulls.

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.

Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage.

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae.

Regulation of ATF1 and ATF2 transcripts by sequences in their 3' untranslated region in cleavage-stage cattle embryos.

The sequence of a 3' untranslated region (3'UTR) of mRNA governs the timing of its polyadenylation and translation in mammalian oocytes and early embryos. The objective of this study was to assess the influence of cis-elements in the 3'UTR of the developmentally important ATF1 and ATF2 transcripts on their timely translation during first cleavages in bovine embryos. Eight different reporter mRNAs (coding sequence of green fluorescent protein [GFP] fused to the 3'UTR of short or long isoforms of cattle ATF1 or -2, with or without polyadenylation) or a control GFP mRNA were microinjected separately into presumptive bovine zygotes at 18 hr post-insemination (hpi), followed by epifluorescence assessment for GFP translation between 24 and 80 hpi (expressed as percentage of GFP-positive embryos calculated from the total number of individuals). The presence of either polyadenine or 3'UTR sequence in deadenylated constructs is required for GFP translation (implying the need for polyadenylation), and all exogenous mRNAs that met either criteria were translated as soon as 24 hpi-except for long-deadenylated ATF2-UTR, whose translation began at 36 hpi. Overall, GFP was more visibly translated in competent (cleaving) embryos, particularly in long ATF1/2 constructs. The current data shows a timely GFP translation in bovine embryos depending on sequences in the 3'UTR of ATF1/2, and indicates a difference between short and long isoforms. In addition, cleaving embryos displayed increased translational capacity of the tested constructs. Functional confirmation of the identification cis-sequences in the 3'UTR of ATF1/2 will contribute to the understanding of maternal mRNA translation regulation during early cattle development.

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

STUDY QUESTION: Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER: The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY: Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION: Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD: RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). LARGE SCALE DATA: The data are available through the GEO series accession number GSE79886. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

Meta-analysis of gene expression profiles in granulosa cells during folliculogenesis.

Folliculogenesis involves coordinated profound changes in different follicular compartments and significant modifications of their gene expression patterns, particularly in granulosa cells. Huge datasets have accumulated from the analyses of granulosa cell transcriptomic signatures in predefined physiological contexts using different technological platforms. However, no comprehensive overview of folliculogenesis is available. This would require integration of datasets from numerous individual studies. A prerequisite for such integration would be the use of comparable platforms and experimental conditions. The EmbryoGENE program was created to study bovine granulosa cell transcriptomics under different physiological conditions using the same platform. Based on the data thus generated so far, we present here an interactive web interface called GranulosaIMAGE (Integrative Meta-Analysis of Gene Expression), which provides dynamic expression profiles of any gene of interest and all isoforms thereof in granulosa cells at different stages of folliculogenesis. GranulosaIMAGE features two kinds of expression profiles: gene expression kinetics during bovine folliculogenesis from small (6 mm) to pre-ovulatory follicles under different hormonal and physiological conditions and expression profiles of granulosa cells of dominant follicles from post-partum cows in different metabolic states. This article provides selected examples of expression patterns along with suggestions for users to access and generate their own patterns using GranulosaIMAGE. The possibility of analysing gene expression dynamics during the late stages of folliculogenesis in a mono-ovulatory species such as bovine should provide a new and enriched perspective on ovarian physiology.

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: 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. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts.

Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development.

Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging.

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P>0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P=0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.

Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.

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.

The effect of energy balance on the transcriptome of bovine granulosa cells at 60 days postpartum.

Dairy cows expend great amounts of energy during the lactation peak to cope with milk production. A state of negative energy balance (NEB) was suggested as a cause for the suboptimal fertility observed during this period, via an interaction with ovarian function. The objective of this study was to identify the impact of NEB on gene expression in granulosa cells of dairy cows at 60 days postpartum and to suggest a potential treatment to improve ovarian function. Dairy cows at 60 days postpartum from 10 typical medium-sized farms were synchronized using a single injection of prostaglandin. Dominant follicles  were collected 42 hours later by transvaginal aspiration. Blood concentrations of beta-hydroxybutyrate (BHB) on the day of aspiration were used to classify animals into two groups: severe NEB (high BHB, n = 12) and mild NEB (low BHB, n = 12). The transcriptomes of granulosa cells from both groups were contrasted using microarrays, and the differentially expressed genes were analyzed using Ingenuity Pathway Analysis to identify affected functions and potential upstream regulators. Genes linked with cellular organization (KRT4 and PPL), proliferation (TACSTD2), and fatty acids metabolism (VNN2) were downregulated in granulosa cells from animals with severe NEB. Several genes linked to decitabine, a hypomethylating agent, and with beta-estradiol, were downregulated in the severe NEB group. Numerous genes linked to vitamins A and D were also downregulated in this group of cows, suggesting a potential deficiency of these vitamins in dairy cows during the postpartum period. This study supports the idea that energy balance has an impact on follicular dynamics which could be detrimental to resumption of fertility after calving.

Global gene expression in granulosa cells of growing, plateau and atretic dominant follicles in cattle.

BACKGROUND: 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. METHODS: Granulosa cells were collected from slaughterhouse dairy cattle follicles with a diameter greater than 9 mm, and were classified at different phases of follicle growth based on flow cytometry profiles of DNA content after staining with propidium iodide. Three phases were identified based on the proportion of cells in -G1 (less than 2n DNA), G0-G1 (2n DNA) or S-M (more than 2n DNA) and follicles were thus allocated to the growing, plateau or atresia group. Between group analysis (BGA) showed clear segregation of the three groups, and the groups were contrasted against each other in a loop design to identify differently expressed genes. Ingenuity Pathway Analysis (IPA) was used to identify the functions and upstream regulators associated with the observed differently expressed genes. RESULTS: Major differences were observed between the growth phases. Granulosa cells from follicles in the plateau phase had increased expression of TYRO3 and downregulation of JAM2 compared to growing follicles, supporting the idea of a shift from proliferation to differentiation. On the other hand, genes regulating the response to oxidative stress (VNN1) and angiogenesis (ANGPT2) were upregulated in granulosa cells from atretic follicles. While the predicted activated functions in cells at the plateau stage compared to cells at the growing stage included synthesis and transport of molecules, the predictions for atretic follicles relative to plateau ones included an increase in apoptosis and cell death. CONCLUSION: Consistent with previous studies, these observations allowed us to match the presence of specific gene transcripts to a particular physiological status and consequently to classify follicles. The results also demonstrated that the plateau phase is not a simple 'in between' status between growth and atresia, as several characteristics are unique to this stage.

Studying bovine early embryo transcriptome by microarray.

Microarrays represent a significant advantage when studying gene expression in early embryo because they allow for a speedy study of a large number of genes even if the sample of interest contains small quantities of genetic material. Here we describe the protocols developed by the EmbryoGENE Network to study the bovine transcriptome in early embryo using a microarray experimental design.

Sexual dimorphism in developmental programming of the bovine preimplantation embryo caused by colony-stimulating factor 2.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes.

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

Rapidly cleaving bovine two-cell embryos have better developmental potential and a distinctive mRNA pattern.

Mammalian embryos that rapidly reach the two-cell stage in culture have a higher probability of becoming viable blastocysts. Our goal was to separate two-cell bovine embryos based on their zygotic cleavage timing, and to assess their global mRNA levels. Following in vitro fertilization, all embryos that cleaved by 29.5 hpi (early) were cultured separately from those that divided at 46 hpi (late). The blastocyst rates were 46.1 ± 3.7% and 6.1 ± 3.4% for early- and late-cleavers, respectively (P < 0.01). Seven replicates of selected two-cell embryos were collected at each time point for microarray characterization (n = 4) and quantitative reverse-transcriptase PCR (n = 3); the rest were left in culture for blastocyst evaluation. A total of 774 and 594 probes were preferentially present in early- and late-cleaving embryos, respectively (fold change ± 1.5, P < 0.05), with important contrasts related to cell cycle, gene expression, RNA processing, and protein degradation functions. A total of 12 transcripts were assessed by quantitative PCR, of which ATM, ATR, CTNNB1, MSH6, MRE11A, PCNA, APC, CENPE, and GRB2 were in agreement with the hybridization results. Since most of these molecules are directly or indirectly associated with cell-cycle regulation, DNA damage response, and transcription control, our results strongly suggest key roles for those biological functions in mammalian preimplantation development.

Evolutionary conservation of the oocyte transcriptome among vertebrates and its implications for understanding human reproductive function.

Cross-phylum and cross-species comparative transcriptomic analyses provide an evolutionary perspective on how specific tissues use genomic information. A significant mRNA subset present in the oocytes of most vertebrates is stabilized or stored for post-LH surge use. Since transcription is arrested in the oocyte before ovulation, this RNA is important for completing maturation and sustaining embryo development until zygotic genome activation. We compared the human oocyte transcriptome with an oocyte-enriched subset of mouse, bovine and frog (Xenopus laevis) genes in order to evaluate similarities between species. Graded temperature stringency hybridization on a multi-species oocyte cDNA array was used to measure the similarity of preferentially expressed sequences to the human oocyte library. Identity analysis of 679 human orthologs compared with each identified official gene symbol found in the subtractive (somatic-oocyte) libraries comprising our array revealed that bovine/human similarity was greater than mouse/human or frog/human similarity. However, based on protein sequence, mouse/human similarity was greater than bovine/human similarity. Among the genes over-expressed in oocytes relative to somatic tissue in Xenopus, Mus and Bos, a high level of conservation was found relative to humans, especially for genes involved in early embryonic development.