Habib A. Shojaei Saadi

Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation

ABSTRACT So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocytes polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

Ovarian Aging-Like Phenotype in the Hyperandrogenism-Induced Murine Model of Polycystic Ovary

There are prominently similar symptoms, effectors, and commonalities in the majority of characteristics between ovarian aging and polycystic ovarian syndrome (PCOS). Despite the approved role of oxidative stress in the pathogenesis of PCOS and aging, to our knowledge, the link between the PCO(S) and aging has not been investigated yet. In this study we investigated the possible exhibition of ovarian aging phenotype in murine model of PCO induced by daily oral administration of letrozole (1 mg/kg body weight) for 21 consecutive days in the female Wistar rats. Hyperandrogenization showed irregular cycles and histopathological characteristics of PCO which was associated with a significant increase in lipid peroxidation (LPO) and reactive oxygen species (ROS) and decrease in total antioxidant capacity (TAC) in serum and ovary. Moreover, serum testosterone, insulin and tumor necrosis factor-alpha (TNF-α) levels, and ovarian matrix metalloproteinase-2 (MMP-2) were increased in PCO rats compared with healthy controls, while estradiol and progesterone diminished. Almost all of these findings are interestingly found to be common with the characteristics identified with (ovarian) aging showing that hyperandrogenism-induced PCO in rat is associated with ovarian aging-like phenotypes. To our knowledge, this is the first report that provides evidence regarding the phenomenon of aging in PCO.

Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates

BackgroundGenome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure.ResultsSeveral PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001).ConclusionIt is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.

Combined methylation mapping of 5mC and 5hmC during early embryonic stages in bovine

BackgroundIt 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.ResultsOver 1.2 million sequencing reads were analyzed, resulting in 151,501 contigs, of which 69,136 were uniquely positioned on the genome. A total of 101,461 putative methylated sites were identified. The output of the three methods differed in genomic coverage as well as in the nature of the identified sites. The classical MspI/HpaII combination of restriction enzymes targeted CpG islands whereas the other methods covered 5mC and 5hmC sites outside of these regions. Data analysis suggests a transition of these methylation marks between Day-7 and Day-12 embryos in specific classes of repeat-containing elements.ConclusionsOur combined strategy offers a genomic map of the distribution of cytosine methylation/hydroxymethylation during early bovine embryo development. These results support the hypothesis of a regulatory phase of hypomethylation in repeat sequences during early embryogenesis.

Meeting the methodological challenges in molecular mapping of the embryonic epigenome.

The past decade of life sciences research has been driven by progress in genomics. Many voices are already proclaiming the post-genomics era, in which phenomena other than sequence polymorphism influence gene expression and also explain complex phenotypes. One of these burgeoning fields is the study of the epigenome. Although the mechanisms by which chromatin structure and reorganization as well as cytosine methylation influence gene expression are not fully understood, they are being invoked to explain the now-accepted long-term impact of the environment on gene expression, which appears to be a factor in the development of numerous diseases. Such studies are particularly relevant in early embryonic development, during which waves of epigenetic reprogramming are known to have profound impacts. Since gametes and zygotes are in the process of resetting the genome in order to create embryonic stem cells that will each differentiate to create one of many specific tissue types, this phase of life is now viewed as a window of susceptibility to epigenetic reprogramming errors. Epigenetics could explain the influence of factors such as the nutritional/metabolic status of the mother or the artificial environment of assisted reproductive technologies. However, the peculiar nature of early embryos in addition to their scarcity poses numerous technological challenges that are slowly being overcome. The principal subject of this article is to review the suitability of various current and emerging technological platforms to study oocytes and early embryonic epigenome with more emphasis on studying DNA methylation. Furthermore, the constraint of samples size, inherent to the study of preimplantation embryo development, was put in perspective with the various molecular platforms described.

Dual targeting of TNF-α and free radical toxic stress as a promising strategy to manage experimental polycystic ovary

Abstract Context: It is now clear that oxidative stress (OS) and chronic low-grade inflammation are two main pathways involved in polycystic ovary syndrome (PCOS) pathogenesis. Therefore, simultaneous targeting of these pathways by means of carvedilol and Semelil (ANGIPARS™), as established medicines with dual anti-cytokine and anti-oxidant potential may be a therapeutic alternative approach to the current treatments. Objective: The objective of this study is to study the protective effects of carvedilol and ANGIPARS™ on inflammatory and oxidative response in hyperandrogenism-induced polycystic ovary (PCO). Materials and methods: The murine model of PCO was induced by letrozole (1 mg/kg/d; orally) and effective doses of carvedilol (10 mg/kg/d; orally) and ANGIPARS™ (2.1 mg/kg/d; orally) were administrated for 21 d in PCO and non-PCO healthy rats. Ovarian folliculogenesis, sex hormones concentrations, OS, inflammatory, and metabolic biomarkers were assessed in serum and ovaries. Results: PCO rats exhibited ovarian cystogenesis which was preserved by the application of carvedilol and ANGIPARS™. In comparison with controls, decreased level of the total antioxidant power (TAP) and higher levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) in serum and ovaries (2.41 ± 0.67 versus 0.72 ± 0.11; and 0.17 ± 0.04 versus 0.05 ± 0.01; 5.48 ± 1.30 versus 10.56 ± 0.77; and 7.06 ± 1.94 versus 17.98 ± 0.98; p < 0.05, respectively) were detected in PCO rats. Moreover, the PCO rats exhibited hyperandrogenism due to a 3.7-fold increase in serum testosterone concentration (35.04 ± 3.17 versus 131.09 ± 13.24; p < 0.05) along with a 2.98-fold decrease in serum progesterone (6.19 ± 0.40 versus 18.50 ± 1.03; p < 0.05) and 5.2-fold decrease in serum estradiol (9.30 ± 0.61 versus 48.3 ± 2.10; p < 0.05) when compared with those of the control group. However, similar to the control group, normal levels of OS markers and sex hormones were detected in ANGIPARS™ and carvedilol co-treated PCO rats. Besides, when compared with controls, increased levels of TNF-α (770.75 ± 42.06 versus 477.14 ± 28.77; p < 0.05) and insulin (1.27 ± 0.10 versus 0.36 ± 0.05; p < 0.05) in PCO rats were significantly inhibited by carvedilol and ANGIPARS™ co-treatment. Discussion and conclusion: We evidenced the beneficial effects of carvedilol and ANGIPARS™ in PCO, which underpin the new alternative approach in using these kinds of medicines in female reproductive disorders.

Responses of bovine early embryos to S-adenosyl methionine supplementation in culture.

AIM There is a growing concern about the potential adverse effects of high dose folic acid (FA) supplementation before and during pregnancy. FA metabolism generates S-adenosyl methionine (SAM) which is an important cofactor of epigenetic programming. We sought to assess the impact of a large dose of SAM on early embryo development. MATERIALS & METHODS In vitro cultured bovine embryos were treated with SAM from the eight-cell stage to the blastocyst stage. In addition to the phenotype, the genome-wide epigenetic and transcription profiles were analyzed. RESULTS Treatment significantly improved embryo hatching and caused a shift in sex ratio in favor of males. SAM caused genome-wide hypermethylation mainly in exonic regions and in CpG islands. Although differentially expressed genes were associated with response to nutrients and developmental processes, no correspondence was found with the differentially methylated regions, suggesting that cellular responses to SAM treatment during early embryo development may not require DNA methylation-driven changes. CONCLUSION Since bovine embryos were not indifferent to SAM, effects of large-dose FA supplements on early embryonic development in humans cannot be ruled out.

Profiling the impact of the embryonic microenvironment through global transcriptomic and DNA methylome surveying

Transcriptomicanalyses have been conducted to study how mammalian early embryos cope with their immediate microenvironments especially when stressed from the application of assisted reproductive technologies, maternal nutrition or any other induced external factors. To study potential long term impacts from these stresses, description of early embryo epigenome is necessary. DNA methylation profiling of mammalian early embryo presents important challenges as they are rare biological material. Very recently, a genome-scale, base-resolution timeline of DNA methylation has been characterized for mouse embryo[1], however, such information has not been reported for treated embryos or for other species. We focus on bovine embryonic development since it follows a similar kinetic and developmental success rate as human. Using our transcriptomic platform[2] studies revealed that several key pathways seems to react when embryos are submitted to stress but it was also observed that long non-coding RNAs (IncRNAs) represent the class of RNA that are the most influenced by the embryonic environment further supporting the involvement of epigenetic mechanisms in the embryonic response. Commercial platform specific to the study of the bovine methylome do not exist yet. We therefore set to develop one with the constraints of enabling to survey the general topology of the genomic methylome using the limited amount of starting material offered by early embryos and also to provide a cost effective platform with a complete data analysis pipeline. We report the development of the Bovine Embryonic Global Methylome platform (BEGMP) which allows global DNA methylation from as little as 7-8 ng of gDNA starting material (representing 10 expanded blastocysts). Samples are processed through a modified HpaII Tiny Fragments (HTFs) enrichment by ligation-mediated PCR (HELP) assay. Initial surveying of the methylome found in Day-7 and Day-12 bovine embryos suggests a transition during development of methylation marks in repeat containing elements in a class-specific manner showing the potential of the platform to describe the ontology of the establishment of methylation marks during early embryogenesis. The platform was also used to study the impact of embryonic culture conditions where both the transcriptome and the methylome were surveyed from the same samples. Treatment of in vitro cultured embryos in presence of the methtyl donor S-adenosyl methionin showed that in addition to protein coding genes, IncRNAs were also impacted by the treatment. At the DNA methylation level, it showed global hypermethylation in treated embryos and parallel analysis revealed some similar function and network between early embryo transcriptome and methylome. Overall, BEGMP is the first developed and functional methylome platform for non-model spices which allows to survey the transcriptome and methylome from very restricted samples.