A. Gad

Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions

ABSTRACT Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENEs bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos.

MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30-40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60-70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction

BACKGROUND In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

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.

Bovine embryo survival under oxidative‐stress conditions is associated with activity of the NRF2‐mediated oxidative‐stress‐response pathway

In present study, we sought to examine the ability of preimplantation bovine embryos to activate the NF‐E2‐related factor 2 (NRF2)‐mediated oxidative‐stress response under an oxidative stress environment. In vitro 2‐, 4‐, 8‐, 16‐cell‐, and blastocyst‐stage embryos were cultured under low (5%) or high (20%) oxygen levels. The expression of NRF2, KEAP1 (NRF2 inhibitor), antioxidants downstream of NRF2, and genes associated with embryo metabolism were analyzed between the embryo groups using real‐time quantitative PCR. NRF2 and KEAP1 protein abundance, mitochondrial activity, and accumulation of reactive oxygen species (ROS) were also investigated in blastocysts of varying competence that were derived from high‐ or low‐oxygen levels. The expression levels of NRF2 and its downstream antioxidant genes were higher in 8‐cell, 16‐cell, and blastocyst stages under high oxygen tension, whereas KEAP1 expression was down‐regulated under the same conditions. Higher expression of NRF2 and lower ROS levels were detected in early (competent) blastocysts compared to their late (noncompetent) counterparts in both oxygen‐tension groups. Similarly, higher levels of active nuclear NRF2 protein were detected in competent blastocysts compared to their noncompetent counterparts. Thus, the survival and developmental competence of embryos cultured under oxidative stress are associated with activity of the NRF2‐mediated oxidative stress response pathway during bovine pre‐implantation embryo development. Mol. Reprod. Dev. 81: 497–513, 2014.

RNA Deep Sequencing Reveals Novel Candidate Genes and Polymorphisms in Boar Testis and Liver Tissues with Divergent Androstenone Levels

Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one) and skatole (3-methylindole). It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq). The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from −4.68 to 2.90 in testis samples and −2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

Transcriptome profile of early mammalian embryos in response to culture environment.

Early embryonic development, the period from maturation until blastocyst formation, is one of the most critical periods of mammalian development involves various morphological, cellular, and biochemical changes related to genomic activity. During the post-fertilization period, several major developmental events occur in the embryo which are regulating by a harmonized expression of genes and strongly influenced by culture conditions. The products of these genes are involved in various biological processes including metabolism, growth factor/cytokine signaling, stress adaptation, transcription and translation, epigenetic regulation of transcription, apoptosis, compaction and blastocyst formation. Post-fertilization culture environment is known to be the most important factor determining the quality of the resulting embryos as indicated in terms of cryo-tolerance and relative abundance of transcripts. However, the exact effect of culture conditions on gene expression and subsequent influences on molecular pathways controlling early development is still unknown. A number of culture environmental factors can influence the gene expression of produced embryos such as media composition, serum supplementation, number of embryos present in the culture drop and gas atmosphere. During the last ten years several studies were concerned with differences in the transcriptome profile of embryos produced under different environmental conditions and its subsequent influence on embryo developmental competence. From these studies, several genes have been determined as candidate genes controlling preimplantation embryo development and affecting its quality. Here we will discuss results of different experiments investigated the effect of different culture conditions on the transcriptome profile of bovine blastocyst. These experiments identified molecular mechanisms and pathways that influenced by culture conditions and this will enable to launch strategies to modify culture conditions to enhance the development of competent blastocyst.

Chemical and biological evaluation of Egyptian Nile Tilapia (Oreochromis niloticas) fish scale collagen.

Collagen is considered to be one of the most useful biomaterials with different medical applications. However, collagen properties differ from one source to another. The aim of this study was to extract, purify, characterize and perform preliminary biological evaluation of type I collagen from scales of Egyptian Nile Tilapia. Pepsin-solubilized collagen (PSC) was successfully prepared from Nile Tilapia fish scale waste. Lyophilized collagen was dissolved in dilute HCl to form acidic collagen solutions (ACS) which was neutralized to form gel. To confirm the biocompatibility of the produced gel, baby hamster kidney (BHK-21) fibroblast cells were seeded onto a 3D collagen gel (0.3% and 0.5%, w/v). The results of an SDS-PAGE test showed that the extracted collagens were type I collagen, with α chain composition of (α1)2α2. Thermal analysis showed that the denaturation temperature was 32 °C. X-ray diffraction (XRD) analysis and Fourier-transform infrared spectra (FTIR) showed that the extracted collagen had a triple helix structure. Active proliferation of BHK-21 cells with no signs of toxicity was evident with both collagen gel concentrations tested. The results show that Nile Tilapia scales can be an effective source of collagen extraction that could be used as a potential biomaterial in biomedical applications.

Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10−3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.