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Abd El Naby, W. S., T. H. Hagos, M. M. Hossain, D. Salilew-Wondim, Y. a Gad, F. Rings, M. U. Cinar, E. Tholen, C. Looft, K. Schellander, et al., "Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos.", Zygote (Cambridge, England), pp. 1-21, 2011. Abstract

SummaryMicroRNAs (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.

Abdelatty, A. M., M. E. Iwaniuk, S. B. Potts, and A. Gad, "Influence of maternal nutrition and heat stress on bovine oocyte and embryo development", International Journal of Veterinary Science and Medicine: Elsevier, 2018. Abstract
a Abdoon, S., N. Ghanem, O. M. Kandil, A. Gad, K. Schellander, and D. Tesfye, "cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos.", Theriogenology, vol. 77, pp. 1240-1251, 2012. Abstract

The retarded development of parthenote embryo could be due to aberrant epigenetic imprinting, which may disrupt many aspects and lead to conceptus demise. The present work was conducted to: 1) compare the development of in vitro produced (IVP) and parthenogenetically developed (P) buffalo embryos from the 2-cell to blastocyst stage; 2) investigate the global gene expression profile and search for new candidate transcripts differing between IVP and P buffalo blastocyst using cDNA microarray analysis (validated by Real Time PCR); 3) follow the particular expression patterns of PLAC8 and OCT4 genes at five different stages of preimplantation development by Real Time PCR; and 4) study the expression of CDX2 at the blastcocyst stage. Cleavage rate was higher (P < 0.05) in P than IVP buffalo embryos, while, progression to blastocyst and number of cells per blastocyst were lower (P < 0.05) in P than IVP blastocysts. Microarray analysis indicate that 56 differentially expressed genes between the two groups, of which 51 genes (91.07%) were up-regulated, and five genes were downregulated in IVP blastocyst, using 1.4 fold-changes as a cutoff. Differentially expressed genes are related to translation, nucleic acid synthesis, cell adhesion and placentation. Validation of candidate genes revealed that the transcript abundance of PTGS2, RPS27A, TM2D3, PPA1, AlOX15, RPLO and PLAC8 were downregulated (7/8) in parthenote blastocyst compared to the IVP blastocyst. PLAC8 gene expression was higher (P < 0.05) at 2-cell, morula and blastocyst stages in IVP embryos compared with parthenote embryos. The OCT4 gene expression was higher (P < 0.05) in 2-cell, 4-cell, 8-cell and blastocysts produced in vitro. In conclusion, the retarded development of parthenogenetic buffalo embryos could be due to downregulation of genes related to translation, nucleic acid synthesis, cell adhesion, and placental development. The low expression of PLAC8 and OCT4 during the different stages of development may be responsible, in part, to the failure of development of parthenote buffalo embryos.

Ahmed, D. A. - E. R., N. Ghanem, S. M. Dessouki, M. S. Faheem, A. Y. Gad, and A. H. Barkawi, "Developmental Competence of Buffalo Oocytes Cultured Under Different Oxygen Tensions after Selection with Brilliant Cresyl Blue", World's Veterinary Journal, vol. 10, issue 2, pp. 246-253, 2020. Abstract
Amin, A., A. Gad, D. Salilew-Wondim, S. Prastowo, E. Held, M. Hoelker, F. Rings, E. Tholen, C. Neuhoff, C. Looft, et al., "Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway", Molecular Reproduction and Development, no. February, pp. n/a–n/a, 2014. AbstractWebsite

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 analysed 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 NRF2 and lower ROS levels were detected in early (competent) blastocysts compared to their late (non-competent) 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.

Ashour, G., A. Gad, A. K. Fayed, N. A. Ashmawy, and A. El-Sayed, "Evaluation of Growth Performance, Blood Metabolites and Gene Expression Analysis in Egyptian Sheep Breeds, in Relation to Age", World’s Veterinary Journal, vol. 10, issue 4, pp. 18-29, 2020. Abstract


Benc, M., F. Strejcek, M. Morovic, A. Bartkova, M. Murin, A. Gad, A. Bonnet-Garnier, F. P. Percinic, and J. Laurincik, "Improving the Quality of Oocytes with the Help of Nucleolotransfer Therapy", Pharmaceuticals, vol. 14, no. 4, 2021. AbstractWebsite

The nucleolus is an important nucleus sub-organelle found in almost all eukaryotic cells. On the one hand, it is known as a differentiated active site of ribosome biogenesis in somatic cells, but on the other hand, in fully grown oocytes, zygotes, and early embryos (up to the major embryonic genome activation), it is in the form of a particular homogenous and compact structure called a fibrillar sphere. Nowadays, thanks to recent studies, we know many important functions of this, no doubt, interesting membraneless nucleus sub-organelle involved in oocyte maturation, embryonic genome activation, rRNA synthesis, etc. However, many questions are still unexplained and remain a mystery. Our aim is to create a comprehensive overview of the recent knowledge on the fibrillar sphere and envision how this knowledge could be utilized in further research in the field of biotechnology and nucleolotransfer therapy.

El-Gendy, E. A., A. Y. Gad, and A. Mostageer, "Sperm-mediated gene transfer in poultry 1. The relationship with cock sperm viability", Arab Journal of Biotechnology, vol. 10, issue 1, pp. 1-12, 2007. Abstract


El-Rashidy, A. A., G. Waly, A. Gad, A. A. Hashem, P. Balasubramanian, S. Kaya, A. R. Boccaccini, and I. Sami, "Preparation and in vitro characterization of silver-doped bioactive glass nanoparticles fabricated using a sol-gel process and modified Stöber method", Journal of Non-Crystalline Solids, vol. 483: North-Holland, pp. 26-36, 2018. Abstract
El-Rashidy, A., G. Waly, A. Gad, J. A. Roether, J. Hum, Y. Yang, R. Detsch, A. Hashem, I. Sami, W. G. H. H. Goldmann, et al., "Antibacterial activity and biocompatibility of zein scaffolds containing silver-doped bioactive glass", Biomedical Materials, 2018. AbstractWebsite

Abstract Composite 3D scaffolds combining natural polymers and bioceramics are promising candidates for bone tissue engineering (BTE). Zein, as a natural plant protein, offers several advantages, including biocompatibility, adequate strength properties, and low/no immunogenicity; however, it lacks bioactivity. Thus, composite zein:bioactive glass (BG) scaffolds are proposed as promising candidate for BTE applications, with silver-doping of bioactive glass providing an antibacterial effect against possible post-implantation infection. Therefore, the aim of this study was to investigate the in vitro antibacterial properties, biocompatibility, bioactivity and compressive strength of zein scaffolds containing silver-doped bioactive glass. BG nanoparticles, undoped and Ag-doped, were fabricated using the sol-gel method. 3D composite zein:BG scaffolds, containing 20 wt.% BG, were prepared and their antibacterial activity against E. coli and S. aureus was assessed using the disk diffusion assay. Human osteoblast-like MG-63 cells were used to evaluate the in vitro biocompatibility of the prepared scaffold groups. In addition, the compressive strength of the scaffolds was determined using uniaxial compression strength testing and the scaffold interconnected porosity was measured using helium pycnometer. Disc diffusion assay showed that only zein scaffolds containing Ag-doped sol-gel BG are antibacterially positive against E. coli and S. aureus. Pure zein scaffolds and zein scaffolds containing sol-gel-derived BG showed no negative influence on the growth of MG-63 cells, as evident by the cells’ ability to survive, proliferate, and function on these scaffolds. Moreover, incorporating sol-gel-derived BG into zein scaffolds at zein:BG of 80:20 ratio showed bioactive properties with adequate porosity without affecting the scaffolds’ compressive strengths, which was similar to that of trabecular bone, suggesting that the new composites have potential for BTE applications in non-loaded bearing areas.

El-Rashidy, A. A., A. Gad, A. E. - H. G. Abu-Hussein, S. I. Habib, N. A. Badr, and A. A. Hashem, "Chemical and Biological Evaluation of Egyptian Nile Tilapia (Oreochromis Niloticas) Fish Scale Collagen", International Journal of Biological Macromolecules, vol. 79: Elsevier, pp. 618–626, 2015. Abstract

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.

El-Sayed, A., G. Ashour, M. Khalifa, and A. Gad, "Vitrification assessment of buffalo (Bubalus bubalis) oocytes: morphological and molecular aspects", Egyptian J. Anim. Prod, vol. 52, pp. 1-7, 2015. Abstract
Faheem, M. S. S., N. Ghanem, A. Gad, R. Procházka, and S. M. M. Dessouki, "Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress Under In Vitro Condition", Animals, vol. 11, no. 3, 2021. AbstractWebsite

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Forde, N., F. Carter, S. di Francesco, J. P. Mehta, M. Garcia-Herreros, A. Gad, D. Tesfaye, M. Hoelker, K. Schellander, and P. Lonergan, "Endometrial response of beef heifers on day 7 following insemination to supraphysiological concentrations of progesterone associated with superovulation.", Physiological genomics, vol. 44, issue 22, pp. 1107-15, 2012. Abstract

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

a. Gad, K. Schellander, M. Hoelker, and D. Tesfaye, "Transcriptome profile of early mammalian embryos in response to culture environment", Animal Reproduction Science, vol. 134, issue 2010: Elsevier B.V., pp. 76-83, 2012. Abstract


Gad, A., J. M. Sánchez, J. A. Browne, L. Nemcova, J. Laurincik, R. Prochazka, and P. Lonergan, "Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle", Scientific Reports, vol. 10, no. 1: Nature Publishing Group UK, pp. 1–16, dec, 2020. AbstractWebsite

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

Gad, A., U. Besenfelder, V. Havlicek, M. Hölker, F. Rings, I. Dufort, M. A. Sirard, K. Schellander, and D. Tesfaye, "In vitro Culture During Oocyte Maturation and Fertilization Influences The Treanscriptome Profiles of Bovine Blastocysts", Reproduction, Fertility and Development, vol. 27, no. 1: CSIRO PUBLISHING, pp. 186–187, 2015. Abstract

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitrofertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivoproduced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivoproduced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared within vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

Gad, A., L. Nemcova, M. Murin, J. Kanka, J. Laurincik, M. Benc, L. Pendovski, and R. Prochazka, "MicroRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles", Molecular Reproduction and Development, vol. 86, issue 4: John Wiley {&} Sons, Ltd, pp. 426–439, feb, 2019. AbstractWebsite

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte‐specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3–6 mm) or small (SO; 1.5–1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR‐205, miR‐16, miR‐148a‐3p, and miR‐125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA‐target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K‐Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development.

Gad, A., M. Hoelker, U. Besenfelder, V. Havlicek, U. Cinar, F. Rings, E. Held, I. Dufort, M. - A. Sirard, K. Schellander, et al., "Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions.", Biology of reproduction, vol. 87, issue 4, pp. 1-13, 2012. 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 (IVP) 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 EmbryoGENE's 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 vs. 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.

Gad, A., U. Besenfelder, F. Rings, N. Ghanem, D. Salilew-Wondim, M. M. Hossain, D. Tesfaye, P. Lonergan, a Becker, U. Cinar, et al., "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.", Human reproduction (Oxford, England), vol. 26, issue 7, pp. 1693-707, 2011. Abstract

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.

Gad, A., L. Nemcova, M. Murin, V. Kinterova, J. Kanka, J. Laurincik, M. Benc, L. Pendovski, and R. Prochazka, "Global transcriptome analysis of porcine oocytes in correlation with follicle size", Molecular Reproduction and Development, vol. 87, issue 1, no. July: John Wiley {&} Sons, Ltd, pp. 102-114, 2020. AbstractWebsite


Gad, A., S. Abu Hamed, M. Khalifa, A. Amin, A. El-Sayed, S. A. Swiefy, and S. El-Assal, "Retinoic acid improves maturation rate and upregulates the expression of antioxidant-related genes in in vitro matured buffalo (Bubalus bubalis) oocytes", International Journal of Veterinary Science and Medicine, vol. 6, issue 2: Elsevier, pp. 279–285, sep, 2018. AbstractWebsite

Retinoic acid, vitamin A metabolite, plays a role in oocyte development and maturation in different ways including gene expression alteration and/or prohibiting oxidative stress. The objective of this study was to examine the effect of 9-cis-retinoic acid (9-cisRA) on the quality and maturation rate of buffalo oocytes. Cumulus-oocyte complexes (COCs, n = 460) were collected from ovaries of slaughtered buffalos. Varying concentrations of 9-cisRA (0, 5, 50, and 200 nM) were added to the maturation medium, and the following parameters were analyzed: (i) maturation and cleavage rates, (ii) mitochondrial activity and reactive oxygen species (ROS) levels, (iii) expression level of antioxidant-related genes (PRDX1, SOD1, CAT, HOMX1, and GPX4) using RT-qPCR. Maturation rate was significantly improved in 5 nM 9-cisRA oocyte group (95.8{%}, P {\textless} .05) compared to control and other treatment groups (86.7{%} in control group). The same oocyte group exhibited significantly higher mitochondrial membrane potential activity and lower ROS accumulation level compared to other treatment groups. Antioxidant-related genes were up-regulated in oocytes matured with 5 or 50 nM 9-cisRA compared to control and 200 nM 9-cisRA groups. In contrast, 200 nM of 9-cisRA showed a clear down-regulation for antioxidant-related genes except for PRDX1. In conclusion, supplementation of 9-cisRA with a lower concentration (5 nM) to the buffalo oocytes maturation media promotes maturation rate through a protection mechanism that maintains adequate levels of antioxidant-related transcripts and improves mitochondrial activity. However, 9-cisRA has no significant effect on the cleavage rate of the treated oocytes.

Gad, A., M. Murin, L. Nemcova, A. Bartkova, J. Laurincik, and R. Procházka, "Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos", Animals, vol. 10, no. 12, 2020. AbstractWebsite

Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

Gebremedhn, S., A. Ali, A. Gad, R. Prochazka, and D. Tesfaye, "Extracellular Vesicles as Mediators of Environmental and Metabolic Stress Coping Mechanisms During Mammalian Follicular Development", Frontiers in Veterinary Science, vol. 7, pp. 961, 2020. AbstractWebsite

Extracellular vesicles are evolutionarily conserved nano-sized phospholipid membraned structures and released from virtually all types of cells into the extracellular space. Their ability to carry various molecular cargos (mRNA, miRNA, proteins, and lipids) from one cell to the other to exert functional impact on the target cells enables them to play a significant role in cell to cell communication during follicular development. As the molecular signals carried by extracellular vesicles reflect the physiological status of the cells of origin, they are expected to mediate any effect of environmental or metabolic stress on the follicualr cells and the growing oocyte. Recent studies have evidenced that reproductive cells exposed to various environmental stressors (heat and oxidative stress) released extracellular vesicles enriched with mRNA and miRNA associated with stress response mechanisms. Moreover, the metabolic status of post-calving cows could be well-reflected in the follicular extracellular vesicle's miRNA profile, which signified the potential role of extracellular cellular vesicle molecular signals in mediating the effect of metabolic stress on follicular and oocyte development. In the present review, the potential role of extracellular vesicles in mediating the effect of environmental and metabolic stress in various reproductive cells and oocytes are thoroughly discussed Moreover, considering the importance of extracellular vesicles in shuttling protective or rescuing molecular signals during stress, their potential usage as means of targeted delivery of molecules to mitigate the effect of stress on oocytes are addressed as the focus of future research.

Gebremedhn, S., A. Gad, H. S. Aglan, J. Laurincik, R. Prochazka, D. Salilew-Wondim, M. Hoelker, K. Schellander, and D. Tesfaye, "Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells", Scientific Reports, vol. 10, no. 1: Nature Publishing Group, pp. 1–19, sep, 2020. AbstractWebsite

Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.