Ali Eroglu

Institute of Molecular Genetics AS CR, Praha, Praha, Czech Republic

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Publications (39)122.98 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me2SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me2SO exposure time, revealing that neither shrinkage nor Me2SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me2SO addition appears to result from interactions between the effects of Me2SO toxicity and osmotic stress. We also investigated Me2SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me2SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me2SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.
    Cryobiology 11/2013; · 2.14 Impact Factor
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    ABSTRACT: Heat shock factor binding protein 1 (HSBP1) is a 76 amino acid polypeptide that contains two arrays of hydrophobic heptad repeats and was originally identified through its interaction with the oligomerization domain of heat shock factor 1 (Hsf1), suppressing Hsf1's transcriptional activity following stress. To examine the function of HSBP1 in vivo, we generated mice with targeted disruption of the hsbp1 gene and examined zebrafish embryos treated with HSBP1-specific morpholino oligonucleotides. Our results show that hsbp1 is critical for preimplantation embryonic development. Embryonic stem (ES) cells deficient in hsbp1 survive and proliferate normally into the neural lineage in vitro; however, lack of hsbp1 in embryoid bodies (EBs) leads to disorganization of the germ layers and a reduction in the endoderm-specific markers (such as α-fetoprotein). We further show that hsbp1-deficient mouse EBs and knockdown of HSBP1 in zebrafish leads to an increase in the expression of the neural crest inducers Snail2, Tfap2α and Foxd3, suggesting a potential role for HSBP1 in the Wnt pathway. The hsbp1-deficient ES cells, EBs and zebrafish embryos with reduced HSBP1 levels exhibit elevated levels of Hsf1 activity and expression of heat shock proteins (Hsps). We conclude that HSBP1 plays an essential role during early mouse and zebrafish embryonic development.
    Developmental Biology 01/2013; · 3.87 Impact Factor
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    ABSTRACT: Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo cryopreservation became an indispensible part of infertility treatments. Currently, ARTs account for up to 3% of annual births in industrialized countries and are considered safe. However, recent studies reported an increased incidence of epigenetic birth defects among children conceived by ARTs. Since the timing of ARTs coincides with re-establishment of epigenetic marks (genomic imprinting) during gametogenesis and embryogenesis, it is likely that suboptimal ARTs may alter normal methylation patterns, and thus expression of imprinted genes, leading to epigenetic birth defects. The objective of this study was to investigate the effect of oocyte cryopreservation on expression of the imprinted genes involved in epigenetic birth defects. To this end, mouse metaphase II (M II) oocytes were cryopreserved by using either standard slow freezing (i.e., cooling to −35 °C at 0.3 °C/min in 1.5 M Me2SO + 0.1 M sucrose, and then plunging into liquid nitrogen [LN2]) or vitrification technique (i.e., plunging of oocytes within a ⩽ 1-ml volume of 15% Me2SO + 15% ethylene glycol + 0.5 M Sucrose into LN2). After warming at 37 °C and removal of cryoprotectants by stepwise dilution, cryopreserved oocytes along with untreated controls were inseminated and cultured to the blastocyst stage to determine their gene expression levels. To do so, blastocysts derived from cryopreserved and control oocytes were subjected first to reverse transcription and subsequently to real-time PCR. Gene expression levels in three replicates of experiments were analyzed by the comparative CT method using Rest Software. So far, our results suggest that both cryopreservation techniques downregulate the expression level of two imprinted genes (i.e., H19, and Ube3a). These findings warrant further studies to address the effect of oocyte cryopreservation on imprinted genes.
    Cryobiology 12/2012; 65(3):341. · 2.14 Impact Factor
  • Ali Eroglu, Lawrence C Layman
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    ABSTRACT: Assisted reproductive technologies (ART) offer revolutionary infertility treatments for millions of childless couples around the world. Currently, ART accounts for 1 to 3% of annual births in industrialized countries and continues to expand rapidly. Except for an increased incidence of premature births, these technologies are considered safe. However, new evidence published during the past decade has suggested an increased incidence of imprinting disorders in children conceived by ART. Specifically, an increased risk was reported for Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Silver-Russell syndrome, and retinoblastoma. In contrast, some studies have found no association between ART and BWS, AS, Prader-Willi syndrome, transient neonatal diabetes mellitus, and retinoblastoma. The variability in ART protocols and the rarity of imprinting disorders complicate determining the causative relationship between ART and an increased incidence of imprinting disorders. Nevertheless, compelling experimental data from animal studies also suggest a link between increased imprinting disorders and ART. Further comprehensive, appropriately powered studies are needed to better address the magnitude of the risk for ART-associated imprinting disorders. Large longitudinal studies are particularly critical to evaluate long-term effects of ART not only during the perinatal period but also into adulthood. An important consideration is to determine if the implicated association between ART and imprinting disorders is actually related to the procedures or to infertility itself.
    Seminars in Reproductive Medicine 04/2012; 30(2):92-104. · 3.21 Impact Factor
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    ABSTRACT: Pluripotent stem cells offer unique opportunities for curing debilitating diseases. However, further comprehensive research is needed to better understand cell signaling during the differentiation of pluripotent cells into different cell lineages and accordingly to develop clinically applicable protocols. One of the limiting steps for differentiation studies is proper culture and expansion of pluripotent stem cells, which is labor intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by successful banking and distribution of embryoid bodies (EBs), which are aggregates of pluripotent stem cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying survival and functionality of cryopreserved EBs. To this end, EBs were formed by culturing mouse 129 embryonic stem (ES) cells in the absence of leukemia inhibitory factor (LIF) in hanging drops and then subjected to different cryopreservation protocols. In a series of experiments, we first tested the postthaw survival of EBs as a function of dimethylsulfoxide (DMSO) and extracellular trehalose concentrations and cooling rates. Next, we studied the functionality of cryopreserved EBs by assessing their postthaw attachment, growth, and differentiation into various cell types. Higher (≥5%) DMSO concentrations alone or in combination with trehalose (0.1 M and 0.2 M) yielded good postthaw survival rates of >80%, whereas cooling of EBs at 1°C/min in the presence of 5% DMSO +0.1 M trehalose gave the best attachment and growth rates, with differentiation into cell lineages of three germ layers. Taken together, our results suggest that EBs are tolerant to cryopreservation-associated stresses and retain their differentiation potential after freezing and thawing. Furthermore, our experiments with dissociated EB cells and nondissociated EBs suggest that the extracellular matrix may play a beneficial role in the cryotolerance of EBs. Overall, our data support the feasibility of EB banking, which would facilitate advancement of cell-based therapies.
    Rejuvenation Research 12/2011; 14(6):641-9. · 2.92 Impact Factor
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    Edyta A Szurek, Ali Eroglu
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    ABSTRACT: The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.
    PLoS ONE 01/2011; 6(11):e27604. · 3.73 Impact Factor
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    ABSTRACT: Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) compose >40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions is not known. Here we show that an LTR retrotransposon of ERV-9 human endogenous retrovirus located 40-70 kb upstream of the human fetal gamma- and adult beta-globin genes serves a long-range, host function. The ERV-9 LTR contains multiple CCAAT and GATA motifs and competitively recruits a high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. The LTR complex transcribes intergenic RNAs unidirectionally through the intervening DNA to loop with and modulate transcription factor occupancies at the far downstream globin promoters, thereby modulating globin gene switching by a competitive mechanism.
    Proceedings of the National Academy of Sciences 07/2010; 107(29):12992-7. · 9.74 Impact Factor
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    Ali Eroglu
    Cryobiology 01/2010; 61(3):361-361. · 2.14 Impact Factor
  • Cryobiology 01/2010; 61(3):385-385. · 2.14 Impact Factor
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    Ali Eroglu
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    ABSTRACT: Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me(2)SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to -196 degrees C in the presence of either 0.3M raffinose and 0.5M Me(2)SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me(2)SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.
    Cryobiology 08/2009; 60(3 Suppl):S54-9. · 2.14 Impact Factor
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    ABSTRACT: Oocyte cryopreservation may avoid many complications of human embryo freezing and provide future fertility for women undergoing cancer therapy. The objective of this study was to explore the application of intra- and extracellular sugars in combination with small amounts of dimethylsulfoxide (DMSO) to human oocyte cryopreservation as an alternative approach. Discarded human oocytes that were obtained from IVF patients under informed consent and IRB approval, were cryopreserved by slow cooling to -196 degrees C after being randomly distributed into three groups: (i) DMSO control without intra- and extracellular sugar; (ii) extracellular sugar (raffinose) + DMSO; and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) + DMSO. Subsequently, all cryopreserved oocytes were thawed rapidly, and their survival was assessed by morphological criteria after 24 h of culture. A total of 71 oocytes were evaluated in three groups with survival rates of 88.5% (23/26), 68.2% (15/22), and 52.2% (12/23) for intra- and extracellular sugar+DMSO, extracellular sugar+DMSO, and DMSO control groups, respectively. These results support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes.
    Journal of Assisted Reproduction and Genetics 07/2009; 26(6):341-5. · 1.82 Impact Factor
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    ABSTRACT: Oocytes of nonhuman primates such as rhesus monkeys are excellent models for diverse studies on developmental biology, epigenetics, human reproduction, and assisted reproductive technologies, as well as on transgenics. Such studies require numerous oocytes that can be retrieved after controlled ovarian stimulation. Currently, most primate centers use laparoscopic aspiration or laparotomy followed by aspiration to collect rhesus oocytes, although the ultrasound-guided needle aspiration is more advantageous due to reduced infection risk, less injury, and a shorter recovery period. Yet, some initial difficulties associated with the ultrasound-guided needle aspiration limit its broader application. The objective of the present study was to address these obstacles. By presenting practical solutions to the initial difficulties, results from our study show that it is possible to collect a mean number of 38 +/- 10 rhesus oocytes per hormonally stimulated female. These results compare favorably to the average number of rhesus oocytes collected using the laparoscopic approach and suggest that when initial obstacles are overcome, the ultrasound-guided oocyte retrieval represents a good alternative to more invasive approaches.
    Molecular Reproduction and Development 07/2009; 76(9):890-6. · 2.81 Impact Factor
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    ABSTRACT: Successful cryopreservation of oocytes of the rhesus monkey (Macaca mulatta) would facilitate the use of this valuable animal model in research on reproduction and development, while providing a stepping stone towards human oocyte cryopreservation and the conservation of endangered primate species. To enable rational design of cryopreservation techniques for rhesus monkey oocytes, we have determined their osmotic and permeability characteristics in the presence of dimethylsulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PROH), three widely used cryoprotectants. Using nonlinear regression to fit a membrane transport model to measurements of dynamic cell volume changes, we estimated the hydraulic conductivity (L(p)) and cryoprotectant permeability (P(s)) of mature and immature oocytes at 23.5 degrees C. Mature oocyte membranes were most permeable to PROH (P(s) = 0.56 +/- 0.05 microm/sec) and least permeable to DMSO (P(s) = 0.24 +/- 0.02 microm/sec); the permeability to EG was 0.34 +/- 0.07 microm/sec. In the absence of penetrating cryoprotectants, mature oocytes had L(p) = 0.55 +/- 0.05 microm/min/atm, whereas the hydraulic conductivity increased to 1.01 +/- 0.10, 0.61 +/- 0.07, or 0.86 +/- 0.06 microm/min/atm when mature oocytes were exposed to DMSO, EG, or PROH, respectively. The osmotically inactive volume (V(b)) in mature oocytes was 19.7 +/- 2.4% of the isotonic cell volume. The only statistically significant difference between mature and immature oocytes was a larger hydraulic conductivity in immature oocytes that were exposed to DMSO. The biophysical parameters measured in this study were used to demonstrate the design of cryoprotectant loading and dilution protocols by computer-aided optimization.
    Molecular Reproduction and Development 11/2008; 76(4):321-33. · 2.81 Impact Factor
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    ABSTRACT: Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.
    Biology of Reproduction 10/2008; 80(1):70-8. · 4.03 Impact Factor
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    ABSTRACT: Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models.
    Transgenic Research 01/2008; 16(6):751-8. · 2.61 Impact Factor
  • Cryobiology 01/2008; 57(3):323-323. · 2.14 Impact Factor
  • Fertility and Sterility - FERT STERIL. 01/2008; 90.
  • Ali Eroglu, Edyta Szurek
    Cryobiology 01/2007; 55(3):341-341. · 2.14 Impact Factor
  • A. Eroglu, E. Szurek
    Fertility and Sterility - FERT STERIL. 01/2007; 88.
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    ABSTRACT: This paper reports the results of an experimental study of the warming and cooling rates achieved using the popular Nakagata Protocol for murine sperm cryopreservation. Problems with the storage and maintenance of the huge number of genetically engineered mouse strains have led to an increased need for murine sperm preservation. Recent studies have begun to focus on optimizing the cryopreservation of murine sperm by carefully studying the effects of cooling and warming rates on sperm survival. In current practice, however, the Nakagata protocol is widely used. The actual cooling and warming rates achieved using the Nakagata protocol have not previously been determined; and the Nakagata protocol has a number of unspecified parameters which we have found can significantly affect cooling rates, warming rates and sperm survival. A detailed study of the thermal response of samples frozen and thawed using the Nakagata protocol reveals that the cooling rates range from 30 to almost 300 degrees C per minute depending on the exact manner in which the Nakagata protocol is implemented. Warming rates range from 160 degrees C/min to about 1000 degrees C/min. Sperm survival depended significantly on the particular cooling rate achieved, and less strongly on the warming rates. Overall, it was found that the particular manner in which the Nakagata protocol was implemented could strongly affect cooling rates and sperm survival; and, consistent with the findings of Mazur and Koshimoto, an optimal cooling rate appears to exist in the range of cooling rates that can be achieved using the Nakagata protocol.
    Cryobiology 03/2006; 52(1):99-107. · 2.14 Impact Factor

Publication Stats

727 Citations
24 Downloads
1k Views
122.98 Total Impact Points

Institutions

  • 2013
    • Institute of Molecular Genetics AS CR
      Praha, Praha, Czech Republic
    • Georgia Regents University
      • Institute of Molecular Medicine and Genetics
      Augusta, Georgia, United States
  • 2008–2013
    • Villanova University
      • Department of Mechanical Engineering
      Norristown, Pennsylvania, United States
  • 2005–2012
    • Georgia Health Sciences University
      • • Institute of Molecular Medicine and Genetics
      • • Medical College of Georgia
      Augusta, GA, United States
  • 2009
    • Mercer University
      • Department of Obstetrics and Gynecology
      Atlanta, Michigan, United States
  • 1996–2004
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1998–2003
    • Massachusetts General Hospital
      Boston, Massachusetts, United States