scholarly journals Glycine Ameliorates Endoplasmic Reticulum Stress Induced by Thapsigargin in Porcine Oocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Sicong Yu ◽  
Lepeng Gao ◽  
Chang Zhang ◽  
Yumeng Wang ◽  
Hainan Lan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Mitochondrial Function ◽  
Caspase 3 ◽  
Polar Body ◽  
Free Calcium ◽  
Voltage Dependent Anion Channel ◽  
Developmental Potential ◽  
First Polar Body

The endoplasmic reticulum (ER) is a multifunctional organelle in the cytoplasm that plays important roles in female mammalian reproduction. The endoplasmic reticulum and mitochondria interact to maintain the normal function of cells by maintaining intracellular calcium homeostasis. As proven by previous research, glycine (Gly) can regulate the intracellular free calcium concentration ([Ca2+]i) and enhance mitochondrial function to improve oocyte maturation in vitro. The effect of Gly on ER function during oocyte in vitro maturation (IVM) is not clear. In this study, we induced an ER stress model with thapsigargin (TG) to explore whether Gly can reverse the ER stress induced by TG treatment and whether it is associated with calcium regulation. The results showed that the addition of Gly could improve the decrease in the average cumulus diameter, the first polar body excretion rate caused by TG-induced ER stress, the cleavage rate and the blastocyst rate. Gly supplementation could reduce the ER stress induced by TG by significantly improving the ER levels and significantly downregulating the expression of genes related to ER stress (Xbp1, ATF4, and ATF6). Moreover, Gly also significantly alleviated the increase in reactive oxygen species (ROS) levels and the decrease in mitochondrial membrane potential (ΔΨ m) to improve mitochondrial function in porcine oocytes exposed to TG. Furthermore, Gly reduced the [Ca2+]i and mitochondrial Ca2+ ([Ca2+]m) levels and restored the ER Ca2+ ([Ca2+]ER) levels in TG-exposed porcine oocytes. Moreover, we found that the increase in [Ca2+]i may be caused by changes in the distribution and expression of inositol 1,4,5-triphosphate receptor (IP3R1) and voltage-dependent anion channel 1 (VDAC1), while Gly can restore the distribution and expression of IP3R1 and VDAC1 to normal levels. Apoptosis-related indexes (Caspase 3 activity and Annexin-V) and gene expression Bax, Cyto C, and Caspase 3) were significantly increased in the TG group, but they could be restored by adding Gly. Our results suggest that Gly can ameliorate ER stress and apoptosis in TG-exposed porcine oocytes and can further enhance the developmental potential of porcine oocytes in vitro.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 169
Author(s):  
H. M. Zhou ◽  
Y. Chen
Keyword(s):  
Skin Fibroblast ◽  
Polar Body ◽  
Subsequent Development ◽  
Fibroblast Cells ◽  
Fetal Skin ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
First Polar Body ◽  
Fetal Fibroblasts

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P > 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.

130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 145
Author(s):  
H. J. Kim ◽  
S. R. Cho ◽  
C. Y. Choe ◽  
S. H. Choi ◽  
D. S. Son ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Non Invasive ◽  
First Polar Body ◽  
Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 199
Author(s):  
L.-Y. Sung ◽  
C.-H. Chen ◽  
T.-A. Lin ◽  
L.-J. Sung ◽  
H.-Y. Su ◽  
...  
Keyword(s):  
Polar Body ◽  
Fluorescent Microscopy ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Adult Rabbit ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
First Polar Body

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

The life span of short-lived plasma cells is partly determined by a block on activation of apoptotic caspases acting in combination with endoplasmic reticulum stress

Blood ◽  
2010 ◽  
Vol 116 (18) ◽  
pp. 3445-3455 ◽  
Author(s):  
Holger W. Auner ◽  
Christine Beham-Schmid ◽  
Niall Dillon ◽  
Pierangela Sabbattini
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Caspase 3 ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Immunoglobulin Secretion ◽  
Effector Caspase ◽  
Induced Apoptosis

Abstract Apoptosis of short-lived plasma cells after a few days of intense immunoglobulin secretion is critical for maintaining a controlled humoral immune response. The mechanisms that regulate this process are poorly understood. Here we report that the key apoptotic caspases, caspase-3 and caspase-9, become resistant to activation by apoptotic stimuli when B cells differentiate into short-lived plasma cells. As a consequence, apoptosis of most short-lived plasma cells in vitro and in vivo is effector caspase-independent. We also show that a triaspartic acid repeat that normally prevents activation of caspase-3 becomes stabilized in short-lived plasma cells and myeloma cell lines. The block on caspase activation occurs before the accumulation of intracellular immunoglobulins and a progressive rise in secretory stress in the endoplasmic reticulum (ER). Plasma cells show increased susceptibility to ER stress–induced apoptosis and activate the ER-associated caspase-12, which is required specifically for nuclear apoptotic events. In nonlymphoid cells that cannot activate effector caspases, programmed cell death is delayed in response to ER stress. These observations suggest that the block on activation of key apoptotic caspases has evolved in short-lived plasma cells to prolong survival under conditions of ER stress resulting from high-level immunoglobulin secretion.

scholarly journals Development of in-vitro maturation protocol for rat oocytes; under simple culture vs co-culture with cumulus cell monolayer and its developmental potential via Parthenogenetic/artificial activation

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Muhammad Joan Ailia ◽  
Yun-Kyong Jin ◽  
Hee-Kyoung Kim ◽  
Goo Jang
Keyword(s):  
In Vitro Maturation ◽  
Disease Modeling ◽  
Polar Body ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Sprague Dawley ◽  
Developmental Potential ◽  
First Polar Body ◽  
Rats And Mice

Abstract Background Murine is the most abundantly used as laboratory animal models. There has been a tremendous amount of research including; their evolution, growth, physiology, disease modeling as well as genomic mapping. Rats and mice are the most widely used among them. Although both rats and mice fall under the same category still both are different a lot too. As regarding in vitro maturation and development mouse studies are well established as compared to rats which still lies in the early phase of development. So, we tried to figure out rat oocytes in vitro maturation and their developmental potential by performing 3 experiments i.e. superovulation, in vitro Maturation as simple culture (COC’s only), and COC’s & cumulus cells co-culture, which later further developed using parthenogenetic activation after IVM. Female Sprague Dawley rat 3–4 week used for these studies, we hyper-stimulated their ovaries using PMSG and hCG 150 IU/kg each. After that, we collected ovaries via dissection and retrieved oocytes. We matured them in TCM 199 supplemented with FSH, Estrogen, EGF, and Pyruvate. After maturation, we activated them using two types of activators i.e. Ethanol 7%, Ionomycin. After that, we saw and compared their developmental potential in vitro. Results Oocytes matured in COC’s and Cumulus cell monolayer co-culture (59% ± 4*) showed significantly more even growth and extrusion of the first polar body as compared to the COC’s only culture (53.8 ± 7%*). While oocytes activated using Ionomycin showed more promising development until 8 cells/blastocyst level compared to ethanol 7%. Conclusion we concluded that COC’s and cumulus monolayer co-culture is better than COC’s only culture. Cumulus monolayer provides extra aid in the absorption of nutrients and supplements thus providing a better environment for oocytes growth. Also, we concluded that matured oocytes showed more developmental capacity after activation via ionomycin compared to ethanol.

156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 192
Author(s):  
L. Cai ◽  
E. Kim ◽  
S. U. Hwang ◽  
J. D. Yoon ◽  
Y. Jeon ◽  
...  
Keyword(s):  
Polar Body ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
First Polar Body

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals MicroRNA-210 Regulates Endoplasmic Reticulum Stress and Apoptosis in Porcine Embryos

Animals ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 221
Author(s):  
Muhammad Rosyid Ridlo ◽  
Eui Hyun Kim ◽  
Geon A. Kim
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Embryo Development ◽  
Caspase 3 ◽  
Formation Rate ◽  
Embryo Production ◽  
Embryo Survival ◽  
Expression Levels ◽  
In Vitro Embryo Production

Endoplasmic reticulum (ER) stress can be triggered during in vitro embryo production and is a major obstacle to embryo survival. MicroRNA (miR)-210 is associated with cellular adaptation to cellular stress and inflammation. An experiment was conducted to understand the effects of miR-210 on in vitro embryo development, ER stress, and apoptosis; to achieve this, miR-210 was microinjected into parthenogenetically activated embryos. Our results revealed that miR-210 inhibition significantly enhanced the cleavage rate, blastocyst formation rate, and total cell number (TCN) of blastocysts, and reduced expression levels of XBP1 (p < 0.05). miR-210 inhibition greatly reduced the expression of ER stress-related genes (uXBP1, sXBP1, ATF4, and PTPN1) and Caspase 3 and increased the levels of NANOG and SOX2 (p < 0.05). A miR-210-mimic significantly decreased the cleavage, blastocyst rate, TCN, and expression levels of XBP1 compared with other groups (p < 0.05). The miR-210-mimic impaired the expression levels of uXBP1, sXBP1, ATF4, PTPN1, and Caspase 3 and decreased the expression of NANOG and SOX2 (p < 0.05). In conclusion, miR-210 plays an essential role in porcine in vitro embryo development. Therefore, we suggest that miR-210 inhibition could alleviate ER stress and reduce apoptosis to support the enhancement of in vitro embryo production.

Xanthohumol Induces Apoptosis in B-CLL In Vitro by Sustained Endoplasmic Reticulum Stress and Inhibition of NFκ-B.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4979-4979
Author(s):  
Sofie Lust ◽  
Barbara Vanhoecke ◽  
Mireille Van Gele ◽  
Mary Kaileh ◽  
Jerina Boelens ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Caspase 3 ◽  
Protein Translation ◽  
Mrna Levels ◽  
Induced Apoptosis ◽  
Sustained Phosphorylation

Abstract Introduction Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells. Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-xL, bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy. Results and conclusion X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-xL, bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.

scholarly journals Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

2021 ◽  
Author(s):  
Sicong Yu ◽  
Lepeng Gao ◽  
Yang Song ◽  
Xin Ma ◽  
Shuang Liang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dysfunction ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Protective Action ◽  
Damage Model ◽  
Developmental Competence ◽  
Free Calcium ◽  
Maturation In Vitro

Abstract Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which glycine affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether glycine could reverse the mitochondrial dysfunction induced by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, induced oxidative stress, which was confirmed by decreased mitochondrial membrane potential (Δ⍦m) and the expression of mitochondrial function-related genes (PGC-1α), and increased reactive oxygen species (ROS) levels and the expression of apoptosis-associated genes (Bax, caspase-3, CytC). More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca 2+]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) distribution. Nevertheless, treatment with glycine significantly ameliorated mitochondrial dysfunction, oxidative stress and apoptosis, glycine also regulated [Ca 2+]i levels and IP3R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes. Taken together, our results indicate that glycine has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.

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