scholarly journals PSVII-22 Trichostatin A as an aging agent in an oocyte

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 214-215
Author(s):  
Haley A Arena ◽  
Emma C Hicks ◽  
Brian Whitaker
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Oocyte Maturation ◽  
Mitochondrial Membrane ◽  
Trichostatin A ◽  
Cumulus Cell ◽  
Potential Gradient ◽  
Electrochemical Potential ◽  
Germinal Vesicle Breakdown ◽  
Prolonged Aging

Abstract As oocytes experience prolonged aging, reactive oxygen species (ROS) typically accumulate, the mitochondrial electrochemical potential gradient dissipates, and cumulus cells undergo expansion. Trichostatin A (TSA), used to stimulate aging, delays oocyte maturation by inhibiting germinal vesicle breakdown during meiosis. The objective of this study was to study the effects of aging during in vitro oocyte maturation in pigs. Oocytes (n=881) were matured with or without TSA (100 ng/mL) for 24 or 48 h followed by an additional 16 h of maturation without TSA. At the end of maturation, oocytes (n=446) were evaluated for cumulus cell expansion (CCE). A portion of the oocytes were stained to determine the relative levels of ROS (n=476) or mitochondrial electrochemical potential gradient dissipation (n=405). Fluorescent images of the oocytes were acquired, images were analyzed using ImageJ, and statistical analysis of the data was performed using ANOVA and Tukey’s test. Oocytes matured with or without TSA for 48 h had significantly less (P < 0.05) CCE compared to oocytes matured without TSA for 24 h. Oocytes matured without TSA for 24 h generated significantly different (P < 0.05) levels of ROS compared to oocytes matured with TSA for 48 h. Oocytes matured without TSA for 48 h had significantly higher (P < 0.05) mitochondrial membrane potential compared to the all other treatments. Results indicate that oocytes experiencing prolonged aging have less CCE and a decrease in mitochondrial membrane potential but no consistent or predictable trends in ROS formation. The use of TSA to stimulate aging in pig oocytes remains a valid and a reliable option.

scholarly journals The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation

2019 ◽  
Vol 25 (11) ◽  
pp. 695-705 ◽  
Author(s):  
Usama AL-Zubaidi ◽  
Jun Liu ◽  
Ozgur Cinar ◽  
Rebecca L Robker ◽  
Deepak Adhikari ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Oocyte Maturation ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Local Level ◽  
Meiotic Spindle ◽  
Mitochondrial Activity ◽  
Wide Range ◽  
Spatio Temporal

Abstract Mitochondria are highly dynamic organelles and their distribution, structure and activity affect a wide range of cellular functions. Mitochondrial membrane potential (∆Ψm) is an indicator of mitochondrial activity and plays a major role in ATP production, redox balance, signaling and metabolism. Despite the absolute reliance of oocyte and early embryo development on mitochondrial function, there is little known about the spatial and temporal aspects of ΔΨm during oocyte maturation. The one exception is that previous findings using a ΔΨm indicator, JC-1, report that mitochondria in the cortex show a preferentially increased ΔΨm, relative to the rest of the cytoplasm. Using live-cell imaging and a new ratiometric approach for measuring ΔΨm in mouse oocytes, we find that ΔΨm increases through the time course of oocyte maturation and that mitochondria in the vicinity of the first meiotic spindle show an increase in ΔΨm, compared to other regions of the cytoplasm. We find no evidence for an elevated ΔΨm in the oocyte cortex. These findings suggest that mitochondrial activity is adaptive and responsive to the events of oocyte maturation at both a global and local level. In conclusion, we have provided a new approach to reliably measure ΔΨm that has shed new light onto the spatio-temporal regulation of mitochondrial function in oocytes and early embryos.

scholarly journals Mitochondria modulate programmed neuritic retraction

2018 ◽  
Vol 116 (2) ◽  
pp. 650-659 ◽  
Author(s):  
Sergei V. Baranov ◽  
Oxana V. Baranova ◽  
Svitlana Yablonska ◽  
Yalikun Suofu ◽  
Alberto L. Vazquez ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Neuronal Death ◽  
Mitochondrial Membrane ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Potential Gradient ◽  
Protein Damage ◽  
Apoptosis Pathway

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon “neuritosis.” Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

scholarly journals Cumulus Cell Mitochondrial Resistance to Stress In Vitro Predicts Oocyte Development During Assisted Reproduction

2016 ◽  
Vol 101 (5) ◽  
pp. 2235-2245 ◽  
Author(s):  
Daniel A. Dumesic ◽  
Annie A. Guedikian ◽  
Vanessa K. Madrigal ◽  
Julia D. Phan ◽  
David L. Hill ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Ovarian Stimulation ◽  
Mitochondrial Membrane ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Energy Utilization ◽  
Oocyte Development ◽  
Resistance To Stress

Abstract Context: Complex cumulus cell-oocyte interactions govern energy utilization during oocyte development. Objective: This study investigates the relationship of cumulus cell mitochondria with oocyte development during ovarian stimulation for in vitro fertilization (IVF). Design: This is a prospective cohort study. Setting: The setting was an academic center. Patients: Thirty women underwent ovarian stimulation for IVF. Intervention(s): Pooled cumulus cells were collected; numbers of total and mature oocytes and two-pronuclear (day 1), six- to eight-cell cleavage (day 3), and blastocyst (day 5) embryos were recorded. Main Outcome Measure(s): A mitochondrial bioassay was developed with Jurkat cells and used with cumulus cells from IVF patients to correlate mitochondrial membrane potential resistance to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) stress with oocyte development and embryogenesis. Results: Adjusting for FSH administered and maternal age, cumulus cell mitochondrial membrane potential resistance to CCCP positively correlated with numbers of total (P < .025) and mature (P < .025) oocytes retrieved. The highest oocyte numbers that correlated with cumulus cell mitochondrial membrane potential occurred in women with the greatest ovarian response to FSH (mitochondrial membrane potential resistance to CCCP-log FSH interactions: total oocytes P < .025; mature oocytes P < .05). Multiple regression modeling of mature oocyte numbers, age, and cumulus cell mitochondrial membrane potential resistance to CCCP showed that numbers of mature oocytes best correlated with numbers of embryos at all stages (P < .0001). Conclusion: During ovarian stimulation for IVF, cumulus cell mitochondrial membrane potential resistance to stress correlates with numbers of total and mature oocytes retrieved, suggesting that cumulus cell–oocyte interactions involving energy facilitate oocyte development.

134 Trichostatin A as an aging agent during invitro fertilization in pig oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 175
Author(s):  
H. A. Arena ◽  
E. C. Hicks ◽  
K. N. Sprungl ◽  
S. B. Reynolds ◽  
B. D. Whitaker
Keyword(s):  
Cell Expansion ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Time Window ◽  
Trichostatin A ◽  
Cumulus Cell ◽  
Potential Gradient ◽  
Optimal Time ◽  
Oxygen Species ◽  
Treatment Groups

An aged oocyte is one that was not fertilized during the optimal time window after ovulation and potentially has diminished fertilization and embryonic development success. Oocytes (n=2562) were matured with or without trichostatin A (TSA; 100ng mg−1), a known meiotic inhibitor, for 24 or 48h (OMI), then for an additional 16h (OMII) without TSA or hormones. Oocytes were measured for meiosis before maturation (n=95), after OMI (n=365), and after OMII (n=230) as well as cumulus cell expansion (CCE). Oocytes (n=800) were fertilized with frozen–thawed boar sperm and potential embryos were evaluated for IVF characteristics and cleavage and blastocyst formation 48 and 144h after IVF, respectively. Apoptosis was determined in the maturing oocytes by levels of reactive oxygen species (ROS; n=476), mitochondrial electrochemical potential gradient dissipation (n=405), caspase 3 (n=405), phosphatidylserine (n=453), and DNA breakdown (n=192). Data were analysed by ANOVA and Tukey’s test. Oocytes matured without TSA for 48h had a significantly higher (P<0.05) percentage of oocytes at the MI stage of meiosis compared to all other treatment groups (16.00±6.80) and oocytes matured without TSA for 24h had a significantly higher (P<0.05) percentage of oocytes at the MII stage of meiosis compared to all other treatment groups (14.7±11.30). Oocytes matured with or without TSA for 48h had significantly less (P<0.05) CCE compared to oocytes matured without TSA for 24h. Supplementation of TSA to OMI significantly decreased (P<0.05) fertilization penetration rates compared with not supplementing TSA for 24h. Percent of embryos cleaved by 48h and those reaching blastocyst by 144h after IVF were significantly higher (P<0.05) in oocytes matured for 24h compared with those matured for 48h. Oocytes matured without TSA for 24h generated significantly different (P<0.05) levels of ROS compared with oocytes matured with TSA for 48h. Oocytes matured without TSA for 48h had significantly higher (P<0.05) mitochondrial membrane potential compared with all other treatments. Levels of caspase 3 activation and phosphatidylserine (which translocates in response to apoptosis) differed (P<0.05) between treatments. Results from the TUNEL assay indicate oocytes matured through OMII with a short OMI and TSA supplemented (23.8±2.99%) and through OMI with a long OMI and TSA supplemented (24.3±1.11%) had significantly greater (P<0.05) percent of oocytes with fragmented DNA than the other treatments, except for oocytes matured through OMII with a long OMI and TSA supplemented (35.0±3.55%), which was significantly greater (P<0.05) than all other treatments. Results indicate that use of TSA to stimulate aging in pig oocytes is a valid and a reliable option.

168 IMPROVED MITOCHONDRIAL MEMBRANE POTENTIAL AND PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS BY DIBUTYRYL cAMP TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 192 ◽  
Author(s):  
J.-S. Kim ◽  
D.-B. Koo ◽  
B.-S. Song ◽  
G. B. Wee ◽  
K.-K. Lee ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Embryonic Development ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Stages ◽  
Treated Group ◽  
Dibutyryl Camp ◽  
Germinal Vesicle Breakdown ◽  
Vitro Fertilization

Mitochondria play a pivotal role in energy metabolism and apoptosis during embryo development. In general, cAMP that exists at high level in GV oocytes inhibits germinal vesicle breakdown (GVBD), and the amount of cAMP in oocyte cytoplasm is gradually decreased for meiotic resumption. We first examined the effects of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryonic development. To determine whether mitocondrial activity is related to embryonic development, mitochondrial membrane potential (ΔΨm) was measured in porcine embryos. Porcine oocytes were cultured in NCSU-23 medium supplemented with 1 mM dbcAMP for 22 h and further cultured without dbcAMP for 22 h. After in vitro fertilization, porcine eggs were cultured in NCSU-23 medium with 4% BSA at 39�C, 5% CO2 in air for 6 d. Porcine embryos were obtained at various developmental stages and stained with the mitochondrial membrane potential-sensitive dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The mitochondrial membrane potential of porcine embryos was quantitatively evaluated by the ratio of green (presumptively alive mitochondria) to red fluorescent signal (presumptively dead mitochondria) using a fluorescence microscope. Acquired images were analyzed using DeltaVision Software (Applied Precision, Inc., Hsin-Chu City, Taiwan, ROC). After completion of IVM, dbcAMP-treated oocytes (91.3 � 0.9%) showed a higher proportion of the metaphase II stage than nontreated ones (72.8 � 2.6%) (P < 0.05). In the dbcAMP-treated group, sperm penetration rate was increased and polyspermic rate was reduced as compared to the nontreated group. Furthermore, the rate (37.3%, 47/126) of blastocyst formation in dbcAMP-treated group was higher than that (19.2%, 28/146) of the nontreated group (P < 0.05). After JC-1 staining, the number of blastomeres having live mitochondria per embryo increased in the dbcAMP-treated group at various developmental stages, whereas the number of blastomeres having dead mitochondria per embryo was enhanced in the nontreated group (43.3% vs. 30.2%). Our results indicate that in vitro maturation of porcine oocytes may affect mitochondrial membrane potential, apoptosis, and embryonic quality during pre-implantation development.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

Sign in / Sign up

Export Citation Format

Share Document